Chapter  150:  Hereditary Nonpolyposis Colorectal Cancer: Diagnostic Strategies and Their Implications

Analytic Validity

Key Question 4: What is known about the analytic (sensitivity, specificity, reproducibility, reliability) and clinical validity of tests that identify HNPCC mutations?

The major laboratory tests used in the evaluation of patients suspected of having HNPCC include testing of tumor tissue using immunohistochemistry (IHC), microsatellite instability (MSI) testing, or germline (generally from peripheral blood mononuclear cells) testing for mismatch repair defects. Analytic validity may also apply to the accuracy of the family history.

Family members generally undergo only germline genetic testing (unless they have also developed a relevant cancer), ideally based upon the genotype of the proband. Detection of a pathogenic mutation (i.e., one known to be associated with HNPCC) in a proband permits testing of at-risk family members for the genotype. Family members with the same genotype have HNPCC while HNPCC can be excluded in those who do not. The situation is more complex when the probands do not have a detectable DNA alteration associated with HNPCC or when an alteration with unclear clinical significance is detected.

There was little published information on the analytic validity of laboratory testing in patients or family members suspected of having HNPCC. Thus, the analytic validity of the tests used to evaluate HNPCC is substantially uncertain. However, there was heterogeneity in the type of testing offered by commercial laboratories, and one study demonstrated variability in the accuracy of protein staining by IHC across facilities.

Additional information that could shed light upon analytic validity is available but would require evaluation of non-published data sources. For example, information has been collected through an external proficiency-testing program for MSI conducted by the College of American Pathologists and through internal testing performed by individual laboratories. Committees of experts could also review the strengths and limitations of specific testing techniques based upon clinical experience with these methods in HNPCC and in other genetically based disorders.

Clinical Validity

Key Questions 2a, 2b and 2c seek to evaluate the prevalence of MMR mutations, suggestive MSI and IHC, respectively, among patients fulfilling the Amsterdam I criteria or the Amsterdam II criteria. We considered several variables in making comparisons across studies such as how populations were selected, the methods used for genetic testing, whether and how mutations were described as being pathogenic, and whether testing for MMR genes other than MLH1 and MSH2 was performed.

Key Question 2a: Assuming a clinical definition of the Lynch Syndrome, what proportion of patients has a mismatch repair mutation?

Most eligible studies evaluated only MLH1 and MSH2 genes; only three studies assessed other MMR genes. Among CRC fulfilling Amsterdam I criteria, the random effects summary prevalence of MLH1 and MSH2 gene mutations was 44% (95% CI: 35, 52; n=19 studies, 464 patients), with evidence for substantial between-study heterogeneity (p<0.01; I²=52%). The six studies that performed sequencing among all Amsterdam I patients had a summary prevalence of 51% (95% CI: 35, 66%).

For patients fulfilling the Amsterdam II criteria, the corresponding prevalence values were 39% (95% CI: 30, 49%; 10 studies 279 Amsterdam II patients) and 40% (95% CI: 30, 52%) based upon two studies that performed sequencing on all 87 Amsterdam II patients.

The three studies examining additional genes (MSH6, PMS1 and PMS2) in Amsterdam I patients did not identify any additional MMR mutations. Two additional MSH6 mutations (in addition to five MLH1 and MSH2 mutations) were found among 20 Amsterdam II patients in a single study.

There were limited studies that performed more comprehensive genetic testing for pathogenic MMR genotypes associated with HNPCC or evaluated all the MMR genes that have been associated with HNPCC. For example, only one study performed sequencing in all samples and tested for large genomic mutations/rearrangements for three MMR genes (MLH1, MSH2, MSH6). The study included only 22 Amsterdam I patients; the prevalence of MMR mutation carriers was 64%.

Limited data suggested that approximately one-fourth to one-third of genotypes associated with HNPCC are related to deletions or rearrangements that would be missed through sequencing alone. As a result, the prevalence of mutations in Amsterdam I or II patients assessed from studies that performed sequencing alone are likely to be underestimates. Accounting for this effect, one may derive a prevalence of MMR mutations of approximately 63% to 67% in Amsterdam I patients. For Amsterdam II patients the corresponding prevalence values would be 50% to 53%.

Furthermore, limited data suggest that approximately 10% of MMR genotypes involve MMR genes other than MLH1 and MSH2. Thus, assuming an additional 10% increase in mutations by assessing more genes would result in an overall prevalence of up to 70% to 75% for Amsterdam I patients and 55 to 59% for Amsterdam II patients.

Because of the limited data, these calculations should be considered as being highly imprecise. Furthermore, other considerations, such as how patients are selected and how genotypes are classified as being pathogenic, may affect these estimates.

Key Question 2b: Assuming a clinical definition of the Lynch Syndrome, what proportion of patients has MSI?

Among patients fulfilling the Amsterdam I criteria, 71% (95% CI: 63, 78%; n=11 studies, 159 patients) of tumors were MSI-H. Among patients who fulfilled the Amsterdam II criteria, the corresponding summary prevalence was 68% (95% CI: 58, 76%; n=4 studies, 102 patients).

Key Question 2c: Assuming a clinical definition of the Lynch Syndrome, what proportion of patients has abnormal protein expression by immunohistochemistry?

Among patients fulfilling the Amsterdam I criteria the overall prevalence of tumors with loss of protein expression by IHC was 40% (95% CI: 28, 53%; n= 6 studies, 63 patients) with no evidence for between-study heterogeneity (p=0.75, I²=0%).

Only one eligible study provided relevant data for 20 patients fulfilling the Amsterdam II criteria. Eight out of 20 tumors had suggestive IHC for the MLH1, MSH2 or MSH6 genes (40% [95% CI: 9, 64%]).

MSI and IHC testing often depended upon practical and logistical considerations (e.g., patient availability and consent and availability of tumor tissue). Thus, not all patients had all tests and it was unclear whether additional bias may have been introduced in selecting patients for testing.

Key Question 2: How accurate are various predictors assuming a genetic definition of the Lynch Syndrome?

Decision Tree Modeling of Genetic Testing Strategies. We evaluated nine strategies for identifying patients with CRC with MMR mutations based upon combinations of clinical features, laboratory testing of tumor tissue (i.e., IHC and MSI testing). These can be conceptually organized into four general strategies:

• Group 1: Perform genetic test on everyone

• Group 2: Screen with a set of clinical criteria

• Group 3: Screen tumor tissue with a laboratory test

• Group 4: Screen using two serial tests: a set of clinical criteria first, and then a laboratory test of tumor tissue

Thus, of the nine clinical strategies, the presence of at least one of three clinical predictors: (i) age less than 50 years old at diagnosis, or ii) a history of colorectal or endometrial cancer in a first degree family member, or iii) the presence of multiple, synchronous or metachronous colorectal or endometrial cancers in the proband, combined with testing of tumor tissue for either IHC or MSI, identified a similar number of patients with colorectal cancer who had mismatch repair mutations associated with HNPCC (and failed to identify a similar number) compared with other, more complex approaches. There were relatively more studies demonstrating test characteristics of MSI testing compared with IHC and thus greater precision in the estimates of sensitivity and specificity of MSI testing.

Benefits and Harms

Key Question 1: Does risk assessment and HNPCC mutation testing in patients with newly diagnosed CRC lead to improved outcomes for the patient or family members, or is it useful in medical, personal, or public health decision making? (Over-arching question)

No study directly addressed Key Question 1.

Key Question 3: What are the harms associated with screening high-risk individuals for HNPCC?

Studies were considered eligible for Key Question 3 if they reported harms of a risk assessment process (e.g., Amsterdam, Bethesda and/or MSI, IHC) used to identify CRC patients at increased risk for HNPCC.

No study described harms of the risk assessment process in CRC patients at increased risk for HNPCC.

Key Question 5: What are the harms associated with screening for high-risk individuals?

Studies were considered eligible for Key Question 5 if they reported the harms associated with testing CRC patients for MMR mutations. Common harms that are thought to be associated with genetic testing are labeling, discrimination in health coverage, and emotional distress.

Three quantitative, comparative studies of quality A and B, and one qualitative study of B quality reported harms associated with MMR mutation testing in CRC patients. One 1-year prospective study (Grade A) compared the psychological impact of MMR mutation testing between mutation carriers and non-carriers. Subjects in this study were CRC probands or relatives from HNPCC families with a prior diagnosis of any cancer (excluding non-melanoma skin cancer). Anxiety, depression, and quality of life measures did not change over time, and there were no differences in these measures between mutation carriers and non-carriers. Distress levels were significantly decreased 2 weeks and 6 months after revealing the genetic testing results but were not significantly different from the baseline at 1-year follow-up. There was no difference in the distress levels between mutation carriers and non-carriers.

Another 1-month prospective study (Grade B) found that three of the 27 probands (11%) had minor depression at 1 month after revealing the genetic testing results, but the prevalence of minor depression was not significantly different compared to the prevalence at baseline or between mutation carriers and non-carriers. Of the six probands who received a positive result, two (33%) felt severe guilt regarding their children.

One prospective study (Grade B) reported changes in the psychological outcomes of CRC patients from self-completed questionnaires pre- and 4–6 weeks post-genetic counseling. There was no genetic testing performed in this study. There was a trend toward greater anticipated ability to cope with a positive gene test after counseling, as reflected by a decrease in anxiety and cancer-specific distress.

The qualitative study of 111 newly diagnosed CRC patients reported a high acceptance and understanding about information on HNPCC. Nineteen percent of participants rated their current level of worry caused by the genetics information at or above the midpoint of 4 on a 1 (not at all) to 7 (all the time) scale.

Key Question 6a: What are the management options for CRC patients who are HNPCC positive?

6b: Does the identification of HNPCC mutations lead to improved patient outcomes in terms of early detection, mortality/morbidity or management decisions (e.g., counseling, surveillance, treatment, other decision making) by patients and providers?

There are three aspects to Key Question 6: 1) Are management options for patients with CRC with a MMR mutation different from those without a MMR mutation? 2) Does the knowledge of MMR mutation status change management decisions by patients and providers? 3) Does changing management options for MMR positive patients with CRC improve outcomes (e.g., prognosis and survival) compared to standard approaches for patients with CRC?

We encountered a variety of surgical and medical management options in patients with CRC who were MMR positive but there were no comparative studies.

Indirect evidence from one study suggested that identification of HNPCC mutations was associated with better prognosis of CRC. However, there were no data on whether management options for CRC differed based on MMR mutation status.

Indirect evidence from one study showed no difference in survival of patients with endometrial cancer, comparing those who were mutation positive to those who were mutation negative.

In five studies with indirect evidence, there was no evidence in favor or against differences in survival, when comparing CRC patients who fulfilled different clinical criteria for HNPCC or screened positive for HNPCC by suggestive laboratory testing with those who did not.

Key Question 7: What are the harms associated with subsequent management options after identification of HNPCC mutations in CRC patients?

No study described harms associated with subsequent management options after identification of HNPCC mutations in patients with CRC or other forms of HNPCC-related cancers.

One study (involving two centers) described the types of colorectal surgery performed on CRC patients who were part of an Amsterdam criteria-positive family, and compared rates of metachronous cancers that followed each type of index operation. The overall rate of second surgeries for metachronous cancer were 23% in patients who underwent right colectomy, 17% in patients who underwent left/sigmoid colectomy or proctosigmoidectomy, 0% in patients who underwent total/subtotal colectomy, and 44% in patients who underwent segmental colectomy. The two centers had significantly different second resection rates for metachronous cancer.

One study described the survival rate of 45 patients with gastric cancer from HNPCC families with MMR mutations. Many of these patients had already had treatments for other HNPCC-related cancers, including CRC. The 5-year survival was higher in patients in whom radical surgery was performed (48%) than in patients in whom radical palliative surgery or explorative laparotomy alone was performed (15%).

Key Question 8b: What is the accuracy of HNPCC testing in family members in predicting the risk of CRC?

8c: Do other factors, such as race/ethnicity, age, gender, or co-morbidities affect the accuracy of the testing?

Only two studies of B quality reported the risk of CRC in family members of probands with positive MMR mutations (the proposed framework). The lifetime risk of CRC was 68.7% for men and 52.2% for women with MMR mutations in one study, and it was 74% and 30% respectively in the other study. Men had higher lifetime risk of CRC than women in both studies.

In a study that reported the risk of CRC in family members of probands with HNPCC based on clinical criteria, the cumulative risk of CRC by age 75 years old was 57% and 41% in families that fulfilled the Amsterdam I and II criteria, respectively. The cumulative risk of CRC by age 75 years old was 42% and 23% for men and women, respectively, from families that fulfilled Bethesda criteria. In another study, family members of CRC probands who were younger than 50 years old at cancer diagnosis had a higher risk of CRC, compared to family members of CRC probands who were 50 years old and older. The risk of CRC was increased three-fold, comparing first-degree relatives of CRC probands who developed a second primary in the HNPCC spectrum with the single primary group in a third report.

In a study that reported the risk of CRC in kindreds with HNPCC based on genetic criteria, the cumulative risk of CRC by age 70 yr was 82% in MLH1/MSH2 mutation carriers. The cumulative incidence of CRC was 100% in men and 54% in women. There was overall a higher risk of CRC in men, but the sex difference was not consistent among HNPCC kindreds with different MMR mutations.

Key Question 9: What are the harms associated with informing/counseling family members or with subsequent testing for HNPCC mutations?

Key Question 8a: What is the efficacy of pre-test genetic counseling for informing family members of potential risks and benefits of testing?

Studies were considered eligible for Key Question 8a if they summarized the efficacy of pre-test genetic counseling immediately after counseling and before performing genetic testing. Studies addressing Key Question 9 were considered together with those addressing Key Question 8a because there was substantial overlap in the studies addressing these questions. The studies addressed the long-term efficacy of pre-test genetic counseling or harms associated with screening high-risk individuals (such as by using clinical criteria, MSI, or IHC), genetic testing, and informing/counseling family members or with subsequent testing for HNPCC mutations.

Four studies addressed the efficacy of pre-test genetic counseling immediately after counseling and before performing MMR genetic testing. Of these, three were of B quality and one was of C quality.

Eight comparative and four qualitative studies addressed the harms associated with genetic testing for HNPCC mutation or with informing/counseling family members or with subsequent testing for HNPCC mutations. Of these, one study was of A quality, six of B quality, and five of C quality.

Overall, pre-test genetic counseling had good efficacy in improving knowledge about HNPCC, and resulted in high likelihood of proceeding with genetic testing, satisfaction in the decision to undergo genetic testing, and decreasing depression and distress levels among family members of HNPCC probands or among asymptomatic individuals from HNPCC families. Family members of HNPCC who received positive MMR mutation test results had higher psychological distress levels and anxiety compared to those who received negative test results, but this difference generally disappeared with time. However, all of these psychological measures were within the normal ranges for the general populations. There were no differences in quality of life, comparing mutation carriers to non-carriers or the general population.

Key Question 10a: What are the management options for family members of CRC patients who have a positive HNPCC test?

b1: Does the identification of HNPCC mutations lead to improved outcomes in terms of decision making by patients, family members and providers, or public health policy?

b2: Does the identification of HNPCC mutations lead to improved outcomes in terms of early detection and mortality/morbidity of patients, family members?

We included studies evaluating all forms of cancer related to HNPCC since family members of CRC probands are potentially at risk for all such cancers.

Six studies examined the impact of mutation testing on the decision to undergo specific management recommendations among family members of CRC patients or asymptomatic individuals from HNPCC families. Of these, four were of B quality and two were of C quality. No study directly examined the impact of HNPCC mutation testing on public health policy or decision making by insurance providers.

Two studies (in three publications) of B and C quality indirectly addressed outcomes of early detection and mortality/morbidity in relation to the identification of MMR mutations in family members of CRC probands or asymptomatic individuals from HNPCC families. These studies were limited by potential selection bias, and/or unclear effects from treatments or subsequent interventions.

Identification of HNPCC mutations was associated with improved outcomes in terms of decision making to undergo screening for cancers in family members of HNPCC. HNPCC family members who took subsequent actions or interventions had a lower risk of developing HNPCC-related cancers and lower mortality rates, compared to those who did not take actions. Most data pertained to screening for CRC with colonoscopy while there was less information about screening for other forms of HNPCC-related cancer.

Key Question 11: What are the harms associated with subsequent actions or interventions for family members?

We included studies that reported any outcome relating to subsequent management options or interventions in HNPCC family members.

Nine studies reported outcomes related to subsequent actions or interventions among family members. Of these, six were of B quality and three were of C quality. Four of these nine studies reported harms or adverse events associated with subsequent actions or interventions for family members. In addition, one study of C quality examined the psychological impacts associated with colonoscopies. Some of these studies did not have a control group of subjects who declined to undergo surveillance and most results did not adjust for potential confounders such as age, personal history of cancer, and educational levels.

Less than 0.5 percent of family members experienced harms associated with screening or surveillance examination or surgical procedures in the studies we evaluated. However, complication rates associated with these interventions in the non-HNPCC setting are probably applicable. There was some negative psychological impact associated with colonoscopies.

Clinical Criteria That Suggest the Diagnosis

The recognition that certain types of cancers cluster in families with HNPCC and that cancer develops at relatively early ages compared with the general population provided the rationale for development of criteria that could be used to aid in the diagnosis. Two sets of criteria (the Amsterdam criteria and Bethesda guidelines) developed by a consensus of experts, have been most widely accepted and best studied, although many similar criteria have been proposed.^{24, 25} Both have been revised since their initial development.^{26, 27} These criteria have applied by healthcare providers and genetic counselors during interviews with the patient and/or their family and/or with assistance of written documents such as a survey.

Table 4

Original and revised Amsterdam criteria

Table 4

Original and revised Amsterdam criteria

Original (Amsterdam I)	Revised (Amsterdam II)
At least 3 relatives with colorectal cancer, one of whom must be a first degree relative of the other two Involvement of 2 or more generations At least 1 case diagnosed before age 50 Familial adenomatous polyposis has been excluded	At least 3 relatives with HNPCC-associated cancer One should be 1st degree relative of other two At least 2 successive generations affected At least 1 diagnosed before age 50 Familial adenomatous polyposis excluded Tumors should be verified by pathologic examination

Table 5

Original and revised Bethesda guidelines

Table 5

Original and revised Bethesda guidelines

Original

Revised

Individuals with cancer in families that meet the Amsterdam criteria Patients with two HNPCC-related cancers, including synchronous and metachronous colorectal cancer or associated extracolonic cancers (endometrial, ovarian, gastric, hepatobiliary, small bowel, or transitional cell carcinoma of the renal pelvis or ureter). Patients with colorectal cancer and a first-degree relative with colorectal cancer and/or HNPCC-related extracolonic cancer and/or a colorectal adenoma with one of the cancers diagnosed before age 45 years, and the adenoma diagnosed before age 40 years. Patients with right-sided colorectal cancer having an undifferentiated pattern (solid/cribriform) on histopathologic diagnosis before age 45 years. Patients with signet-ring cell type colorectal cancer diagnosed before age 45. Patients with adenomas diagnosed before age 40.

Colorectal cancer (CRC) diagnosed in a patient <50 Presence of synchronous, metachronous colorectal or other HNPCC-associated tumors regardless of age CRC with the MSI-H-like histology diagnosed in a patient less than 60 CRC diagnosed in a patient with one or more 1st degree relatives with an HNPCC-related tumor, with one of the cancers being diagnosed under age 50 CRC in a patient with two or more 1st or 2nd degree relatives with HNPCC-related tumors, regardless of age

Although the Bethesda guidelines and Amsterdam criteria continue to be used widely, several studies evaluating them (both the original and revised) have underscored the limitations of their accuracy in predicting the presence of mismatch repair mutations.^{11, 12, 20, 28, 29} A 2006 review of the literature reported that the sensitivity of the original Amsterdam criteria ranged from 54 to 91%.⁷ Such a wide range of estimates leaves substantial uncertainty as to the role of the Amsterdam criteria as a screening test for mismatch repair mutations. As described, above, there are many potential explanations for the variability across studies. In this report, we attempt to clarify important differences across studies that may help explain the variability.

In addition to the limitations regarding their predictive accuracy, there are practical problems with policies based on the implementation of these clinical criteria. Patients' report of the family history may not be accurate, particularly for cancers other than colorectal that are potentially related to HNPCC.³⁰ Issues of uncertain paternity may also be relevant in some families while some families may be too small (or have insufficient contact among family members) to obtain a clinically meaningful family history. In addition, the criteria are not always remembered or practical to obtain; as a result many caregivers (including oncologists) fail to obtain a detailed family history, and among those who do, many do not act appropriately upon it.^{30, 31}

Testing All Cancers Regardless of the Family History

Because of the limitations of relying on clinical criteria to guide testing, some authorities have proposed that tumors from patients with colorectal cancer be evaluated for markers of HNPCC regardless of the family history.^{8, 32} One of the largest studies evaluating this approach⁸ included 1066 patients with colorectal cancer whose tumors were tested for MSI. Patients with suggestive MSI results were tested for germ-line mutations in the mismatch repair genes (MSH2, MLH1, MSH6, and PMS2) by IHC, genomic sequencing, and deletion studies. A mutation causing HNPCC was detected in 23 patients (2.2 percent) of whom ten were older than 50 and five did not meet the Amsterdam criteria or Bethesda guidelines.

These data suggest that the Amsterdam or Bethesda criteria alone may miss as many as 22 percent of patients with HNPCC. However, only five additional individuals from the cohort of 1066 subjects (one-half of 1 percent) would have been identified by routine molecular analysis of all colon cancers fulfilling the Bethesda criteria, making such an approach impractically expensive for routine clinical use. Furthermore, the detailed laboratory analysis the authors performed on tumor tissue is not widely available. Despite these considerations, we include a strategy of testing all patients with colorectal cancer for mismatch repair mutations for comparison against alternative strategies.

Combinations of Family History and Laboratory Testing

Most expert guidelines on HNPCC suggest a combination of sequential laboratory testing in patients who fulfill the Amsterdam criteria or Bethesda guidelines to minimize costs and maximize test accuracy.^{1, 33} Approaches based on such a strategy have been considered to be cost-effective.^{19, 34} However, the exact methods and order of testing are unsettled. Proposed strategies include initial testing of tumors for MSI with or without IHC for loss or expression of mismatch repair proteins, with germline gene sequencing reserved for patients with suggestive results. Certain histologic features of HNPCC-related tumors may also raise clinical suspicion, but none is sufficiently specific to establish the diagnosis.^{35, 36} One report identified a specific oral manifestation (Fordyce granules) as highly predictive of mismatch repair mutations, but this observation has not yet been confirmed.³⁷ Several other strategies for selecting patients for genetic testing have been described, but none has been widely adopted. Thus, this report focuses mainly on the predictive accuracy of testing tumor tissue for MSI and IHC.

Microsatellite instability occurs as a result of “slippage” of DNA polymerase during DNA replication of microsatellite DNA sequences (short dinucleotide or mononucleotide repeats).¹ These are normally repaired by DNA repair mechanisms. In the presence of deficient mismatch repair functions (such as in HNPCC) these errors are not corrected, leading to a state that is referred to as microsatellite instability. The United States National Cancer Institute (NCI) defines the MSI-high (MSI-H) when two of five microsatellite markers from a standard panel display instability and the MSI-low phenotype when only one marker is unstable. Tumors without instability are labeled as microsatellite stable (MSS).

The NCI panel has been widely adopted in recent years, although some centers use additional or different markers. MSI-H tumors are generally more predictive of mismatch repair mutations than MSI-low tumors. However, approximately 10 to 20 percent of spontaneous colorectal cancer test positive for MSI-H, not all laboratories test for the full panel of microsatellite markers suggested by the National Cancer Institute, MSI-testing is not widely available, and archived tissue may not be readily available to perform such testing.

In some studies, MSI-H and MSI-L tumors are combined and compared to MSS, while in others each category has been considered separately. In this report, we record the specific methods used for determining MSI status and how tumors were categorized to permit valid comparisons among studies.

IHC techniques can identify the expression of mismatch repair proteins.^{38, 39} Testing for other mismatch repair proteins has not been performed routinely, although it may be important in some families. This approach is less costly than MSI testing and is technically much easier. Mutations associated with HNPCC generally lead to the absence of a detectable gene product, although some may lead to a dysfunctional protein that can still be detected (and hence cause a false negative result).

Some studies have suggested that almost all tumors in which MSH2 or MLH2 is absent by IHC demonstrate MSI-H, while approximately 8 percent of MSI-H tumors will demonstrate retained immunostaining.³⁹ However, the extent to which IHC and MSI correlate with one another is not known precisely. Nevertheless, some authorities have proposed that IHC may be a suitable alternative to MSI testing while others consider the two to be complementary. In this report we attempt to clarify these issues by providing test characteristics of each approach used alone or in combination.

Studies Related to Analytic Validity

We required the following for studies of analytic validity:

• The study evaluated biological material from patients with colorectal cancer considered to be at risk for HNPCC. This criterion was selected to choose studies evaluating laboratory techniques that were directly applicable to the spectrum of genotypes associated with HNPCC. We did not consider it feasible to conduct a systematic review on the individual laboratories tests used for other conditions.

• Reported any of the following (these criteria were selected to choose studies evaluating the most common tests performed in HNPCC):

  • 1

    Proportion of tumors that were MSI-H with National Cancer Institute (NCI) panel of markers (i.e., BAT-25, BAT-26 D2S123, DS346 and D17S250) versus other markers

  • 2

    Sensitivity or specificity of MSI-H using NCI markers compared with a reference standard that the study claimed was better

  • 3

    Sensitivity or specificity of IHC compared with an immunohistochemical standard that study claimed was better

  • 4

    Sensitivity or specificity of a genetic technique compared with a reference standard (or combination of standards)

  • 5

    Reliability of MSI, IHC or genetic methods across laboratories or within a laboratory.

• Data were extractable into 2×2 tables.

We excluded studies that included the index test in the reference standard. For example, some studies attempted to calculate sensitivity of a specific NCI MSI marker by comparing it with a reference standard that included the marker plus additional markers. Such an approach produces an invalid estimate of sensitivity since the calculation includes the index test (i.e., the specific NCI marker) in the numerator and the denominator.

Studies Related to Clinical Validity

We required all three of the following criteria to be met for studies of clinical validity with extractable data:

• Enrolled patients with colorectal cancer

• Compared an index test to genetic testing (at least one of the following: suggestive family history, MSI, or IHC)

• Sought mutations using DNA sequencing (or other similar genetic approaches) for a minimum of MLH1 and MSH2.

HNPCC has been defined clinically and genetically as described in Chapter 1. We used both approaches depending upon which Key Questions were being addressed. In all cases, the definitions used are described explicitly in the tables and figures.

Key Questions 2a–2c focus on the prevalence of MMR mutations, suggestive MSI or ICH among patients with a clinical definition of HNPCC. We defined Lynch Syndrome I and Lynch Syndrome II using the Amsterdam I and II criteria, respectively. Studies that reported this proportion or provided sufficient data for it to be calculated were eligible.

We required that studies evaluated all available patients who fulfilled the Amsterdam criteria or a representative (random) sample thereof. We excluded studies that did not evaluate all patients because they selected a non-random patient sample (e.g., patients who fulfilled the Amsterdam criteria who were also younger than a certain age).

Key Question 2 pertains to the predictive accuracy of clinical or laboratory features for determining the presence of mismatch repair mutations. We assessed the performance of different clinical and laboratory predictors (preliminary tests) to identify carriers of MMR gene mutations. We considered all studies that provided relevant data. When the primary study reported proportions rather than actual counts, we reconstructed the 2×2 tables using the information conveyed by the proportions and their 95% confidence intervals.

We analyzed the following predictors:

Laboratory predictors:

• 1

  MSI high versus MSI stable.

• 2

  MSI high and low versus MSI stable.

• 3

  Suggestive IHC versus non suggestive IHC.

Clinical predictors specified a priori:

• 1

  Amsterdam I criteria fulfilled versus not fulfilled.

• 2

  Amsterdam II criteria fulfilled versus not fulfilled.

• 3

  Modified Amsterdam criteria fulfilled versus not fulfilled.

• 4

  Bethesda guidelines fulfilled versus not fulfilled.

• 5

  Revised Bethesda guidelines fulfilled versus not fulfilled.

• 6

  Young age of onset (<50 years) versus later age of onset

• 7

  Presence of CRC or HNPCC related cancer in first degree family versus sporadic CRC cases.

• 8

  Presence of CRC or HNPCC related cancer in family (any definition) versus sporadic CRC cases (irrespectively of how they were selected).

Clinical predictors specified a posteriori:

• 9

  Presence of multiple tumors in a CRC proband versus probands without multiple tumors.

• 10

  Presence of young age of onset (<50 years) or suggestive family history of cancer or multiple tumors in a CRC proband (i.e., predictors #6 or #7 or #8) versus absence of all three characteristics.

We focused only on patients with CRC who received at least some form of genetic testing to minimize the effects of verification bias. Verification bias occurs when patients with a negative test result are not evaluated with the reference test.⁴⁷ We generally did not accept screening with MSI or IHC as a substitute for genetic testing. A single exception pertained to studies assessing clinical predictors among newly diagnosed, unselected, non-referral CRC, because this was of particular clinical importance and there were few studies available for analysis. For such studies we accepted the authors' assumption that patients who were not tested for mutations based on MSI and IHC test results were indeed mutation negative.

Studies Related to Benefits and Harms

For studies related to benefits and harms, we accepted studies of virtually any design and using any definition of HNPCC that reported any outcomes or other findings pertinent to patients or families with HNPCC, or provided insights into these issues from a public health perspective. We did not confine our inclusion criteria to studies of patients with colorectal cancer even though this report focuses on patients with colorectal cancer. Benefits and harms of genetic testing, counseling and other management approaches are pertinent to the full spectrum of tumors associated with HNPCC. For example, a woman with colorectal cancer who is found to have HNPCC is at risk for other forms of HNPCC-related cancer (such as endometrial cancer). Thus, the benefits and harms related to genetic testing may not only be relevant to clinical issues related to colorectal cancer management (and prevention of metachronous cancers) but also management (and prevention) of other HNPCC-related cancer in the patient or her family members. We expanded the scope of all pertinent Key Questions to provide as comprehensive a view on these issues as possible.

Although we attempted to be as comprehensive as possible in including studies related to benefits and harms, we occasionally encountered studies that did not report any outcomes or other findings that appeared to be relevant to the Key Questions and thus excluded them. As noted above, the reasons that specific studies were excluded are summarized in the Appendix D ^*. List of Excluded Studies.

Questions Related to Analytic Validity

As will be discussed in Chapter 3, we encountered only a few studies of analytic validity, and thus these are described in the text rather than in tables. There were insufficient data for a pooled analysis.

Questions Related to Clinical Validity

Studies relevant to each Key Question were grouped together, organized according to their applicability to the specific Key Question and their overall quality score. In some cases, Key Questions that had substantive overlap in the type of findings reported were grouped together to provide a comprehensive yet succinct overview of the literature.

Questions related to clinical validity: For questions related to clinical validity, sensitivity and specificity were based upon the definitions summarized in Chapter 1. We presented studies by categorizing them for important features (such as use of similar definitions and selection criteria) so that similarities and differences would be as transparent as possible.

Prevalence of MMR Mutations, MSI or Suggestive IHC Among Patients Fulfilling Amsterdam I and II Criteria. For each study we estimated the proportion (and exact binomial 95% confidence interval) of Amsterdam I and II patients with MMR mutations, MSI or suggestive IHC. We estimated the corresponding summary prevalence values with random effects meta-analyses.^{52, 53} For the quantitative syntheses, proportions from each study were logit transformed to stabilize variances, and then back-transformed to their natural scale. Analyses using the more drastic Tuckey-Freeman arcsin transform yielded largely similar estimates, and thus are not reported.

Diagnostic Ability of Predictors (Preliminary Tests) To Identify MMR Mutation Carriers. As mentioned above, the preliminary tests that identify MMR mutation carriers are either sets of clinical criteria, or laboratory tests (namely MSI and IHC). Different considerations are applicable to the analysis of clinical and laboratory predictors across a selection of studies.

Considerations on study populations for clinical predictors (preliminary tests). In HNPCC the clinical preliminary tests are highly susceptible to spectrum effects across populations that have been selected with eligibility criteria of a clinical nature. Although the detailed analysis of this statement is cumbersome, it can be intuitively evaluated through an illustrative example. Assume that the preliminary test of interest is the clinical criterion of “age less than 50 years at diagnosis of CRC”. It is expected that the sensitivity and specificity of this criterion will be different among unselected people with CRC compared to CRC patients who were less than 55 years at diagnosis. The second population has already been “pre-selected” based on a clinical criterion (less than 55 years at CRC diagnosis) that is quite similar to the evaluated “preliminary test” (less than 50 years at CRC diagnosis). This pre-selection alters the composition of the population with respect to the clinical predictor in a systematic and non-random way, both among MMR mutation carriers and among non-carriers. The net effect is a change in the apparent sensitivity and specificity across populations with increasing prevalence of MMR mutation carriers (spectrum effects).

Because of the aforementioned consideration we decided a priori not to synthesize the sensitivity and specificity of clinical predictors across populations that had been defined using different clinical eligibility criteria. Identifying such homogeneous populations is challenging, especially given the incomplete descriptions of the studied populations in many of the assessed studies.

Thus we decided to focus on populations fulfilling sets of criteria that are most frequently used for HNPCC, are well known, and presumably assessed identically by different research teams. In order of increasing prevalence of MMR mutation carriers these homogeneous populations were: unselected, incident CRC, patients selected by the Bethesda guidelines and revised Bethesda guidelines, modified Amsterdam criteria, and Amsterdam II and I criteria. We also created separate estimates for the sensitivity and specificity for patients who were pre-selected based on suggestive MSI and/or IHC results.

Considerations on study populations for laboratory predictors (preliminary tests). The above consideration may not be as important for laboratory predictors (e.g., IHC) because there is no strong reason to assume that pre-selection with a clinical eligibility criterion (e.g., “age less than 55 at CRC diagnosis”, as above) in a study would be correlated with IHC results both in MMR carriers and in those who are not MMR mutation carriers. However, it is possible that some laboratory predictor tests (e.g., MSI analysis) may be vulnerable to spectrum effects.⁵⁴ Thus, for laboratory predictors, we did not require the strict similarity of populations across studies as above, and considered all studies together.

Separate analyses of sensitivity and specificity. For each study we estimated the sensitivity and specificity (and exact binomial 95% confidence intervals thereof) for the clinical and laboratory predictors (preliminary tests) of interest. As described before, we derived summary sensitivity and specificity estimates with random effects syntheses using logit-transformations of proportions for the meta-analyses. As a general rule, this approach tends to underestimate the diagnostic performance, because it ignores the correlation between the sensitivity and the corresponding specificity from the same study. However, it provides a pooled estimate (and 95% confidence interval) of sensitivity and specificity that can be useful for providing overall appraisal of test performance.

Summary receiver operating characteristic curve analyses. For laboratory predictors only, we summarize test performance graphically using summary receiver operating characteristic curve (SROC) analyses. SROC curve analysis can be used to graphically describe the tradeoff between sensitivity and specificity across studies. The sensitivity and specificity of each study are represented in the graph, thereby depicting the operating characteristics of a group of studies.

The tradeoff between sensitivity and specificity generally reflects the threshold for calling a test positive or negative. Such a tradeoff can be understood easily when considering a single study that assessed various cutoff values of a diagnostic test. By contrast, the threshold implied in an SROC curve analysis is not always apparent. Different estimates of sensitivity and specificity across studies may be due to several considerations such as variations in how the tests were implemented (e.g., differences in the number of microsatellite markers used) or in interpretation of results. With regard to the latter, the same specimen (e.g., a tumor stained for IHC) may be interpreted differently when the interpreter believes the test is being used for screening versus confirmation of a high-risk patient. Thus, by grouping studies according to specific study features, SROC analysis can be used to help explain differences in test characteristics across studies that seemingly used the same diagnostic test. However, it does not necessarily define an explicit threshold effect.

The area under a receiver operating curve has been used to describe the overall diagnostic ability of a test; the greater the area, the better the test. However, calculating the area under a SROC curve requires extrapolation outside the range of the sensitivity and specificity values of the analyzed studies and thus may not produce a valid appraisal of the test's accuracy. As a result, we did not attempt to calculate the area under the SROC curve.

Subgroup and Sensitivity Analyses. We defined a priori factors that might explain heterogeneity of pooled estimates. These included:

Overall quality. We compared results from high and low quality studies (as defined by the A, B or C classification described above).

Study size. For the questions relating to the proportion of patients with the Lynch Syndrome with mutations or abnormal MSI or IHC results, we used a cutoff of 20 patients (≥20 versus <20) to distinguish larger and smaller studies. Although this was a largely arbitrary cutoff, a single misclassification in smaller studies would result in more than a 5% misclassification rate, which we considered clinically important. Similarly, we used a cutoff of at least 40 people in the 2 by 2 table for studies that were used to assess sensitivity and specificity.

Characteristics of how genetic testing was performed. Studies were categorized in groups according to the comprehensiveness of the genetic testing strategy they used. As least comprehensive, we classified studies that used only gene screening methods to detect MMR mutations, and did not perform sequencing on any sample; performed sequencing on samples with suggestive gene screening analyses; or performed sequencing on all available samples. As most comprehensive we considered studies that performed sequencing and analyses for large genomic deletions/rearrangements in all samples. The application of more advanced techniques such as conversion analysis or mono allelic mutation analysis (MAMA) was only sporadic or in the context of demonstrating their feasibility in only a few samples, and thus did not comprise a separate category. Examples of genetic screening strategies are single-stranded conformation polymorphism (SSCP), conformation sensitive gel electrophoresis (CSGE), denaturing gradient gel electrophoresis (DGGE), and denaturing high-pressure liquid chromatography (DHPLC). Examples of methods to detect large genomic deletions are: southern blotting and multiplex ligation-dependent probe amplification (MLPA). These methods are described in more detail on Chapter 3 in the section on Analytic Validity.

Whether and how the study defined pathogenic mutations. We determined if and how studies defined mutations as being pathogenic (i.e., known to be associated with HNPCC versus a variant of unknown significance). It is possible that the same mutations might have been defined as pathogenic in one study and non pathogenic in another.⁵⁵ Furthermore, not all definitions are equally valid and the methods used to define pathogenicity may not have been conducted with equal rigor. For example evidence from functional studies (i.e., in which the function of the mutated gene was assessed) may provide strong support that the mutation is pathogenic, whereas absence of the mutation in a small sample of healthy controls is not as strong.

Characteristics of MSI testing. We assessed whether studies that used the panel of markers recommended by the NCI and whether they performed microdissection. Microdissection helps assure that the sample analyzed was from malignant tissue and did not contain DNA from surrounding, healthy colonic tissue. Microdissection is not pertinent to germline MMR mutation testing or IHC. Germline mutations are typically assessed in blood samples (non-malignant tissue). For IHC, a pathologist studies a tissue section to evaluate MMR protein expression in regions of malignancy (microdissection it typically not needed).

The sample selection process. We attempted to identify studies that had a biased sample selection process. Studies with transparent sample selection process clearly stated that they selected their samples among all available patients using a set of eligibility criteria. Studies with non-transparent sample selection process did not report that they applied the same criteria to all available patients. For example, they may have used a convenience sample of cases from various sources.

Despite our efforts, we caution that studies that applied seemingly transparent eligibility criteria may still have a highly biased selection process. For example, studies from a referral center may have recruited patients with a relatively higher prevalence of familial cancer compared with studies that recruited consecutive patients with colorectal cancer in the community.

Whether the study used consecutive, unselected patients with CRC. We identified studies that used consecutive CRC (including retrospective studies that assessed all unselected patients that were diagnosed during a specific time period) that were otherwise nonselective or representative of the general population. Populations evaluated in studies from specialized centers or studies that imposed clinical or other criteria to select their populations were not considered to be representative of the general population.

Decision Tree Model Methods. We performed analyses using decision trees to model the expected outcomes with different testing strategies from the payers'/third party perspective. The outcomes were the number of incident CRC with positive diagnosis for HNPCC, and the number of tests (MMR, MSI, or IHC) needed to detect them. We also assessed how many patients found to be mutation carriers with each strategy would be truly positive.

The decision trees pertain to a cross-section in time. We used a hypothetical population of 100,000 incident cases of CRC. This number is in the order of incident cases expected annually in the US (approximately 150,000, given an annual incidence of 50/100,000 and a population of 300 million) and is a number convenient for calculations.

We assessed nine different strategies, which were most commonly represented in the studies summarized in this review. The strategies used clinical criteria, MSI, IHC or a combination of clinical criteria with MSI or IHC to select patients for MMR mutation testing. The probabilities that were used in the decision trees were derived from the eligible studies. See Chapter 3 for a complete description of the modeled strategies and the probabilities that were used.

Questions Related to Benefits and Harms

For studies related to benefits and harms, we grouped studies according to their relevance to the Key Questions, presenting comparative trials and higher quality studies first followed by qualitative and other types of descriptive studies. Statistical comparisons made within each study are presented; we did not attempt to recalculate these comparisons; however, specific comments about study methods are included in footnotes.

The majority of studies related to benefits and harms were qualitative. Among the comparative trials, there were insufficient studies of similar design to allow for meta-analysis.

We present major findings that were considered to be clinically important or addressed (directly or indirectly) the Key Questions and subset questions. We described together studies that used similar endpoints (e.g., quality of life instruments or depression scales) and interventions (e.g., surveillance colonoscopy) whenever possible.

Evidence and Summary Tables

Evidence tables offer a detailed description of the studies that addressed each of the Key Questions. Each study appears once regardless of how many interventions or outcomes were reported. We did not attempt to construct evidence tables for all the extracted studies but all data extracted for each study are included in data extraction forms that are available in Appendix C ^*.

Summary tables succinctly report summary measures of the main study features and outcomes evaluated. They are designed to facilitate comparisons and synthesis across studies. Individual studies may appear more than once if the data they contain are relevant to more than one Key Question. The summary tables are featured in sections corresponding to each Key Question in Chapter 3.

Key Question 4: What is Known About the Analytic (Sensitivity, Specificity, Reproducibility, Reliability) and Clinical Validity of Tests That Identify HNPCC Mutations?

Definition

The analytic validity of a laboratory test refers to its accuracy and reliability for identifying a finding of interest (such as a genotype). There are four general elements of analytic validity (see http://www.cdc.gov/genomics/gtesting/ACCE.htm for further details):

• 1)

  Sensitivity (i.e., the ability to detect a finding when it is present)

• 2)

  Specificity (i.e., the ability to exclude a finding when it is absent)

• 3)

  Laboratory quality control (i.e., the procedures used in a testing laboratory to ensure that results fall within specified limits)

• 4)

  Assay robustness (i.e., how resistant the assay is to changes in pre-analytic and analytic variables).

Laboratory Tests Used in HNPCC

The major laboratory tests used in the evaluation of patients suspected of having HNPCC include testing of tumor tissue using IHC, MSI testing, or germline (generally from peripheral blood mononuclear cells) testing for mismatch repair defects. Family members generally undergo only germline genetic testing (unless they have also developed a relevant cancer), ideally based upon the genotype of the proband. Detection of a pathogenic mutation (i.e., one known to be associated with HNPCC) in a proband permits testing of at-risk family members for the genotype. Family members with the same genotype have HNPCC while HNPCC can be excluded in those who do not. The situation is more complex when the proband does not have a detectable DNA alteration associated with HNPCC or when an alteration with unclear clinical significance is detected. In such cases, it may not be possible to exclude HNPCC in family members; as a result, they may be empirically offered enhanced cancer surveillance.

Table 11

Genetic testing strategies used by studies included (more...)

Table 11

Genetic testing strategies used by studies included in quantitative analyses

Study, year	Brief description of genetic strategy	MMR genes other than MLH1, MSH2	Sequencing	Gene screening	Deletion analysis
Syngal, 2000 Wahlberg, 2002 Rossi, 2002 Wolf, 2005 Lee, 2005 Moslein, 1996 Debniak, 2000 Peel, 1999	PCR → Sequencing	No	All samples	X	X
Farrington, 1998	PCR → IVSP → Sequencing	No	All samples	√	X
	PCR → Sequencing
Barnetson, 2006	PCR → Sequencing (MSH6, some MLH1 and MSH2 exons)	MSH6	All samples	√	X
	PCR → DHPLC → Sequencing
Wang, 1999	PCR → IVSP → Sequencing	MSH6	Practically all samplesa	√	√
	PCR → HD → Sequencing	PMS1
	in vivo MLH1 expression analysis	PMS2
	PCR → Sequencing (of all other samples)
Durno, 2005	[used PTA and Sequencing]	No	Some samples?b	√	X
Aaltonen, 1998	MSI → PCR → DGGE (some samples) → Sequencing;	No	Some samples	√	√
	MSI → PCR → Sequencing (all other samples)
	PCR for founder mutations in MLH1 (all samples)
Salovaara, 2000	MSI → PCR → Sequencing	No	Some samples	X	√
	PCR for founder mutations in MLH1 gene
Liu, 2004 Colombino, 2005 Yuan, 2004 De Abajo, 2005	PCR → DHPLC → Sequencing	No	Some samples	√	X
Southey, 2005	PCR → DHPLC → Sequencing	MSH6	Some samples	X	√
	MLPA (in only 10 tumors)c	PMS2
Curia, 1999 de Leon, 1999 Dieumegard, 2000 Yuan, 1998	PCR → SSCP → Sequencing	No	Some samples	√	X
Katballe, 2002 Christensen, 2002	PCR → SSCP & HD →Sequencing	No	Some samples	√	X
Luce, 1995	PCR → IVTT → Sequencing	No	Some samples	√	X
Zhu, 2005	MLPA → Sequencing	No	Some samples	X	√
Park, 1999	PCR → SSCP	PMS1	None	√	√
	Southern blotting	PMS2d
Callistri, 2000	PCR → SSCP	No	None	√	X
Lamberti, 1999	PCR → SSCP	No	None	√	X
	RT-PCR, PTA
Samowitz 2001	MSI → PCR → Sequencing	No	Some samples	X	√
	MSI → PCR for founder mutations in MLH1
Raedle, 2001 Pistorius, 2000	MSI → PCR → Sequencing	No	Some samples	X	X
Terdiman, 2001	MSI → PCR → DGGE → Sequencing	No	Some samples	√	X
Nakahara, 1997	MSI → PCR→ SSCP → Sequencing	No	Some samples	√	X
Casey, 2005	MSI, IHC → PCR → Sequencing	No	All availablee	X	√
	MSI, IHC → Conversion analysis
	MSI, IHC → Deletion analysis
Pinol, 2005	MSI, IHC → PCR → Sequencing	No	Some samples	X	√
	MSI, IHC → MLPA
Stormorken, 2001	Not described	MSH6	Unclear	Unclear	Unclear

DHPLC: denaturating high performance liquid chromatography; DGGE: Denaturating gradient gel electrophoresis; HD: heteroduplex formation; IHC: immunohistochemistry; IVSP: in vitro synthesized protein test; IVTT: in vitro transcription translation; MLPA: multiplex ligation-dependent probe amplification; MSI: Microsatellite instability testing; PCR: polymerase chain reaction; SSCP: Single-stranded conformation polymorphism.

Shown are the genetic strategies used in the various studies that were assessed in the quantitative analyses of this report. All studies assessed MLH1 and MSH2 at minimum.

a

In Wang 1999 essentially all patients were sequenced, but not completely; however, complete sequencing was performed for those testing negative with other methods.

b

Durno 1999 does not describe the used strategy in detail.

c

The 10 tumors were MSI positive, IHC negative and MMR negative with the usual strategy.

d

In Park 1999 PMS1 was assessed in 27 samples and PMS2 on 24 samples out of 123 samples for MLH1 and MSH2 (all were negative, unclear how the 27 and 24 were selected).

e

All available patients were tested with full sequencing. However, the sample was assembled mainly on the basis of suggestive MSI testing (85 out the 89 patients had suggestive MSI testing).

Immunohistochemistry. Pathogenic mutations in mismatch repair proteins usually lead to the absence of a detectable gene product providing the rationale for IHC techniques used to detect underexpression. Tumors from patients suspected of having HNPCC (based upon clinical or pathologic findings) can be stained for mismatch repair proteins. The surrounding normal colonic tissue can be used as a positive control.

As noted in the table below, testing is available commercially for MLH1, MSH2, MSH6 and PMS2, although the extent to which various laboratories stain for all of these proteins is unclear. Furthermore, such staining is relatively easy to perform and available in kits. As a result, local pathologists, who may select to stain for some or all of these proteins, can perform it.

Knowledge of how the mismatch repair proteins interact during DNA repair can help interpret the results of such testing and be useful for guiding germline genetic testing. For example, MSH2 forms a heterodimer with MHS6; MLH1 complexes with PMS2 and binds to the MSH2–MSH6 heterodimer. When MSH2 is not expressed in a tumor, MSH6 is also not expressed. Because MSH2 and MLH1 are the most common mismatch repair proteins implicated in HNPCC, a patient who has a tumor that does not stain for MSH2 (and whose normal surrounding colonic mucosal demonstrates preserved staining) is most likely to have a mutation in MSH2 (but could also possibly have a mutation in MHS6). Similarly, MSH6 may be the likely gene involved in a patient with a tumor that expresses MLH1 and not MSH6 (again with normal staining patterns in surrounding colonic mucosal). The situation is more complex with lack of expression of MLH1; promoter hypermethylation of MLH1 is common with sporadic colorectal cancer and may lead to its underexpression. Some laboratories offer methylation analysis to help determine whether lack of expression is due to promoter hypermethylation. Such an approach has been suggested in various reviews of HNPCC.

Immunohistochemistry has an advantage over other techniques (particularly MSI testing) since it is much easier to perform and is less expensive. However, the technique is vulnerable to the quality of tissue preparation, staining, and interpretation. This concern is not merely, hypothetical; our literature search revealed that sensitivity to detect loss of MSH2 expression ranged from 84 to 100 percent in a study of 18 participating centers performing such testing.⁵⁶ Variability in specificity was even greater (see below).

Microsatellite Instability. Microsatellite instability (MSI) refers to a variety of patterns of microsatellite repeats observed when DNA is amplified from a tumor with defective mismatch repair compared with DNA amplified from surrounding normal colonic tissue. Repetitive mono- or dinucleotide DNA regions are particularly vulnerable to defective mismatch repair. For example, the mononucleotide sequence “AAAAAAAAAAAAAAAAAAAAAAAAAA” is located on chromosome 2P, near the MSH2 gene locus. This sequence is referred to as the “Big A Tract-26” (BAT26). As a result, a tumor suspected to result from mismatch repair defects can be tested for the presence of these repeats.

MSI testing involves amplification of a standardized panel of DNA markers; five markers were agreed upon by a consensus panel convened by the National Institutes of Health in 1997 (BAT25, BAT26, D2S123, D5S345, and D17S250) as described in Chapter 1. Three categories of MSI have been recognized based upon these panels: MSI-high (instability of two or more markers), MSI-low (instability of one marker), and MS-stable (no instability). More recently, some laboratories have begun using ten or more markers. In such cases MSI is defined as “stable” when fewer than 10% of markers are unstable, “low” when 10 to 30% of markers are unstable, and “high” when greater than 30% of markers are unstable (some laboratories use 40%). We recorded the markers used in each study included in this review (see section on clinical validity and accompanying tables).

There are several pitfalls of MSI testing. First, it is labor intensive, relatively costly (compared with IHC), and requires expert pathologic services. In addition, tissue to be amplified should ideally be microdissected to avoid amplifying DNA from normal colonic mucosa. We systematically recorded whether microdissection was performed for all studies related to clinical validity. As a practical consideration, tissue may not always be available since the diagnosis of HNPCC may not be suspected when the cancer was first diagnosed.

Genetic Testing. Multiple methods have been used for genetic testing in HNPCC. The methods used should ideally be able to detect the many potential genotypes associated with HNPCC (e.g., nonsense, missense, and frameshift mutations, genomic deletions, duplications, and rearrangements). We recorded the specific methods and order of testing used in all studies included in this report and, in the case of clinical validity, attempted to discern whether the specific testing methods were associated with the accuracy of HNPCC testing (see sections and corresponding tables on clinical validity).

A detailed review of these methods is beyond the scope of this report. However, the following summarizes major categories of testing that are used currently, and that were reported in the studies included in this report.

High Output Screening Techniques. High output screening techniques include single stranded conformation polymorphisms (SSCP), conformation sensitive gel electrophoresis (CSGE), denaturing gradient gel electrophoresis (DGGE) and denaturing high-pressure liquid chromatography (DHPLC). These methods all take advantage of the observation that alteration of DNA (due to a polymorphisms or mutation) confers chemical properties that allow it to be differentiated from normal DNA. These approaches can be performed relatively rapidly and allow more detailed studies (such as DNA sequencing) to be targeted to specific regions of DNA.

DNA Sequencing. DNA sequencing can be used following a high output screening technique or as a primary approach (particularly when IHC patterns allow for targeting of a specific mismatch repair gene). It is considered the method of choice for detecting most mismatch repair gene mutation. However, it does not reliably allow for detection of deletions or rearrangements, which are important in HNPCC. DNA sequencing has become automated in recent years, greatly reducing the required time, costs, and expertise.

Conversion analysis. Conversion analysis involves converting diploid cells to haploid cells so that only a single allele is analyzed at a time. The rationale is based upon the observation that a wild-type allele can mask the presence of a mutant allele when performing DNA sequencing (thereby obscuring the presence of a mutation). Conversion testing can increase the yield of genetic testing in HNPCC but is technically complicated, expensive, and, as a result, not widely available. We recorded whether conversion analysis was performed in all studies included in this report.

Methods To Detect Large Structural DNA Abnormalities. Large structural DNA abnormalities (such as large genomic deletions, rearrangements) are potentially important in HNPCC but are not detected by the high output screening techniques or DNA sequencing. There are several methods for detecting these defects. We recorded the specific methods used for all studies included in this report.

Southern blotting involves digestion of genomic DNA (which breaks it into pieces), separation of fragments using electrophoresis, transfer of the fragments to a membrane, and hybridization using probes to recognize deletions, duplications or rearrangements. Southern blotting has not yet been automated to the extent of DNA sequencing and is time-consuming.

Multiplex ligation-dependent probe amplification (MLPA) is a newer technique. It involves measurement of the relative copy number of DNA sequences. MLPA has evolved to become a standard approach for analyzing mismatch repair genes for deletions.

Family History. The family history (and related risk assessment tools such as the Amsterdam criteria) can also be considered as a type of laboratory testing, which can be applied to both the probands and their family members. The accuracy of the family history in predicting the presence of germline mismatch repair mutations is described in the sections on clinical validity. By contrast, the analytic validity of the family history can be considered the accuracy with which individuals are able to report their family history.

We did not identify any studies that assessed the analytic validity of the family history in patients or families with HNPCC. However, we confined our literature search specifically to HNPCC, while there are several studies that have assessed the validity of the family history for a variety of tumor types, including those associated with HNPCC such as colorectal or endometrial cancer.⁵⁷–⁶⁰ A systematic review of these data found that (in individuals without a personal history of cancer) the positive and negative likelihood ratios of a family history for colon cancer in a 1^st degree relative were 23.0 (95% CI 6.4–81.0) and 0.25 (95% CI 0.1–0.63), respectively.⁵⁹ These values were 14.0 (95% CI 2.2–83.4) and 0.68 (95% CI 0.31–1.52), respectively for endometrial cancer. In another report, the degree of relationship to the probands, type of cancer, age at diagnosis of the probands, and source of ascertainment of probands were all statistically significant predictors of the accuracy of reporting.⁶⁰ The extent to which these data can be generalized to patients and families with HNPCC is unclear.

Limitations of a Literature-Based Approach to Analytic Validity

Table 12

North American laboratories offering clinical testing (more...)

Table 12

North American laboratories offering clinical testing for Hereditary Nonpolyposis Colon Cancer (Lynch Syndrome)*

Clinical laboratories Location	Analysis of entire coding region: Sequence analysis	Sequence analysis of select exons	Analysis of entire coding region; Mutation scanning	Targeted mutation analysis	Linkage analysis	Microsatellite instability testing (MSI)	Deletion/duplication analysis	Mutation scanning of select exons	Immuno-histochemistry	Sequence analysis of RNA
ARUP Laboratories, Inc. Salt Lake City, UT	---	---	---	---	---	PCR: BAT25, BAT26, D2S123, D5S346, D17S250 in MSI low (instability<30%) samples, additional markers available: BAT40, MYCL1, TGS-beta-R2, D10S197, D18S58	---	---	---	---
Baylor College of Medicine Medical Genetics Laboratories Houston, TX	PCR-based assay: MLH1, MSH2, MSH6 aMLH1 promoter methylation assay (in combination with MSI analysis): MLH1, MSH2, MSH6	---	---	---	---	Multiplex PCR-based assay (in combination with MSI analysis): BAT25, BAT26, NR21, NR22, D2S123, D17S250, D5S346, D18S35, DIS2883	PCR-based assay: MLH1, MSH2	---		---
Boston University School of Medicine Center for Human Genetics Boston, MA	MLH1, MSH2, MSH6	MLH1, MSH2, MSH6	---	---	---	---	MLPA	---	---	---
Children's Hospital of Eastern Ontario Molecular Genetics Diagnostic Laboratory Ottawa, Ontario, Canada	MLH1, MSH2	---	---	---	---	---	MLH1, MSH2	---	---	---
City of Hope Clinical Molecular Diagnostic Laboratory Duarte, CA	√	---	Fluorescent sequencer: MLH1, MSH2, MSH6	√	---	Amplification ≥5 markers by denaturing polyacrylamide gel electrophoresis: MSI ≥2 markers, low MSI = 1 marker	---	---	MLH1, MSH2, MSH6	---
Creighton University Medical Center Creighton Medical Laboratories Omaha, NE	---	---	---	---	---	√	---	---	---	---
Fox Chase Cancer Center Clinical Molecular Genetics Laboratory Philadelphia, PA	---	---	---	---	---	√	---	√	---	---
Huntington Medical Research Institutes Molecular Oncology & Cancer Genetics Laboratory Pasadena, CA	MSH2, MLH1, MSH6	MSH2, MLH1	---	---	MSH2, MLH1	PCR: MSH2, MLH1	√	---	---	√
London Health Sciences Centre Molecular Diagnostic Laboratory London, Ontario, Canada	MSH2, MLH1	---	---	---	---	√	√	---	MLH1 and MSH2 in combination with MSI analysis	---
Mayo Clinic Molecular Genetics Laboratory Rochester, MN	MLH1, MSH2, MSH6	---	---	---	---	---	MLPA: MLH1, MSH2	---	PCR: MLH1, MSH2, MSH6, PMS2	---
Memorial University of Newfoundland Molecular Genetics Laboratory St. John's, Newfoundland, Canada	---	---	---	MISH2, Del 50 CODONS	---	---	---	---	---	---
Myriad Genetics Laboratory Salt Lake City, UT	MLH1, MSH2, MSH6 (Southern Blot available for MLH1 and MSH2	---	---	√	---	---	√	---	---	---
North York General Hospital Molecular Genetics Laboratory North York, Ontario, Canada	---	---	---	---	---	√	---	---	---	---
Ohio State University Molecular Pathology Laboratory Columbus, OH	---	---	---	---	---	BAT 25, BAT 26	---	---	---	---
Quest Diagnostics, Inc. Molecular Genetics Laboratory San Juan Capistrano, CA	MLH1, MSH2, MSH6	---	---	---	---	Multiplex polymerase chain reaction (PCR): BAT 25, BAT 26, D2S123, D5S346, D17S250	√	---	---	---
Saint Louis University Health Science Center DNA Diagnostic Laboratory Saint Louis, MO	---	---	---	---	---	√	---	---	---	---
UCLA Medical Center Diagnostic Molecular Pathology Laboratory Los Angeles, CA	---	---	---	---	---	√	---	---	---	---
University of Alberta Molecular Diagnostic Laboratory Edmonton, Alberta, Canada	---	---	MSH2, MLH1	---	---	---	√	---	---	---
University of Pennsylvania School of Medicine The Genetic Diagnostic Laboratory Philadelphia, PA	MSH2, MLH1 (If negative, Southern blot optional)	---	√	---	√	---	√	---	---	---

*

May include Muir-Torre Syndrome, Turcot Syndrome.

√ = Genetests.org indicates offered by laboratory, but no additional information given.

Abbreviations: PCR, Polymerase Chain Reaction/Fragment Analysis.

a

due to space limitations promoter methylation assay was reported in this column.

• A)

  Many of the laboratory techniques (e.g., DNA sequencing) are used not only in testing for HNPCC but also in testing for a variety of genetic disorders. As a result, a comprehensive search of issues related to analytic validity should consider the entire literature evaluating such testing, an undertaking that is neither practical nor feasible. A search in MEDLINE^® on DNA sequencing, for example, will yield more than 416,000 citations.

  As noted in the methods section, we confined our search to studies that used biological materials from patients or family members suspected of having HNPCC to make the results as applicable to the population of interest as possible. The accuracy of these methods used for other disorders may not be generalizable to HNPCC. The extent to which such sources of indirect evidence may be applicable to HNPCC was beyond the scope of this review.

• B)

  The methods used in laboratory testing evolve rapidly. The specific methods used are commercialized by companies or individuals who developed them. As a result, the published literature may not (and often does not) reflect the actual testing performed in clinical laboratories. It is customary for commercial laboratories to assess at least some components of analytic validity for these contemporary tests, but laboratories are under no obligation to publish the results.

  A partial list of laboratories offering testing services for HNPCC is available through “Genetests” an organization funded through the National Institutes of Health (see http://www.genetests.org/servlet/access?id=8888891&key=AmmF6UdFjuxHB&fcn=y&fw=vDlR&filename=/). A summary of the tests listed on this Website is presented in Table 12 below. We attempted to contact one of the laboratories (chosen at random) to determine whether they would share information related to analytic validity. Our attempt had limited success. Such a review of “gray” literature may be helpful for understanding the analytic validity of these tests but would require appropriate resources, including incentives, for participating laboratories.

  Some commercial laboratories participate in proficiency testing, but the results are generally not published. Proficiency testing involves distributing samples of tissue that are known to be positive or negative for findings of interest. Laboratories participating in such programs can obtain a benchmark for how they are performing in detecting (or excluding) such findings (e.g., genotypes of interest). Proficiency testing also helps understand issues related to reliability.

  A major limitation of proficiency testing is that it does not consider the pre-analytic components of testing; for example, the accuracy of a test may be greatly affected by the methods used to acquire tissue. In addition, proficiency testing does not allow for a detailed understanding of the spectrum of abnormalities (e.g., various genotypes) that may be clinically relevant.

  Proficiency testing for MSI was introduced in 2006 and is being conducted by the College of American Pathologists. Further information is available at their Website (see http://www.cap.org/apps/cap.portal) but the initial results of their program have not been made public.

• C)

  Studies evaluating analytic validity often suffer from serious methodologic limitations that potentially invalidate the results. Particularly problematic was the inappropriate use of reference standards or lack of clear definitions of references standards altogether. For example, a common pitfall was the inclusion of the test under evaluation as part of the reference standard.

  We chose inclusion and exclusion criteria carefully to select for high quality studies while excluding studies whose results are misleading or clinically irrelevant. There were remarkably few high-quality studies directly addressing analytic validity using biological materials from probands or family members proven or suspected to have HNPCC.

Eligible Studies. We identified only three studies that fulfilled eligibility criteria for analytic validity.

• One study compared conversion analysis (in which alleleles are separated into hybrids prior to mutation screening) with DNA sequencing alone to detect heterogeneous germline mutations in MLH1, MSH2 and MSH6 in patients with colorectal cancer.⁶¹ The authors estimated that conversion analysis provided an increased yield of 56% (35/63 cases) compared with DNA sequencing alone. The study was rated a methodologic quality B because the investigators were not blinded to the method used.

• Another study included 20 patients with CRC with known mutations in MLH1 or MSH2.⁵⁶ A set of two unstained slides from each case were sent to participating medical centers with capability of performing immunoperoxidase assays for MLH1 and MSH2.

Of 18 participating centers 2 were excluded: one because slides were damaged in transit and the other because of insufficient staining. Sensitivity for detecting loss of MSH2 expression ranged from 84 to 100%; 10 centers identified all six. Five out of six false positive results were in the same case suggesting that staining or interpretation were not random. Fourteen out of 16 laboratories showed 100% specificity (one laboratory had 93% specificity due to staining failure on one slide and one lab demonstrated 45% specificity due to weak or absent staining in most cases.)

Re-review of returned MSH2 slides showed lack of internal positive control staining in at least 2 of the 6 MSH2-negative cases from 8 of 16 centers. The other 8 centers had 100% sensitivity and 93–100% specificity on re-review. The slides that lacked internal positive control staining were largely accounted for by two cases.

The study was rated a methodologic quality B because of its small sample size, and because it did not describe quality control methods or whether microdissection had been performed when preparing the specimens.

• A third study evaluated the sensitivity and specificity of DHPLC analysis compared with DNA sequencing in 46 patients with colorectal cancer from families with HNPCC.⁶² DHPLC analysis identified 19 changes previously identified by DNA sequencing and 16 new alterations not previously described. DHPLC was considered to be highly sensitive, detecting a mutation in all patients with no false negative results. The study was rated methodologic quality C because of several deficiencies in reporting.

Summary. In summary, the analytic validity of the tests used to evaluate HNPCC is substantially uncertain. However, there is heterogeneity in the type of testing offered by commercial laboratories and the available data suggest that there may be variability across testing facilities. Additional information that could shed light upon analytic validity is available but would require evaluation of non-published data sources. Committees of experts could also review the strengths and limitations of specific testing techniques based upon clinical experience, with particular reference given to experience with these methods in other genetically-based disorders.

Key Question 2 and Pertinent Subquestions

HNPCC has been defined clinically and genetically as described in Chapter 1. The following sections present analyses using either the clinical or the genetic definition of the condition, depending on which Key Question was being addressed.

Key Questions 2a to 2c seek to evaluate the prevalence of MMR mutations and suggestive MSI and IHC among patients with the Lynch Syndrome. Thus, they assume a clinical definition of the condition.

Key Question 2 pertains to the ability of clinical and laboratory predictors to identify the presence of MMR mutations. Thus, it assumes a genetic definition of the condition.

We first present analyses based on a clinical definition of HNPCC. In these analyses we estimated the frequency of mismatch repair gene mutations and tumors with MSI and IHC among CRC patients fulfilling the Amsterdam I and II criteria.

We present subsequent analyses based on the genetic definition of the Lynch syndrome. These help define test characteristics of various strategies (such as the combination of a clinical history with laboratory testing of tumor tissue) for predicting the presence of mismatch repair mutations.

Finally, we used a decision tree model to calculate the expected number of patients with MMR mutations among unselected patients presenting with CRC using various predictive strategies. The parameters used in the calculations are based on our best estimates derived from this systematic review.

Key Question 2a: Assuming a Clinical Definition of the Lynch Syndrome, What Proportion of Patients has a Mismatch Repair Mutation?

Among CRC fulfilling Amsterdam I criteria, the random effects summary prevalence of MLH1 and MSH2 gene mutations was 44% (95% CI: 35, 52; n=19 studies, 464 CRC patients), with evidence for substantial between-study heterogeneity (p<0.01; I²=52%). The six studies that performed genetic testing among all Amsterdam I patients had a summary prevalence of 51% (95% CI: 35, 66%; 84 CRC patients).

For patients fulfilling the Amsterdam II criteria, the corresponding prevalence values were 39% (95% CI: 30, 49%; 10 studies, 279 CRC patients) and 40% (95% CI: 30, 52%) based upon 2 studies that performed sequencing on all 87 Amsterdam II patients.

Only three studies examined other MMR genes (MSH6, PMS1 and PMS2) in Amsterdam I patients, without identifying any additional mutations. Two additional MSH6 mutations were found among 20 Amsterdam II patients in a single study.

Frequency of MMR Gene Mutations Among Patients Fulfilling Amsterdam Criteria I

Table F-1

Prevalence of mismatch repair gene mutations among (more...)

Table F-1

Prevalence of mismatch repair gene mutations among colorectal cancer probands who fulfill Amsterdam I criteria

Study, year (Ref ID);	A. Comments on sampling	NAm1/Ntotal	MLH1 and MSH2 mutations		Quality
Country	B. Genetic testing strategy		Positive/ Analyzed	Proportion [%](95% CI)
Single-/multi-center	C. Definition of deleterious mutations
De Abajo 2005;	A. Selection among referrals to a specialized center	56/132	32/56	57 (42, 70)	B
Spain	B. PCR → DGGE → sequencing (MSH6 assessed in MLH1/MSH2 negative cases, included in the counts)
Single-center	A. Predicted non-conservative transcription alteration; literature; comparison with healthy controls
Syngal 2000 (1672) &	C. Selection among referrals to a specialized center	28/70	11/28	39 (22, 59)	B
Wahlberg, 2002 (1158);	D. PCR, sequencing
US	E. Predicted non-conservative transcription alteration; literature
Single-center
Wang, 1999 (1939);	A. Sampled among referrals to a genetic consultation center	22/70	14/22	64 (41, 83)	B
France	B. ØRT-PCR → IVSP; ♦ PCR → HD; in vivo hML1 expression
Single-center	C. Predicted transcription alteration; literature
Curia, 1999 (1959);	A. Sampled from pathology registries, unclear selection criteria	15/30	1/15	7 (0, 32)	B
Italy	B. ♦RT-PCR → SSCP → Sequencing of abnormal patterns
Single-center	C. Predicted non-conservative transcription alteration; literature; comparison with non-cancer controls
Luce, 1995 (2703);	A. Selection among referrals to a specialized centera	12/19a	6/12	50 (21, 79)	B
US	B. ♦PCR → IVTT → Sequencing of abnormal peptides
Single-center	C. Unclear
Katballe, 2002 (1310) &	A. Selection from a population of 1514 incident CRC	11/45	5/11	45 (17, 77)	B
Christensen, 2002 (1038);	B. ♦PCR → SSCP & HD →Sequencing of abnormal patterns
Denmark	C. Predicted non-conservative transcription alteration; literature
Single-center
Dieumegard, 2000 (1791);	A. Sample assembled with unclear selection process	10/34	6/10	60 (26, 88)	B
France	B. ♦PCR → SSCP → Sequencing of abnormal patterns
Multi-center	C. Predicted non-conservative transcription alteration; literature
Aaltonen, 1998 (2282);	A. Selection of all incident unrelated CRC from 9 hospitals between 05/1994 and 04/1996	4/509	4/4	100 (40, 100)	B
Finland	B. ♦PCR → DGGE (for some samples) → Sequencing; PCR → Sequencing (for all other samples); Ø and PCR for founder mutations (all samples)
Multi-center	C. Literature; and comparison with healthy controls
Rossi, 2002 (1146);	A. Selected from consecutive CRC referrals	4/25	1/4	25 (0, 81)	B
Brazil	B. PCR → Sequencing
Single-center	C. Unclear
Park, 1999 (2007);	A. Study sample assembled from the ICG-HNPCC database	154/277	77/154b	50 (42, 58)	C
7 countries	B. ♦PCR → SSCP; and Ø southern blotting (no details)
Multi-center (8 centers)	C. Unclearb
Lamberti, 1999 (2036);	A. Study sample assembled with unclear selection process	57/160	15/57	26 (16,40)	C
Italy	B. ♦PCR → SSCP, and RT-PCR → PTA
Single-center	C. Literature
de Leon, 1999 (2012);	A. Sample selected from CRC patient registries	18/36	3/18	17 (4, 41)	C
Italy	B. ♦PCR → SSCP → Sequencing of abnormal patterns
Single-center	C. Unclear
Liu, 2004 (445);	A. Study sample assembled with unclear selection process	15/28	5/15	33 (12,62)	C
China	B. ♦PCR → DHPLC → Sequencing of abnormal patterns
Single center	C. Unclear
Moslein 1996 (2545);	A. Sample assembled from various databases; only cases stated to have had CRC are analyzedc	14/46	6/14c	43 (18,71)	C
US & Germany	B. PCR → Sequencing
Multi-center	C. Predicted non-conservative transcription alteration; literature
Callistri, 2000 (1797);	A. Study sample assembled with unclear selection process	13/45	7/13	54 (25,81)	C
Italy	B. ♦PCR → SSCP
Multi-center	C. Unclear
Colombino, 2005 (1058);	A. Selection from consecutive CRC cases enrolled over 3 yearsa	13/362a	10/13	77(46,95)	C
Italy	B. ♦PCR → DHPLC→Sequencing of abnormal patterns
Single-Center	C. Compared mutations to 103 people without cancer
Stormorken, 2001 (721)	A. First 56 families in the Norwegian Radium hospital registry	12/56	3/12	25 (5,57)	C
Norway	B. Not described (was already done) (MSH6 was assessed also, included in the counts)
Single-center	C. Not described
Debniak, 2000 (1784);	A. Sampled from consecutive CRC, selection process not transparent	3/168	1/3	33 (0,91)	C
Poland	B. PCR → Sequencing
Single-center(?)	C. Unclear
Yuan, 2004 (303);	A. Sampled with unclear selection process; all fulfill Chinese HNPCC criteriad	3/14	1/3	33 (0,91)	C
China	B. ♦PCR → DHPLC → Sequencing of abnormal products
Single-center	C. Predicted non-conservative transcription alteration; literature; comparison with non-cancer controls

Studies are ordered by quality and then by decreasing number of patients fulfilling Amsterdam I criteria. Demographic data (on age and gender distributions) were not available for probands fulfilling Amsterdam I criteria. Primary studies did not describe how many of the MMR mutations were MLH1 or MSH2 for the subset of CRC fulfilling Amsterdam I criteria.

CI: confidence interval; CRC: colorectal cancer; DHPLC: denaturating high performance liquid chromatography; HD: heteroduplex formation; IVSP: in vitro synthesized protein assay; IVTT: in vitro transcription-translation; N_Am1: number fulfilling Amsterdam I criteria; N_total: total number of studied CRC; PCR: polymerase chain reaction; PTA: protein truncation assay; RT-PCR: reverse transcriptase PCR; SSCP: Single-stranded conformation polymorphism

Conversion analysis was not used in any study.

♦: Gene screening as been used

Ø: Analysis for large deletions has been used

a

Unclear if all probands come from unrelated families.

b

This study probed for hPSM1 or hPSM2 mutations in some but not all patients; none was found

c

Although the paper states that 20 patients fulfilled Amsterdam 1 criteria, only 14 were described to have had CRC in the pertinent data table. The other had other cancers.

d

The Chinese HNPCC criteria ≥2 relatives with histologically proven CRC (≥2 must be first degree relatives) and one of the following: multiple colorectal tumors, one CRC diagnosed at age younger than 50 years, or development of extracolonic HNPCC-related cancer in family members.

Description of studies. Nineteen studies (described in 21 papers ⁶³–⁸³) provided data for the prevalence of MMR mutations among CRC probands who fulfilled the Amsterdam I criteria. The median number of patients with relevant data in each study was only 13 (interquartile range: 10, 22), and the actual number ranged between 3 and 154 patients. However, study sizes (all included patients) ranged between 19 and 509. Only five studies included at least 20 probands fulfilling the Amsterdam I criteria.^{67, 72, 76, 79}–⁸¹ Nine studies were rated grade B for their overall methodologic quality^{63, 65}–^{67, 70, 71, 74, 77, 79}–⁸¹ while the rest were rated grade C (Appendix F-1 ^*).

Six studies performed bidirectional sequencing on all patients who fulfilled the Amsterdam I criteria.^{69, 75, 77, 79, 81, 83} (Colombino 2005⁸³ performed full sequencing only among familial CRC cases, which included Amsterdam I patients). Ten studies performed full sequencing only on patients who were selected by gene screening methods such as DHPLC or SSCP.^{63, 66}–^{68, 70, 71, 73, 74, 76, 82} The remaining three studies did not describe the presence of any mutation with bidirectional sequencing in any patient^{64, 72} or did not provide any details on the genetic testing strategy they used.⁷⁸ None of these 19 studies used conversion analysis to detect mutations.

Assessment of mutations in MMR genes other than MLH1 and MSH2. All reports assessed mutations in the MLH1 and MSH2 genes. Additional MMR genes were assessed in three studies.^{76, 78, 81} Park 1999⁷⁶ assessed the PMS1 and PMS2 genes in a minority of their Amsterdam I patients (24 and 27 patients respectively, less than 20% of their sample), without identifying any additional mutations(Appendix F-1 ^*). Wang 1999⁸¹ also tested for MSH6, PMS1 and PMS2, but found no additional mutations among the subgroup of patients fulfilling the Amsterdam I criteria. Stormorken 2001⁷⁸ also tested for MSH6 mutations, but found none among Amsterdam I patients.

Table 13

Summary estimates of the prevalence of MMR mutations (more...)

Table 13

Summary estimates of the prevalence of MMR mutations among CRC fulfilling Amsterdam I criteria

Summary	Number of studies (CRC patients fulfilling AM1)	% with mutations(95% CI)	Heterogeneity P-value, (I2[%])	Between-subgroup heterogeneity, P-value
Overall	19 (464)	44 (35, 52)	<0.01 (52)	NA
Overall quality scale
B a	9 (162)	49 (37, 61)	0.09 (41)	0.12
C	10 (302)	39 (28, 51)	0.01 (58)
Sequencing
All samples	6 (84)	51 (36, 66)	0.17 (35)	0.02
Some b	10 (298)	44 (33, 55)	0.04 (50)
None	3 (82)	33 (19, 51)	0.16 (46)
Deletion analysis and sequencing in all samples
Yes	1 (22)	64 (41, 83)	NA	0.09
No	18 (442)	42 (34, 51)	0.03 (51)
Assessment of additional MMR genes (other than MLH1 and MSH2)
Yes	3 (188)	49 (34, 65)	0.12 (54)	0.12
No	16 (276)	42 (32, 52)	0.01 (51)
Any definition for pathogenic mutations
Yes	11 (233)	47 (34, 60)	<0.01 (63)	0.90
No	8 (231)	40 (30, 51)	0.13 (33)
Total number of patients fulfilling Amsterdam I criteria
≥20	5 (317)	47 (35, 59)	<0.01 (73)	0.28
<20	14 (147)	41 (30, 53)	0.06 (40)
Sampling among unselected, non-referral CRC
Yes	3 (28)	66 (35, 88)	0.17 (44)	0.09
No	16 (436)	41 (31, 50)	0.01 (52)

AM1: Amsterdam I criteria; CI: confidence interval; CRC: colorectal cancer patients.

None of the studies was rated A in the overall quality scale.

Gene sequencing was performed only to those selected by gene screening methods.

Figure 3. Prevalence of mismatch repair gene mutations (more...)

   Figure 3. Prevalence of mismatch repair gene mutations among colorectal cancer patients fulfilling the Amsterdam I criteria

Analyses. Overall, the random effects summary estimate from the 19 studies was 44% (95% CI: 35, 52%), with evidence for substantial between-study heterogeneity (p<0.01; I²=52%) (Table 13, Figure 3). The six studies that performed full sequencing in all available samples identified mutations in 51% (95% CI: 35, 66%) of 84 patients fulfilling the Amsterdam I criteria (Table 13). The corresponding prevalence estimate was 44% when sequencing was performed after suggestive SSCP, DHPLC or other gene screening methods, and only 33% among studies that used screening methods without further confirmation by sequencing (Table 13). The prevalence of MMR mutations did not differ beyond chance with respect to overall study quality score, whether the study was small (i.e., analyzed fewer than 20 patients), or whether the authors described how they classified mutations as being pathogenic.

Finally, three studies (n=28 eligible patients) that sampled unselected, nonreferral patients with CRC found a higher prevalence of MMR mutations (66%) among patients who fulfilled the Amsterdam I criteria, compared to studies that sampled among referral or otherwise selected cases (prevalence 41%; Table 13). The precision of these estimates is uncertain because of the very small number of eligible patients.

Frequency of MMR Gene Mutations Among Patients Fulfilling Amsterdam II Criteria

Table F-2

Prevalence of MLH1 and MSH2 mutations among colorectal (more...)

Table F-2

Prevalence of MLH1 and MSH2 mutations among colorectal cancer probands who fulfill Amsterdam II criteria

Study, year (Ref ID);	A. Comments on sampling	NAm2/Ntotal	MLH1 and MSH2 mutations		Quality
Country	B. Genetic testing strategy		Positive/A nalyzed	Proportion [%] (95% CI)
Single-/multi-center	C. Definition of deleterious mutations
De Abajo 2005;	A. Selection among referrals to a specialized center	67/132	36/67	54 (41, 66)	B
Spain	B. PCR → DGGE → sequencing (MSH6 assessed in MLH1/MSH2 negative cases)
Single-center	C. Predicted non-conservative transcription alteration; literature; comparison with healthy controls
Katballe, 2002 (1310) &	A. Selection from a population of 1514 incident CRC	18/45	8/18	44 (22, 69)	B
Christensen, 2002 (1038);	B. ♦PCR → SSCP & HD → Sequencing of abnormal patterns
Denmark	C. Predicted transcription alteration; literature
Single-center
Wolf, 2005 (23)	A. Retrospective cohort of referral cancer patients fulfilling the modified Bethesda criteria	35/81	13/35	37 (21, 55)	B
Austria	B. PCR → Sequencing
Single-center	C. Predicted transcription alteration; literature; comparison with healthy controls
Syngal 2000 (1672) &	A. Selection among referrals to specialized center	34/70	14/34	41 (25, 59)	B
Wahlberg, 2002 (1158);	B. PCR → Sequencing
US	C. Predicted transcription alteration; literature
Single-center
Zhu, 2005 (138)	A. Study sample assembled with unclear selection process	21/78	4/21	19 (5, 42)	B
China	B. Ø MLPA → Sequencing of detected aberrations
Single-center	C. Unclear
Lee, 2005 (105)	A. Sample selected from referrals to a tertiary center	5/46	3/5	60 (15, 95)	B
Singapore	B. PCR → Sequencing
Single-center	C. Predicted transcription alteration; literature
Salovaara, 2000 (1740)	A. Selection of all new unrelated CRC from 9 hospitals between 03/1996 and 06/1998	5/535	4/5	80 (28, 99)	B
Finland	B. PCR → Sequencing Ø PCR for founder mutations in MLH1 gene
Multi-center	C. Literature; comparison with non-cancer controls
Rossi, 2002 (1146)	A. Selected from consecutive CRC referrals	5/25	2/5	40 (5, 85)	B
Brazil	B. ♦PCR, → Sequencing
Single-center	C. Unclear
Lamberti, 1999 (2036);	A. Study sample assembled with unclear selection process	69/160	17/69	25 (15, 36)	C
Italy	B. ♦PCR → SSCP; and RT-PCR, PTA
Single-center	C. Literature
Stormorken, 2001 (721)	A. First 56 families in the Norwegian Radium hospital registry	20/56	7/20a	35 (15, 59)	C
Norway	B. Not described (was already done) (MSH6 was assessed also)
Single-center	C. Not described

Studies are ordered by quality and then by decreasing number of patients fulfilling Amsterdam II criteria. Demographic data (on age and gender distributions) were not available for probands fulfilling Amsterdam II criteria. Primary studies did not describe how many of the MMR mutations were MLH1 or MSH2 for the subset of CRC fulfilling Amsterdam II criteria.

Conversion analysis was not used in any study.

♦: Gene screening as been used

Ø: Analysis for large deletions has been used

CI: confidence interval; CRC: colorectal cancer; HD: heteroduplex formation; MLPA: Multiple ligation-dependent probe amplification; NAm2: number fulfilling Amsterdam II criteria; Ntotal: total number of studied CRC; PCR: polymerase chain reaction; PTA: protein truncation assay; RT-PCR: reverse transcriptase PCR; SSCP: Single-stranded conformation polymorphism

a

2/7 mutations shown are MSH6

Description of studies. Ten studies assessed the prevalence of MMR mutations among 271 CRC probands who fulfilled the Amsterdam II criteria (Appendix F-2 ^*). They were described in 12 papers.^{65, 67, 71, 72, 77}–^{80, 84}–⁸⁷

The total number of patients in the various study populations ranged from 45 to 535 patients with CRC. However, the median number of patients fulfilling Amsterdam II criteria was 19.5 (interquartile range: 5, 35). Only five studies assessed more than 20 probands from families fulfilling the Amsterdam II criteria.^{67, 72, 79, 80, 86, 87} Eight out of ten studies received grade B in the overall quality rating while two^{72, 78} received grade C.

Four studies performed bidirectional sequencing in all patients fulfilling Amsterdam II criteria.^{77, 79, 84, 86} Four studies performed full sequencing only to patients who were selected by gene screening methods.^{65, 67, 71, 85, 87} The remaining two studies did not describe the presence of any mutation with bidirectional sequencing in any patient⁷² or did not provide any details on the genetic testing strategy they used.⁷⁸ None of these 10 studies used conversion analysis.

Assessment of mutations in MMR genes other than MLH1 and MSH2. All studies tested for MLH1 and MSH2 mutations. Only Stormorken 2001⁷⁸ also tested for MSH6 mutations. In this study five out of 20 Amsterdam II patients had deleterious MLH1 and MSH2 mutations (25% [95% CI: 9, 49%]) and another two had deleterious MSH6 mutations (10% [95% CI: 1, 32%]).

Table 14

Summary estimates of the prevalence of MMR mutations (more...)

Table 14

Summary estimates of the prevalence of MMR mutations among CRC fulfilling Amsterdam II criteria

Summary	Number of studies (patients fulfilling AM2)	% with mutations (95% CI)	Heterogeneity P-value, (I2 [%])	Between-subgroup heterogeneity, P-value
Overall	10 (279)	39 (30, 49)	0.03 (53)	NA
Overall quality scale
B a	8 (190)	45 (38, 52)	0.16 (34)	0.01
C	2 (89)	27 (19, 37)	0.36 (0)
Sequencing
All samples	2 (87)	40 (30, 52)	0.82 (0)	0.02
Some b	4 (111)	45 (26, 65)	0.03 (66)
None	2 (89)	27 (19, 37)	0.36 (0)
Deletion analysis and sequencing in all samples
Yes	0 (0)	NA	NA	NA
No	10 (279)	39 (30, 49)	0.03 (53)
Assessment of additional MMR genes (other than MLH1 and MSH2)
Yes	1 (20)	35 (15, 59)	NA	0.68
No	9 (259)	40 (30, 51)	0.02 (57)
Any definition for pathogenic mutations
Yes	7 (233)	43 (31, 55)	0.02 (60)	0.14
No	3 (46)	29 (18, 44)	0.45 (0)
Total number of patients fulfilling Amsterdam criteria II
≥20	5 (226)	36 (24, 49)	0.01 (73)	0.41
<20	5 (53)	44 (31, 58)	0.51 (0)
Sampling among unselected, non-referral CRC
Yes	2 (23)	57 (23, 85)	0.19 (43)	0.28
No	8 (256)	37 (28, 48)	0.03 (56)

AM2: Amsterdam II criteria; CI: confidence interval; CRC: colorectal cancer patients.

a

None of the studies was rated A in the overall quality scale.

b

Gene sequencing was performed only to those selected by gene screening methods.

Figure 4. Prevalence of mismatch repair gene mutations (more...)

   Figure 4. Prevalence of mismatch repair gene mutations among colorectal cancer patients fulfilling the Amsterdam II criteria

Analyses. As expected, the overall prevalence of MMR gene mutations was lower compared with patients who fulfilled the stricter Amsterdam I criteria (39% [95% CI: 30, 49%]; Table 14, Figure 4). Between-study heterogeneity was statistically significant.

Studies that performed sequencing in all Amsterdam II patients estimated similar prevalence of MMR mutations compared to studies that performed sequencing only after suggestive gene screening with SSCP, DHPLC, or other methods (40% versus 45%, respectively; Table 14). Studies that were rated C for their overall quality estimated significantly lower prevalence rates (27%; Table 14) compared to studies that received B for their overall quality. In the latter, the summary estimate was 43% (95% CI: 34, 54%) with no evidence for heterogeneity.

Studies that mentioned how pathogenic mutations were characterized as such, contributed fewer than 20 patients, or sampled unselected, non-referral patients with CRC, tended to provide higher estimates for the prevalence for MMR mutations. However, all of these studies were small and thus it is possible that the observed variability could have been due to chance (Table 14).

Interpretation of the MMR Prevalence Eestimates. The summary prevalence was 44% among all studies on Amsterdam I patients, and 51% for studies that sequenced all patients. For Amsterdam II the corresponding numbers were 39% and 40%. These estimates pertain mainly to MLH1 and MSH2 mutations, because most studies tested only these two genes. The true prevalence of MMR mutations among Amsterdam I (or Amsterdam II) patients may be different (possibly higher rather than lower) than these estimates for several reasons:

• Genes other than MLH1 and MSH2: Only a minority of studies tested for mutations in other MMR genes (especially MSH6, the gene most commonly implicated in patients without mutations in MLH1 or MSH2⁸⁸).

  • Stormorken 2001 and Wang 1999 did not find any deleterious MSH6 mutations among 34 Amsterdam I patients in total. The upper boundary of the exact binomial 95% CI for the frequency of MSH6 mutations in these two studies is approximately 10%.

  • Stormorken 2001 found two deleterious MSH6 mutations in addition to five MLH1 and MSH2 mutations out of 20 Amsterdam II patients. Thus, MSH6 mutations resulted in an increment of 10% (95% CI: 1, 32%).

  Presumably, if other studies had assessed MSH6 routinely, the total prevalence of MMR mutations would be higher. However, there were insufficient data to provide robust estimates.

• Comprehensiveness of genetic testing - sequencing of all samples and testing for large deletions/rearrangements: Studies that performed sequencing in all samples estimated higher frequency of MMR mutations among Amsterdam I patients compared to other studies. This was not true among studies that focused on Amsterdam II patients. Thus, more comprehensive testing resulted in more identified MMR mutations (at least in Amsterdam I studies), as expected.

• Role of founder mutations: The degree to which a search for mutations other than MLH1 and MSH2 will identify mutations in other MMR genes depends upon the population being studied. The spectrum of MMR mutations is variable and some are specific to certain populations. An example of the latter is the presence of founder mutations in the MLH1 genes that are common among patients of Finnish origin, but are not prevalent in patients of different descent. The Finnish study⁶³ included only 4 patients fulfilling the Amsterdam criteria I, all of whom were found to have MMR mutations. An American founder mutation in the MSH2 gene has been described in one kindred, probably originating from German immigrants, but it is unlikely to have a large impact in the general population.^{90, 91} The Finnish founder mutations are probably not applicable to the US population. Nevertheless, the summary estimates are practically identical if the Finnish study⁶³ is excluded.

• Identifying deleterious mutations: As mentioned in the methods, only mutations that were characterized by the primary studies as deleterious or pathogenic were analyzed. Misclassifications may have occurred in both directions:

  • Some deleterious missense mutations may have been erroneously misclassified as non-pathogenic.

  • The opposite is also likely, especially when pathogenicity is inferred on the basis that the mutation was absent from cancer-free controls. This was done in five studies.^{63, 66, 67, 82, 83} However, most of the studies included only small numbers of cancer-free controls (only around 100 patients were used as controls in the five studies). Absence of a mutation in such a small population of cancer-free controls does not necessarily prove that the mutation is not pathogenic.

  There were insufficient data to provide precise estimates of how many missense mutations were found among the subgroup of Amsterdam I (or Amsterdam II) patients in the studies that performed comprehensive genetic testing.

• Sampling of tested Amsterdam I and Amsterdam II cases: The strategy used for selecting patients with CRC can have a substantial impact on the prevalence estimates (see comments on additional studies that were suggested for consideration by the TEP, below). Only three studies on Amsterdam I patients studied unselected patients with CRC, but they identified a total of only 28 patients. By contrast, studies that identified cases that had been included in cancer databases (e.g., the ICG-HNPCC database that was used in the Park 1999 study) represent a highly selected population,⁸⁸ or may pertain almost entirely to individuals with a high probability of having mutations (e.g., because of suggestive MSI testing prior to their registration in the database).⁶¹ Biases resulting from sampling are not only a theoretical concern (see below comments on additional studies that were suggested for consideration by the TEP).

Additional studies suggested for consideration by the TEP. Several additional studies were considered by the TEP during the peer review process as important and potentially relevant that had not been included in the draft report. Although none of these studies was eligible for these analyses according to the eligibility criteria, they provide insight and complementary data, directly supporting the analyses and their interpretation. Most importantly, the findings from these studies are in accordance with the notion that sampling of Amsterdam I or II patients from different sources may result in very different prevalence estimates, even when the genetic testing strategies are comprehensive.

• Balmana 2006⁹² reported the prevalence of MLH1 and MSH2 gene mutations among 1914 unrelated people who had been tested for MMR mutations in the Myriad Genetic Laboratories. Genetic analysis was performed using a combination of sequencing and southern blotting. The cohort included 534 unrelated people fulfilling the Amsterdam II criteria (although it was unclear if all of them had CRC). Amsterdam I patients were not reported separately. Only half of the Amsterdam II patients (274/534=51%) were tested for large deletions or rearrangements.

  In total, 180 Amsterdam II participants were found to be carriers of MLH1 or MSH2 mutations (34% [9%% CI: 30, 38%]). This estimate includes large deletions or rearrangements and pathogenic and non-pathogenic mutations. About one fourth (27% [95% CI: 20, 35%] of the mutations were due to large deletions/rearrangements in a subgroup of the 1016 participants who underwent such testing. Extrapolating the proportion of large deletions to all Amsterdam II patients would result in a total prevalence of approximately 38%, very close to the summary estimate provided above. This study was excluded from the analyses because it was unclear whether all Amsterdam II participants had CRC.

• Wagner 2003⁹³ evaluated 49 individuals from the Henry Lynch Cohort fulfilling the Amsterdam criteria (presumably Amsterdam I criteria), at least 9 of whom were not CRC patients. They used DGGE to screen for MMR mutations in the MLH1, MSH2 and MSH6 genes, and performed sequencing on exons with altered migration patterns. They tested for large deletions, and performed mono-allelic mutation analysis (MAMA) in very few samples, mainly to demonstrate the feasibility of the technique.

  Overall, 25 out of 49 had deleterious MLH1 or MSH2 mutations, 1 had deleterious MSH6 mutations, and 14 had genomic rearrangements. Thus, the overall proportion was 82% (95% CI: 68, 91%). In addition, 5 missense mutations were found; were they to be considered pathogenic, the overall proportion would be 92% (95% CI: 80, 98%). The detection of large deletions/rearrangements resulted in 29% increase in the estimated prevalence of MMR mutations. The degree to which the Henry Lynch Cohort is representative of the whole Amsterdam I population is unclear.

• Liu 1996⁹⁴ assessed the MLH1, MSH2 and PMS2 genes 48 families of Amsterdam I patients who had suggestive MSI (note that more than one person per family was tested for mutations -i.e, reporting was at the family not the individual level). They found a prevalence of “drastic” (deleterious) mutations of 54%. Counting all identified mutations, drastic or not, 71% (95% CI: 56, 83%) of the families carried a mutation. Fifteen families had large genomic deletions/rearrangements (31%), but many families had more than one type of mutations.

  It is unclear how many unrelated people had only large deletions or rearrangements in this study. There might be some overlap with the Wanger 2003⁹³ families for patients from North America. The authors note that their sample was not representative of the general Amsterdam I population.

Caveat on true overall prevalence of MMR mutation carriers. Limited data suggested that approximately one-fourth to one-third of genotypes associated with HNPCC are related to deletions or rearrangements that would be missed through sequencing alone. As a result, the prevalence of mutations in Amsterdam I (or II patients) assessed from studies that performed sequencing alone are likely to be underestimates.

Accounting for this effect, one may derive a prevalence of MMR mutations of approximately 63% to 67% in Amsterdam I patients. For Amsterdam II patients the corresponding prevalence values would be 50 to 53%.

Furthermore, limited data suggest that approximately 10% of MMR genotypes involve MMR genes other than MLH1 and MSH2. Thus, assuming an additional 10% increase in mutations by assessing more genes would result in an overall prevalence of up to 70% to 75% for Amsterdam I patients, and 55 to 59% for Amsterdam II patients.

Key Question 2b: Assuming a Clinical Definition of the Lynch Syndrome, What Proportion of Patients has MSI?

Among patients fulfilling the Amsterdam I criteria (data from 11 studies, n=159 patients fulfilling the criteria), 71% (95% CI: 63, 78%) of tumors were found to be MSI-H (p=0.51 for heterogeneity; I²=0%). Among patients who fulfilled the Amsterdam II criteria (data from four studies, n=102 patients fulfilling the criteria), the corresponding summary prevalence was 68% (95% CI: 58, 76%) without evidence for between-study heterogeneity (p=0.91, I²=0%).

Figure 5. Prevalence of MSI in colorectal cancer tumors (more...)

   Figure 5. Prevalence of MSI in colorectal cancer tumors from patients fulfilling the Amsterdam I criteria

Figure 5

Table F-3

Prevalence of microsatellite instability among colorectal (more...)

Table F-3

Prevalence of microsatellite instability among colorectal cancer probands who fulfill Amsterdam I criteria

Study, year (Ref ID);	A. Comments on sampling	NAm1/ Ntotal	Tumor microsatellite instability				Micro-dissection	NCI 5 marker set	Quality
Country	B. Definition of MSI-H		MSI-H/Analyzed	MSI-H [%](95% CI)	MSI-H&L/Analyzed	MSI-H&L [%](95% CI)
Single-/multi-center
Wahlberg, 2002 (1158) & Syngal 2000 (1672);	A. Selection among referrals to specialized center	28/70	15/19	79 (54, 94)	18/19	95 (74, 100)	√	√	B
US	B. ≥2 out of 5 dinucleotide repeats (NCI set)
Multi-center
Curia, 1999 (1959);	A. Sampled from pathology registries, unclear selection criteria	15/30	12/15	80 (52, 96)	ND	ND	√	X	B
Italy	B. Screened with 3 markers and if none was unstable up to a total of 7. MSI-H defined as ≥2 markers
Single-center
Katballe, 2002 (1310) & Christensen, 2002 (1038);	A. Selected from a population of 1514 incident CRC	11/45	7/10	70 (34, 93)	7/10	70 (34, 93)	√	X	B
Denmark	B. ≥2 out of 5 dinucleotide repeats
Single-center
Dieumegard, 2000 (1791);	A. Sample assembled with unclear selection process	10/34	9/10	90 (55, 100)	9/10	90 (55, 100)	X	X	B
France	B. ≥10% of up to 23 markers (But all were ≥30%)
Multi-center
Aaltonen, 1998 (2282);	A. Selection of all new unrelated CRC from 9 hospitals between 05/1994 and 04/1996	4/509	4/4	100 (40, 100)	4/4	100 (40, 100)	X	X	B
Finland	B. ≥30% of 16 markers for tumor analyzed with fluorescence methods or ≥2 out of 7 (≈30%) markers analyzed with radioactive technique
Multi-center
Lamberti, 1999 (2036);	A. Study sample assembled with unclear selection process	57/160	35/49	71 (57, 83)	37/49	76 (61, 87)	X	X	C
Italy	B. ≥40% out of up to 10 mono- di- and tetranucleotide repeats
Single-center
de Leon, 1999 (2012);	A. Sample selected from CRC patient registries	18/36	11/18	61 (36, 83)	ND	ND	√	X	C
Italy	B. ≥2 out of ≥5 markers
Single-center
Callistri, 2000 (1797);	A. Study sample assembled with unclear selection process	13/45	11/13	85 (55, 98)	11/13	85 (55, 98)	√	√	C
Italy	B. ≥2 out of 13 microsatellite markers
Multi-center
Peel, 1999 (1660);	A. Referral HNPCC cases, other than the 1134 CRC probands assessed for other purposes	11/11	4/9	44 (14, 79)	4/9	44 (14, 79)	√	X	C
US	B. Unclear; at least 5 markers were used
Multi-center
Moslein, 1996 (2545);	A. Sample assembled from various databases; we present only cases stated to have CRCa	14/46a	5/9	56 (21, 86)	ND	ND	X	X	C
US & Germany	B. ≥30% of 9 to 34 markers analyzed (unclear which exactly)
Multi-center
Debniak, 2000 (1784);	A. Sampled from consecutive CRC, selection not transparent	3/168	2/3	67 (9, 99)	ND	ND	X	X	C
Poland	B. ≥2 out of 5 dinucleotide repeats (panel I), or ≥3 out of 10 dinucleotide repeats (panel II, used if only 1 positive in panel I)
Single-center(?)

Studies are ordered by overall quality and then by decreasing number of patients fulfilling the Amsterdam I criteria. Demographic data (on age and gender distributions) were not available for probands fulfilling Amsterdam I criteria.

CI: confidence interval; CRC: colorectal cancer; MSI-H/H&L: microsatellite instability high/combined high and low; ND: Not described; N_Am1: number fulfilling Amsterdam I criteria;

N_total: total number of studied CRC; NCI: National cancer institute

X: Was not used/not stated

√: Was used

a

Although the paper states that 20 patients fulfilled Amsterdam I criteria, only 14 were described to have a CRC in the pertinent data table. The other had other cancers. Only the 14 with CRC are analyzed.

Table 15

Summary estimates of the prevalence of MSI-H in colorectal (more...)

Table 15

Summary estimates of the prevalence of MSI-H in colorectal cancer tumors from patients fulfilling Amsterdam I criteria

Summary	Number of studies (CRC patients fulfilling AM1)	% with MSI-H (95% CI)	Heterogeneity P-value, (I2 [%])	Between-subgroup heterogeneity, P-value
Overall	11 (159)	71 (63, 78)	0.51 (0)	NA
Overall quality scale
B a	5 (58)	79 (67, 88)	0.81 (0)	0.10
C	6 (101)	66 (56, 75)	0.44 (0)
Microdissection
Yes	6 (84)	70 (58, 80)	0.32 (15)	0.89
No	5 (75)	71 (59, 81)	0.50 (0)
Use of the NCI-recommended marker sets
Yes	2 (32)	81 (64, 91)	0.69 (0)	0.17
No	9 (127)	68 (59, 76)	0. 52 (0)
Total number of patients fulfilling Amsterdam criteria I
≥20	1 (49)	71 (57, 83)	NA	0.89
<20	10 (110)	70 (60, 79)	0.42 (2)
Sampling among unselected, non-referral CRC
Yes	2 (14)	70 (62, 78)	0.39 (5)	0.81
No	9 (145)	74 (44, 91)	0.39 (0)

AM1: Amsterdam I criteria; CI: confidence interval; CRC: colorectal cancer patients.

a

None of the studies was rated A in the overall quality scale.

Table F-4

Prevalence of microsatellite instability among colorectal (more...)

Table F-4

Prevalence of microsatellite instability among colorectal cancer probands who fulfill Amsterdam II criteria

Study, year (Ref ID);	A. Comments on sampling	NAm2/ Ntotal	Tumor microsatellite instability				Micro-dissection	NCI 5 marker set	Quality
Country	B. Definition of microsatellite instability		MSI-H/ Analyzed	MSI-H [%] (95% CI)	MSI-H&L/ Analyzed	MSI-H&L [%](95% CI)
Single-/multi-center
Wolf, 2005 (23)	A. Retrospective cohort of referral cancer patients fulfilling the modified Bethesda criteria	35/81	16/24	67 (45, 84)	ND	ND	√	X	B
Austria	B ≥30% out of up to 10 markers
Single-center
Katballe, 2002 (1310) & Christensen, 2002 (1038);	A. Selected from a population of 1514 incident CRC	17/45	10/16	63 (35, 85)	11/16	69 (41, 89)	√	X	B
Denmark	B. ≥2 out of 5 dinucleotide repeats
Single-center
Salovaara, 2000 (1740)	A. Selection of all new unrelated CRC patients from 9 hospitals between 03/1996 and 06/1998	5/535	4/5	80 (28, 99)	NA	NA	X	X	B
Finland	B. Single marker (BAT26) analyzed in all samples
Multi-center
Lamberti, 1999 (2036);	A. Study sample assembled with unclear selection process	69/160	39/57	68 (55, 80)	41/57	72 (58, 83)	X	X	C
Italy	B. ≥40% out of up to 10 mono- di- and tetranucleotide repeats
Single-center

Studies are ordered by quality and then by diminishing number of patients fulfilling the Amsterdam II criteria. Demographic data (on age and gender distributions) were not available for probands fulfilling Amsterdam II criteria.

CI: confidence interval; CRC: colorectal cancer; MSI-H/H&L: microsatellite instability high/combined high and low; NA: Not applicable; ND: Not described; N_Am2: number fulfilling Amsterdam II criteria; N_total: total number of studied CRC; NCI: National cancer institute

X: Was not used/not stated

√: Was used

Figure 6. Prevalence of MSI-H in colorectal cancer (more...)

   Figure 6. Prevalence of MSI-H in colorectal cancer tumors from patients fulfilling the Amsterdam II criteria

Figure 6

A pooled estimated for the three fair quality (B overall quality) studies was 66% (95% CI: 51, 79%) with no evidence for heterogeneity. Estimates from the two studies with more than 20 patients that fulfilled the Amsterdam II were similar to the overall estimate. The results were also similar for studies that used the markers sets recommended by the National Cancer Institute.

Interpretation of the MSI Prevalence Estimates. Some of the biases that may affect the calculated prevalence for MMR mutations are also applicable in this section, specifically biases that pertain to the selection/sampling of Amsterdam I and Amsterdam II patients who were studied. In addition, even among the selected Amsterdam I or Amsterdam II patients in each study, MSI testing often depended upon practical and logistical considerations (e.g., patient availability and consent and availability of tumor tissue). Thus, not all patients had all tests and it was unclear whether additional bias may have been introduced in selecting patients for testing.

Key Question 2c: Assuming a Clinical Definition of the Lynch Syndrome, What Proportion of Patients has Abnormal Protein Expression by Immunohistochemistry?

Among patients fulfilling the Amsterdam I criteria, the overall prevalence of tumors with loss of protein expression was 40% (95% CI: 28, 53%; n= 6 studies, 63 patients) with no evidence for between-study heterogeneity (p=0.75, I²=0%).

Only one eligible study provided relevant data for 20 patients fulfilling the Amsterdam II criteria. Eight out of 20 tumors had suggestive IHC for the MLH1, MSH2 or MSH6 genes (40% [95% CI: 9, 64%]).

Patients Fulfilling Amsterdam I Criteria. Only six studies that included a total of 63 eligible patients (median 12 tumors in each study) provided relevant data; no study contributed more than 15 tumors in this analysis. The total number of patients included in the pertinent studies ranged from 30 to 168. These were described in eight publications.^{65, 66, 69}–^{71, 78}–⁸⁰.

Table F-5

Prevalence of negative immunostaining for MLH1 or MSH2 (more...)

Table F-5

Prevalence of negative immunostaining for MLH1 or MSH2 among colorectal cancer probands who fulfill Amsterdam I criteria

Study, year (Ref ID);	A. Comments on sampling	NAm1/Ntotal	MLH1 and MSH2 immunostaining		Quality
Country	B. Antibodies used for immunostaining		Negative/Analyzed	Proportion [%] (95% CI)
Single-/multi-center
Curia, 1999 (1959)	A. Sampled from pathology registries, but selection process is not transparent	15/30	7/15	47 (21, 73)	B
Italy	B. Anti-MSH2: FE11 (Oncogene Research Products); anti-MLH1: clone 14 (Oncogene Research Products)
Single-center
Wahlberg, 2002 (1158) & Syngal 2000 (1672);a	A. Selection among referrals to specialized center	28/70	5/14	36 (13, 65)	B
US	B. Anti-MSH2: FE11 (Oncogene Research Products); anti- MLH1: G168–728 (PharMingen)
Single-center
Katballe, 2002 (1310) & Christensen, 2002 (1038);b	A. Selection from a population of 1514 incident CRC	11/42	4/11	36 (11, 69)	B
Denmark	B. Anti-MSH2: Ab-1, Ab-2 (Oncogene Research Products); anti-MLH1: G168-15 (PharMingen)
Single-center
Dieumegard, 2000 (1791)	A. Sample assembled without a transparent selection process	10/34	4/8	50 (16, 84)	B
France	B. Anti-MSH2: FE11 (Oncogene Research Products); anti- MLH1: Ab-1 (Oncogene Research Products)
Multi-center
Stormorken, 2001 (721)	A. First 56 families in the Norwegian Radium hospital registry	12/56	3/12	25 (5, 57)	C
Norway	B. Anti-MSH2: FE11 (Calbiochem); anti-MLH1: G168-15 (Pharmingen); anti-MSH6: Clone 44 (transduction laboratories)c
Single-center
Debniak, 2000 (1784);	A. Sampled from consecutive CRC, but selection process is not transparent	3/168	2/3	67 (9, 99)	C
Poland	B. Not stated
Single-center(?)

Studies are ordered by overall quality and then by decreasing number of patients fulfilling Amsterdam I criteria. Demographic data (on age and gender distributions) were not available for probands fulfilling Amsterdam I criteria. None of these studies assessed any other mismatch repair genes apart from MLH1 and MSH2.

CI: confidence interval; CRC: colorectal cancer; N_Am1: number fulfilling Amsterdam I criteria; N_total: total number of studied CRC

a

Data from the Wahlberg et al. 2002 paper; comments on sampling from the Syngal et al. 2000 paper.

b

Data from the Christensen et al. 2002 paper; comments on sampling from the Katballe et al. 2002 paper.

c

2 MSH6 MMR mutations were also picked up by the anti-MSH2 IHC exam.

Figure 7. Prevalence of suggestive IHC in colorectal (more...)

   Figure 7. Prevalence of suggestive IHC in colorectal cancer tumors from patients fulfilling the Amsterdam I criteria

Studies are ordered by decreasing number of patients.

Figure 7

Patients Fulfilling Amsterdam II Criteria. Only Stormorken 2001⁷⁸ assessed the prevalence of suggestive IHC among 20 patients fulfilling Amsterdam criteria II. This study was rated C for its overall methodologic quality. The expression of three MMR genes was sought, namely MLH1, MSH2 and MSH6. Eight out of 20 Amsterdam II patients had suggestive IHC (40% [95% CI: 19, 64%]).

Tumors that have suggestive IHC for MSH2 may also have suggestive IHC for MSH6, because in the absence of functional MSH2 protein the MSH2/MSH6 heterodimer is not formed correctly. Two out of 20 Amsterdam II patients had deleterious mutations in the MSH6 gene in Stormorken 2001.⁷⁸ None had suggestive IHC only for the MSH6 antibody.

Interpretation of the IHC Prevalence Estimates. In contrast to MSI testing that detects replication errors, and is generally not MMR-gene specific, IHC uses antibodies that target specific MMR genes. Only one of the eligible studies used antibodies against MSH6.⁷⁸ The MSH6 gene is the third most common gene with pathogenic mutations in HNPCC patients (approximately 6% of the mutations in the ICG-HNPCC database are in MSH6⁸⁸). Thus, the prevalence of suggestive IHC would probably be higher if anti-MSH6 or additional antibodies were used.

Some of the biases that may affect the calculated prevalence for MMR mutations are also applicable in this section, specifically biases that pertain to the selection/sampling of Amsterdam I and Amsterdam II patients who were studied. In addition, even among the selected Amsterdam I or Amsterdam II patients in each study, IHC testing often depended upon practical and logistical considerations (e.g., patient availability and consent and availability of tumor tissue). Thus, not all patients had all tests and it was unclear whether additional bias may have been introduced in selecting patients for testing.

Key Question 2: How Accurate Are Various Predictors Assuming a Genetic Definition of the Lynch Syndrome?

We examined a variety of clinical and laboratory predictors as described in Chapters 1 and 2. A central question of this report relates to the accuracy of various predictors in unselected patients with CRC. Because testing for MMR mutations is expensive, most studies performed testing only in patients who had been pre-selected based on clinical or laboratory features. Thus, it is critically important to describe the specific CRC population when attempting to make comparisons among studies.

Figure 8. Relationship between the CRC populations (more...)

   Figure 8. Relationship between the CRC populations defined by different clinical predictors

Figure 8

Table 16

Overview of available evidence on the sensitivity and (more...)

Table 16

Overview of available evidence on the sensitivity and specificity of various predictors for MMR mutation rates

Predictor	All studies	Specific CRC populations defined by increasingly selective criteria
		Unselected CRC probands	Revised Bethesda guidelines	Bethesda guidelines	Modified Amsterdam criteria	Amsterdam II criteria	Amsterdam I criteria
Amsterdam I criteria	F-6	F-7	ND	F-8	ND	F-9
	(n=19)	(n=2)		(n=2)		(n=5)
Amsterdam II criteria	F-10	F-11	F-12	F-13	ND
	(n=12)	(n=2)	(n=1)	(n=3)
Modified Amsterdam criteria	F-14	ND	ND	ND
	(n=2)
Bethesda guidelines	F-15	F-16	ND
	(n=5)	(n=1)
Revised Bethesda guidelines	F-17	F-17
	(n=1)	(n=1)
Young age of onset	F-18	F-19	ND	ND	ND	ND	ND
	(n=5)	(n=4)
Family history	F-20	F-21	ND	ND	ND
	(n=9)	(n=4)
Multiple tumors in the same patient	F-22	F-22	ND	ND	ND	ND	ND
	(n=3)	(n=3)
Age <50 years, family history, or multiple tumors in same patient	F-23	F-23	ND	ND	ND	ND	ND
	(n=3)	(n=3)
Suggestive MSI (MSI-H; MSI-H and MSI-L)	F-24	F-25	F-26	ND	ND	ND	F-27
	(n=16)	(n=2)	(n=1)				(n=3)
Suggestive IHC	F-28	ND	ND	ND	ND	ND	F-29
	(n=9)						(n=2)

Table rows represent different predictors; the table columns describe the populations addressed in the summary tables. Each cell in the table refers to a corresponding Summary Table of the appendix. For example, the second cell of the second row shows that there were 19 studies that described the ability of Amsterdam I criteria to predict MMR status. The studies are described in Appendix F-6. The third cell of the second row shows that of these 19 studies, only two performed MMR testing in unselected patients; these studies are described in Appendix F-7, and so on.

ND: No data

Figure 8

Table 17

Sensitivity and specificity of various predictors for (more...)

Table 17

Sensitivity and specificity of various predictors for detecting MMR gene mutations

Predictor	Unselected CRC probands			Revised Bethesda guidelines			Bethesda guidelines			Amsterdam II criteria			Amsterdam I criteria
	N	Sens [%](95% CI)	Spec [%](95% CI)	N	Sens [%](95% CI)	Spec [%](95% CI)	N	Sens [%](95% CI)	Spec [%](95% CI)	N	Sens [%](95% CI)	Spec [%](95% CI)	N	Sens [%](95% CI)	Spec [%](95% CI)
Amsterdam I criteria	2	45 (29, 63)	99 (74, 100)		ND		2	57 (46, 68)	48 (35, 62)	5	80 (70, 88)	24 (12, 43)
Amsterdam II criteria	2	28 (15, 47)	99 (97, 100)	1	68 (43, 87)	65 (51, 76)	3	84 (74, 91)	62 (53, 69)
Modified Amsterdam criteria		ND			ND			ND
Bethesda guidelines	1	73 (39, 94)	82 (80, 84)		ND
Revised Bethesda guidelines	1	91 (59, 100)	77 (75, 79)
Age <50 years	3	31 (18, 47)	95 (94, 96)		ND			ND			ND			ND
1st degree family history of CRC or EC	4	76 (50, 91)	87 (86, 89)		ND			ND
Multiple CRC or EC tumors in the same patient	3	38 (25, 54)	97 (91, 99)		ND			ND			ND			ND
Age <50 years, family history of CRC or EC, or multiple tumors in same patient	3	88 (60, 97)	77 (74, 81)		ND			ND			ND			ND
Suggestive MSIa	2	100 (88, 100)	90 (88, 92)	1	100 (75, 100)	79 (63, 90)		ND			ND		3	100 (62, 100)	69 (20, 95)
Suggestive IHC		ND			ND			ND			ND		2	50 (20,80)	72 (15, 97)

CRC: colorectal cancer; EC: endometrial cancer; N: Number of studies; Sens: (summary) sensitivity; Spec: (summary) specificity.

a

Estimates are the same for combined MSI-H and MSI-L versus MSS and for MSI-H versus MSS.

We first present an overview of the clinical predictors among unselected CRC patients. We then present each one of the clinical predictors separately.

Overview of Clinical Predictors Among Unselected Patients With CRC

Table F-11

Ability of Amsterdam II criteria to identify MLH1 and (more...)

Table F-11

Ability of Amsterdam II criteria to identify MLH1 and MSH2 mutation carriers among unselected colorectal cancer probands

Study, year (Ref ID);	A. Study sample characteristics	A. Genetic testing	NAm2/Ntotal	Mutation		No mutation		Diagnostic performance (95% confidence interval)		Quality
Country	B. Verification bias	B. MLH1/MSH2		Am2 (+)	Am2 (-)	Am2 (+)	Am2 (-)	Sensitivity [%]	Specificity [%]
Single-/multi-center	C. Mean age (y); Males (%)	C. Definition of deleterious mutations
Genetic testing irrespectively of MSI or IHC test results
Salovaara, 2000 (1740)	A. Selection of all new unrelated CRC from 9 hospitals between 03/1996 and 06/1998	A. PCR → Sequencing Ø PCR for founder mutations in MLH1 gene	5/535	4	14	1	516	22 (6, 48)	100 (99, 100)	B
Finland	B. Yes: patients without MSI were screened only for MLH1 founder mutations	B. 17/1
Multi-center	C. 67, ND	C. Literature; comparison with non-cancer controls
Genetic testing only in patients selected after MSI and/or ICH
Pinol, 2005 (52)	A. Selection of newly diagnosed CRC from 25 centers.	A. CRC selected by MSI/IHC: PCR → Sequencing Ø also MLPA	22/1222	Assuming no mutations in the absence of MSI-H tumors or tumors with negative immunostaining:						B
Spain	B. Yes: patients without MSI or with negative immunostaining were not sequenced	B. 4/7		4	7	18	1197	36 (11, 69)	99 (98, 99)
Multi-center	C. 70, 60	C. Predicted transcript alteration; literature and databases

As mentioned in the methods, especially for unselected CRC populations we accepted that patients with MSI negative tumors would be negative for mutations.

Am2: Amsterdam II criteria; CRC: colorectal cancer; MLPA: Multiple ligation-dependent probe amplification; MSI(-H): microsatellite instability (high); ND: Not described;

N_Am2:Number fulfilling Amsterdam II criteria; N_total: total number of studied CRC; PCR: polymerase chain reaction

Conversion analysis or gene screening were not used in these two studies.

Ø: Analysis for large deletions has been used

Table F-16

Ability of Bethesda guidelines to identify MLH1 and (more...)

Table F-16

Ability of Bethesda guidelines to identify MLH1 and MSH2 mutation carriers among unselected patients with colorectal cancer

Study, year (Ref ID);	A. Comments on sampling characteristics	A. Genetic testing	NBeth/ Ntotal	Mutation		No mutation		Diagnostic performance (95% confidence interval)		Quality
Country	B. Verification bias	B. MLH1/MSH2		Beth (+)	Beth (-)	Beth (+)	Beth (-)	Sensitivity [%]	Specificity [%]
Single-/multi-center	C. Mean age (y); Males (%)	C. Definition of deleterious mutations
Pinol, 2005 (52)	A. Selection of newly diagnosed CRC from 25 centers.	A. CRC selected by MSI/IHC: PCR → Sequencing Ø also MLPA	224/1222	Assuming no mutations in the absence of MSI-H tumors or tumors with negative immunostaining:						B
Spain	B. Yes: patients without MSI or with negative immunostaining were not sequenced	B. 4/7		8	3	216	995	73 (39, 94)	82 (80, 84)
Multi-center	C. 70, 60	C. Predicted transcript alteration; literature and databases

Beth: Bethesda guidelines; CRC: colorectal cancer; IHC: immunohistochemistry; MLPA: Multiple ligation-dependent probe amplification; MSI: microsatellite instability; ND: Not described; N_total: total number of studied CRC; PCR: Polymerase chain reaction

Ø: Deletion analysis (detection of large genomic deletions) was used

Pinol et al. did not use gene screening methods or conversion analysis. They assessed only the MLH1 and MSH2 genes.

Table F-17

Ability of revised Bethesda guidelines to identify (more...)

Table F-17

Ability of revised Bethesda guidelines to identify MLH1 and MSH2 mutation carriers among unselected patients with colorectal cancer

Study, year (Ref ID);	A. Comments on sampling characteristics	A. Genetic testing	NBeth/Ntotal	Mutation		No mutation		Diagnostic performance (95% confidence interval)		Quality
Country	B. Verification bias	B. MLH1/MSH2		Beth Rev (+)	Beth Rev (-)	Beth Rev (+)	Beth Rev (-)	Sensitivity [%]	Specificity [%]
Single /multi-center	C. Mean age (y); Males (%)	C. Definition of deleterious mutations
Pinol, 2005 (52)	A. Selection of newly diagnosed CRC from 25 centers.	A. CRC selected by MSI/IHC: PCR → Sequencing Ø also MLPA	287/1222	Assuming no mutations in the absence of MSI-H tumors or tumors with negative immunostaining:						B
Spain	B. Yes: patients without MSI or with negative immunostaining were not sequenced	B. 4/7		10	1	277	934	91 (59, 100)	77 (75, 79)
Multi-center	C. 70, 60	C. Predicted transcript alteration; literature and databases

Beth Rev: Revised Bethesda guidelines; CRC: colorectal cancer; IHC: immunohistochemistry; MLPA: Multiple ligation-dependent probe amplification; MSI: microsatellite instability; ND: Not described; N_total: total number of studied CRC; PCR: Polymerase chain reaction

Ø: Deletion analysis (detection of large genomic deletions) was used

Pinol et al. did not use gene screening methods or conversion analysis. They assessed only the MLH1 and MSH2 genes.

Table F-19

Ability of Ability of young age at diagnosis (early (more...)

Table F-19

Ability of Ability of young age at diagnosis (early disease onset) to identify MLH1 and MSH2 mutation carriers among unselected colorectal cancer probands

Study, year (Ref ID);	A. Definition of early onset	A. Genetic testing	Ntotal	Mutation		No mutation		Diagnostic performance (95% confidence interval)		Quality
Country	B. Study sample characteristics	B. MLH1/MSH2		Early onset	No early onset	Early onset	No early onset	Sensitivity [%]	Specificity [%]
Single-/multi- center	C. Verification bias	C. Definition of deleterious mutations
	D. Mean age (y); Males (%)
Genetic testing irrespectively of tumor microsatellite instability status
Salovaara, 2000 (1740)	A. <50 y	A. PCR → Sequencing Ø PCR for founder mutations in MLH1 gene	535	5	13	40	477	28 (10, 53)	92 (90, 94)	B
Finland	B. Selection of all new unrelated CRC from 9 hospitals between 03/1996 and 06/1998	B. 17/1
Multi-center	C. Yes: patients without MSI were screened only for MLH1 founder mutations	C. Literature; comparison with non-cancer controls
	D. 67, ND
Aaltonen, 1998 (2282)	A. <50 y	A. Ø All CRC: PCR for founder mutations 1 & 2 in MLH1 ♦CRC with MSI: (some) PCR → DGGE → Sequencing (Remaining) PCR → Sequencing	509	4	6	12	487	67 (22, 96)	98 (96, 99)	B
Finland	B. Selection of all new unrelated CRC from 9 hospitals between 05/1994 and 04/1996	B. 9/1
Multi-center	C. Yes: patients without MSI were screened only for MLH1 founder mutations	C. Literature; comparison with healthy controls
	D. ND, ND
Genetic testing only in patients selected after MSI and/or ICH
Pinol, 2005 (52)	A. <50 y	A. CRC selected by MSI/IHC: PCR → Sequencing Ø also MLPA	1222	Assuming no mutations in the absence of MSI-H tumors or tumors with negative immunostaining:						B
Spain	B. Selection of newly diagnosed CRC from 25 centers	B. 4/7		3	8	55	1156	27 (6, 61)	95 (94, 97)
Multi-center	C. Yes: patients without MSI or with negative immunostaining were not sequenced	C. Predicted transcript alteration; literature and databases
	D. 70, 60
Samowitz 2001, (34);	A. <55y	A. CRC selected by MSI:bPCR → Sequencing Ø Also PCR for founder mutations in the MLH1 gene	1066	Assuming no mutations in the absence of MSI-H tumors:						B
US	B. Selection among incident CRC	B. 5/3c		4	3	154	863	57 (18, 90)	85 (83, 87)
Multicenter	C. Yes: patients without suggestive MSI results were not sequenced	C. Predicted transcript alteration; literature
	D. ND, ND

Studies are ordered by quality and then by decreasing number of patients available for the calculation of sensitivity and specificity (2 by 2 tables). None of the studies used conversion analysis to detect mismatch repair gene mutations.

Am1/2: Amsterdam I criteria/II; CRC: colorectal cancer; DGGE: Denaturing gradient gel electrophoresis; DHPLC: denaturating high performance liquid chromatography; MLPA: Multiple ligation-dependent probe amplification; MSI: microsatellite instability; ND: Not described; N_total: total number of studied CRC; PCR: polymerase chain reaction

♦: Gene screening method

Ø: Detection of large genomic deletions

Table F-21

Ability of familial history of malignancy to identify (more...)

Table F-21

Ability of familial history of malignancy to identify MLH1 and MSH2 mutation carriers among unselected colorectal cancer probands

Study, year (Ref ID);	A. Definition of familial history of cancer	A. Genetic testing	Ntotal	Mutation		No mutation		Diagnostic performance (95% confidence interval)		Quality
Country	B. Definition of sporadic cancer	B. MLH1/MSH2		FamilialCancer Hx	Sporadic Cancer	Familial Cancer Hx	Sporadic Cancer	Sensitivity [%]	Specificity [%]
Single-/multi-center	C. Comments on sample characteristics	C. Definition of deleterious mutations
	D. Verification bias
	E. Mean age (y); Males (%)
Genetic testing irrespectively of tumor microsatellite instability status
Salovaara, 2000 (1740);	A. 1st degree relative with CRC or endometrial cancer	A. PCR → Sequencing Ø PCR for founder mutations in MLH1 gene	535	15	3	62	455	83 (59, 96)	88 (85, 91)	B
Finland	B. All other CRC	B. 17/1
Multi-center	C. Selection of all new unrelated CRC from 9 hospitals between 03/1996 and 06/1998	C. Literature; comparison with non-cancer controls
	D. Yes: patients without MSI were screened only for MLH1 founder mutations
	E. 67, ND
Aaltonen, 1998 (2282);	A. 1st degree relative with CRC or endometrial cancer	A. Ø All CRC: PCR for founder mutations 1 & 2 in MLH1 ♦CRC with MSI: (some) PCR → DGGE → Sequencing (Remaining) PCR → Sequencing	509	9	1	71	428	90 (55, 100)	86 (82, 89)	B
Finland	B. All other CRC	B. 9/1
Multi-center	C. Selection of all new unrelated CRC from 9 hospitals between 05/1994 and 04/1996	C. Literature; comparison with healthy controls
	D. Yes: patients without MSI were screened only for MLH1 founder mutations
	E. ND, ND
Genetic testing only in patients selected after MSI and/or ICH
Pinol, 2005 (52);	A. 1st degree relative with CRC of endometrial cancer	A. CRC selected by MSI/IHC: PCR → Sequencing Ø also MLPA	1222	Assuming no mutations in the absence of MSI-H tumors or tumors with negative immunostaining:						B
Spain	B. All other CRC	B. 4/7		6	5	151	1060	55 (23, 83)	88 (86, 89)
Multi-center	C. Selection of newly diagnosed CRC from 25 centers.	C. Predicted transcript alteration; literature and databases
	D. Yes: patients without MSI or with negative immunostaining were not sequenced
	E. 70, 60
Samowitz 2001, (34);	A. Family history of CRC	A. CRC selected by MSI:aPCR → Sequencing Ø Also PCR for founder mutations in the MLH1 gene	1066	Assuming no mutations in the absence of MSI-H tumors:						B
US	B. All other patients	B. 5/3b		4	3	147	870	57 (18,90)	86 (83,88)
Multicenter	C. Selection among incident CRC	C. Predicted transcript alteration; literature
	D. Yes: patients without suggestive MSI results were not sequenced
	E. ND, ND

Studies are ordered by quality and then by decreasing number of patients available for the calculation of sensitivity and specificity (2 by 2 tables). None of the studies used conversion analysis to detect mismatch repair gene mutations.

Am1: Amsterdam I criteria; CRC: colorectal cancer; DGGE: Denaturing gradient gel electrophoresis; Hx: History; IHC: immunohistochemistry; MSI: microsatellite instability; ND: Not described; N_total: total number of studied CRC; PCR: polymerase chain reaction

♦: Gene screening method

Ø: Detection of large genomic deletions

a

130 out of 171 of people with tumors with MSI instability could be genetically tested.

b

One patient had mutations both in the MLH1 and in the MSH2 gene

Table F-22

Ability of presence of multiple tumors to identify (more...)

Table F-22

Ability of presence of multiple tumors to identify MLH1 and MSH2 mutation carriers among unselected colorectal cancer probands

Study, year (Ref ID);	A. Definition of multiple tumors	A. Genetic testing	Ntotal	Mutation		No mutation		Diagnostic performance (95% confidence interval)		Quality
Country	B.Comments on sample characteristics	B. MLH1/MSH2		Multiple tumors	No multiple tumors	Multiple tumors	No multiple tumors	Sensitivity [%]	Specificity [%]
Single-/multi-center	C. Verification bias	C. Definition of deleterious mutations
	D. Mean age (y); Males (%)
Genetic testing irrespectively of tumor microsatellite instability status
Salovaara, 2000 (1740);	A. Synchronous or metachronous endometrial cancer or CRC	A. PCR → Sequencing Ø PCR for founder mutations in MLH1 gene	535	7	11	5	512	64 (31, 89)	99 (98, 100)	B
Finland	B. Selection of all new unrelated CRC from 9 hospitals between 03/1996 and 06/1998	B. 17/1
Multi-center	C. Yes: patients without MSI were screened only for MLH1 founder mutations	C. Literature; comparison with non-cancer controls
	D. 67, ND
Aaltonen, 1998 (2282);	A. Synchronous or metachronous endometrial cancer or CRC	A. Ø All CRC: PCR for founder mutations 1 & 2 in MLH1 ♦ CRC with MSI: (some) PCR → DGGE → Sequencing (Remaining) PCR → Sequencing	509	4	6	12	487	40 (12,74)	98 (96,99)	B
Finland	B. Selection of all new unrelated CRC from 9 hospitals between 05/1994 and 04/1996	B. 9/1
Multi-center	C. Yes: patients without MSI were screened only for MLH1 founder mutations	C. Literature; comparison with healthy controls
	D. ND, ND
Genetic testing only in patients selected after MSI and/or ICH
Pinol, 2005 (52);	A. Synchronous or metachronous endometrial cancer or CRC	A. CRC selected by MSI/IHC: PCR → Sequencing Ø also MLPA	1222	Assuming no mutations in the absence of MSI-H tumors or tumors with negative immunostaining:						B
Spain	B. Selection of newly diagnosed CRC from 25 centers	B. 4/7		4	7	90	1121	36 (11, 69)	93 (91, 94)
Multi-center	C. Yes: patients without MSI or with negative immunostaining were not sequenced	C. Predicted transcript alteration; literature and databases
	D. 70, 60

Studies are ordered by quality and then by decreasing number of patients available for the calculation of sensitivity and specificity (2 by 2 tables). None of the studies used conversion analysis to detect mismatch repair gene mutations.

CRC: colorectal cancer; DGGE: Denaturing gradient gel electrophoresis; Hx: History; IHC: immunohistochemistry; MSI: microsatellite instability; ND: Not described; N_total: total number of studied CRC; PCR: polymerase chain reaction

♦:Gene screening method

Ø:Detection of large genomic deletions

Table F-23

Ability of presence of combined family history of colorectal (more...)

Table F-23

Ability of presence of combined family history of colorectal cancer, young age at onset or presence of multiple tumors to identify MLH1 and MSH2 mutation carriers among unselected colorectal cancer probands

Study, year (Ref ID);	A. Definition of combined criteria	A. Genetic testing	Ntotal	Mutation		No mutation		Diagnostic performance (95% confidence interval)		Quality
Country	B. Comments on sample characteristics	B. MLH1/MSH2		Combined criteria	Other	Combined criteria	Other	Sensitivity [%]	Specificity [%]
Single-/multi-center	C. Verification bias	C. Definition of deleterious mutations
	D. Mean age (y); Males (%)
Genetic testing irrespectively of tumor microsatellite instability status
Salovaara, 2000 (1740);	A. Age <50 or CRC or endometrial cancer in 1st degree family or personal history of CRC or endometrial cancer	A. PCR → Sequencing Ø PCR for founder mutations in MLH1 gene	535	17	1	100	417	94 (73, 100)	81 (77, 84)	B
Finland	B. Selection of all new unrelated CRC from 9 hospitals between 03/1996 and 06/1998	B. 17/1
Multi-center	C. Yes: patients without MSI were screened only for MLH1 founder mutations	C. Literature; comparison with non-cancer controls
	D. 67, ND
Aaltonen, 1998 (2282);	A. Age <50 or CRC or endometrial cancer in 1st degree family or personal history of CRC or endometrial cancer	A. Ø All CRC: PCR for founder mutations 1 & 2 in MLH1 ♦ CRC with MSI: (some) PCR → DGGE → Sequencing (Remaining) PCR → Sequencing	509	10	0	112	387	100 (69, 100)	78 (74, 81)	B
Finland	B. Selection of all new unrelated CRC from 9 hospitals between 05/1994 and 04/1996	B. 9/1
Multi-center	C. Yes: patients without MSI were screened only for MLH1 founder mutations	C. Literature; comparison with healthy controls
	D. ND, ND
Genetic testing only in patients selected after MSI and/or ICH
Pinol, 2005 (52);	A. Age <50 or CRC or endometrial cancer in 1st degree family or personal history of CRC or endometrial cancer	A. CRC selected by MSI/IHC: PCR → Sequencing Ø also MLPA	1222	Assuming no mutations in the absence of MSI-H tumors or tumors with negative immunostaining:						B
Spain	B. Selection of newly diagnosed CRC from 25 centers.	B. 4/7		8	3	306	905	73 (39, 94)	75 (72, 77)
Multi-center	C. Yes: patients without MSI or with negative immunostaining were not sequenced	C. Predicted transcript alteration; literature and databases
	D. 70, 60

Studies are ordered by quality and then by decreasing number of patients available for the calculation of sensitivity and specificity (2 by 2 tables). None of the studies used conversion analysis to detect mismatch repair gene mutations.

CRC: colorectal cancer; DGGE: Denaturing gradient gel electrophoresis; Hx: History; IHC: immunohistochemistry; MSI: microsatellite instability; ND: Not described; N_total: total number of studied CRC; PCR: polymerase chain reaction

♦:Gene screening method

Ø:Detection of large genomic deletions

Table F-25

Ability of microsatellite instability to identify MLH1 (more...)

Table F-25

Ability of microsatellite instability to identify MLH1 and MSH2 mutation carriers among unselected colorectal cancer probands

Study, year (Ref ID);	A. Comments on sample characteristics	Ntotal	Mutation		No mutation		Diagnostic performance (95% confidence interval)		Micro-dissection	NCI 5 marker set	Quality
Country	B. Verification bias		MSI-H	MSS	MSI-H	MSS	Sensitivity [%]	Specificity [%]
Single-/multi-center	C. Mean age (y); Males (%)
	D. Definition of MSI
Salovaara, 2000 (1740)	A. Selection of all new unrelated CRC from 9 hospitals between 0.3/1996 and 06/1998	535	18	0	48	469	100 (81, 100)	91 (88, 93)	X	X	B
Finland	B. Yes: patients without MSI were screened only for MLH1 founder mutations
Multi-center	C. 67, ND
	D. MSI based on BAT26 only
Aaltonen, 1998 (2282)	A. Selection of all new unrelated CRC from 9 hospitals between 05/1994 and 04/1996	509	10	0	53	446	100 (69, 100)	89 (86, 92)	X	X	B
Finland	B. Yes: patients without MSI were screened only for MLH1 founder mutations
Multi-center	C. ND, ND
	D. ≥30% of 16 markers for tumor analyzed with fluorescence methods or ≥ 2 out of 7 (≈30%) markers analyzed with radioactive technique (MSI-H)

Note that studies that assessed genetic mutations only in patients with MSI cannot be used to construct 2 by 2 tables for the ability of MSI testing to detect MMR mutations. Thus such studies are not included in this table.

CRC: colorectal cancer; MSI(-H): microsatellite instability (-high); MSS: microsatellite-stable (no instability); ND: Not described; N_total: total number of studied CRC