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Bast RC Jr, Kufe DW, Pollock RE, et al., editors. Holland-Frei Cancer Medicine. 5th edition. Hamilton (ON): BC Decker; 2000.

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Holland-Frei Cancer Medicine. 5th edition.

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Chapter 29Principles of Cancer Pathology

, MD, , MD, , MD, ,  MD, and , MD.

Pathologists are physicians who are concerned primarily with the study of disease in all its aspects; that is, causation, diagnosis, pathogenesis, mechanisms, natural history, anatomic and biochemical features, progression, and prognosis. There is a great deal of truth in the old adage that pathologists are “doctors’ doctors,” consultants with specialized knowledge that can be helpful to the clinician who is caring directly for the patient. Nowhere in medicine is this adage more true than in the care of patients with cancer.

Pathologists engage in three major types of activity: anatomic pathology, which includes surgical pathology, cytology, and autopsy pathology; clinical pathology, also known as laboratory medicine, that is, the direction of clinical laboratories; and experimental pathology or basic investigations of the pathogenesis of disease. While oncologists are apt to interact most closely, most consistently, and on a more personal level with anatomic pathologists in the course of their practice, they need to be aware of the roles played by pathologists of all three types if they are to provide optimal patient care. This is particularly true as the distinctions between several traditional types of pathologists have become blurred as advances in technology such as immunohistochemistry, flow cytometry, and molecular biologic approaches to cancer diagnosis, have moved from the research laboratory into the clinic.

This chapter reviews some of the basic principles of pathology as they apply to neoplastic disease. Primary emphasis is on solid tumors, though much of what is said also applies to neoplasms of other types, such as lymphomas, and leukemias. The goal is to provide for oncologists a better understanding of what pathologists do, how they arrive at diagnoses, what tools, especially what modern ones, they have at their disposal, and how the oncologist can interact most productively with the pathologist to achieve the greatest benefit for the patient.

Solid Tumor Structure and Tumor Stroma Generation

Structure of Solid Tumors

What is a tumor? Although physicians know very well what they mean when they use the term, the question is not a simple one to answer in a concise and comprehensive manner. The word “tumor” is of Latin origin and means “swelling.” But not all swellings (e.g., the swellings of inflammation and repair) are tumors in the modern sense of the term. The distinguished pathologist Wallace H. Clark1 has offered an excellent definition, paraphrased as follows: a tumor (fully evolved) is a population of abnormal cells characterized by temporally unrestricted growth and the ability to grow in at least three different tissue compartments—the original compartment; the mesenchyme of the primary site (tumor invasion); and a distant mesenchyme (tumor metastasis). This definition usefully emphasizes the progressive nature of tumor growth, the common (though not exclusive) origin of tumors as benign growths, their gradual acquisition of autonomy, and, at some stage, their ability to grow in new tissues distant from their site of origin, that is, to metastasize.

While some tumors (e.g., leukemias, ascites tumors) grow as cell suspensions, most tumors grow as solid masses of tissue. Solid tumors have a distinct structure that mimicks that of normal tissues2–4 and comprises two distinct but interdependent compartments: the parenchyma (neoplastic cells) and the stroma that the neoplastic cells induce and in which they are dispersed. In many tumors, including those of epithelial cell origin, a basal lamina generally separates clumps of tumor cells from stroma. However, the basal lamina is often incomplete, especially at points of tumor invasion.

Stroma is interposed between malignant cells and normal host tissues and is essential for tumor growth. Stroma is largely a product of the host and is induced as the result of tumor cell-host interactions. Thus, it comprises nonmalignant supporting tissue and includes connective tissue, blood vessels, and, very often, inflammatory cells. Stroma includes as one of its components the vascular supply that tumors require for obtaining nutrients, gas exchange, and waste disposal. Solid tumors, regardless of their type or cellular origin, require stroma if they are to grow beyond a minimal size of 1 to 2 mm.5 The stroma of solid tumors may also limit the influx of inflammatory cells or may limit the egress of tumor cells (invasion). Stroma, therefore, at once provides a lifeline that is necessary for tumor growth and imposes a barrier that inhibits and may regulate interchange of fluids, gases, and cells with the host.*

The singular importance of new blood vessel formation to tumor survival and growth has rightly led to an emphasis on angiogenesis; however, this emphasis has been accompanied by an unfortunate tendency to undervalue other tumor stromal components. Blood vessels are only one component of tumor stroma. In fact, in many tumors, the bulk of stroma comprises interstitial connective tissue, and blood vessels are only a minor component of the stromal mass. For the most part, tumor stroma is formed by elements that are derived from the circulating blood and from adjacent host connective tissues.6 Plasma components include water and plasma proteins, together with various types and numbers of inflammatory cells.* Almost any element found in normal connective tissues may be represented in tumor stroma, including even bone and cartilage. Generally speaking, the major components of tumor stroma include, in addition to new blood vessels, leaked plasma and plasma proteins; proteoglycans and glycosaminoglycans; interstitial collagens (types I, III, and, to a lesser extent, type V); fibrin (Plate 5, Fig. 29.1); fibronectin; and cells of two general types, fixed tissue cells, such as fibroblasts, that reside in normal connective tissue and inflammatory cells that are derived from the blood.6

Although the same basic building blocks comprise all tumor stroma, pathologists have long recognized that tumors differ markedly from each other in stromal content. Sometimes these differences are primarily quantitative. At one extreme are desmoplastic tumors, such as many carcinomas of the breast, stomach and pancreas, in which up to 90% or more of the total tumor mass consists of stroma. At the other extreme are tumors such as medullary carcinomas of the breast and many lymphomas in which only minimal stroma is deposited.

In other cases, differences in stromal content among different tumors are largely qualitative. For example, some carcinomas of the breast provoke the deposition of abundant elastic tissue along with collagen, whereas others (e.g., medullary carcinoma of the breast) induce an extensive lymphocytic infiltrate and little else in the way of stroma. Even within a single tumor, there may be significant variations in stromal composition from one area to another. This stromal heterogeneity should not be surprising in view of the well-recognized heterogeneity of the parenchymal cells present within individual tumors.

Tumor Stroma Generation

Steps in Tumor Stroma Generation

Studies of transplantable tumors have yielded important information concerning the pathogenesis of tumor stroma generation (Fig. 29.2).3,4,6–10 Among the earliest steps in this process is local vascular hyperpermeability to circulating macromolecules. Increased vascular permeability is attributable to vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), a multi-functional cytokine that is synthesized and secreted by the great majority of animal and human tumors.11,12

Figure 29.2. Actuarial rates of regional nodal failure and distant metastases for 776 patients with infiltrating ductal breast cancer related to histologic grade of the tumor.

Figure 29.2

Actuarial rates of regional nodal failure and distant metastases for 776 patients with infiltrating ductal breast cancer related to histologic grade of the tumor. Grading was performed using Elston’s modification of the Bloom and Richardson grading (more...)

Among other activities, VPF/VEGF renders the microvasculature hyperpermeable to plasma and plasma proteins with a potency some 50,000 times that of histamine and ranks among the most powerful vascular permeabilizing substances known (Fig. 29.3).6,7,9,10,13–15 When injected into skin or other normal tissues, VPF/VEGF, like mediators of inflammation, such as histamine, provokes the extravasation of a protein-rich plasma exudate; also like histamine, the primary target of VPF/VEGF action is postcapillary venules and small veins whose lining endothelial cells express the two VPF/ VEGF tyrosine kinase receptors, VEGFR-1 (flt-1) and VEGFR-2 (KDR, flk-1).11,12

Figure 29.3. Lewis lung carcinoma growing in flank of a syngeneic C57BI/6 mouse that had received a macromolecular tracer, 70 kD fluoresceinated dextran, 15 minutes previously.

Figure 29.3

Lewis lung carcinoma growing in flank of a syngeneic C57BI/6 mouse that had received a macromolecular tracer, 70 kD fluoresceinated dextran, 15 minutes previously. Bright-staining apple-green fluorescence forming a rim around the tumor represents extensive (more...)

An important and almost immediate consequence of VPF/VEGF action is leakage of plasma proteins, including fibrinogen and other clotting factors. For reasons that are not yet totally clear, vascular hyperpermeability and extravasation of plasma proteins leads to activation of the coagulation system.16 As a result, extravasated plasma fibrinogen is rapidly clotted to form an extravascular gel of crosslinked fibrin (see Fig. 29.2).4,7,9 Extravascular fibrin deposits are important because they dramatically alter the local microenvironment, transforming the erstwhile inert extravascular matrix of normal adult tissues into a proangiogenic provisional matrix that favors and apparently stimulates inward migration of host mesenchymal cells.11,17 Indeed, fibrin implanted in animals without tumor cells induces the invasion of new blood vessels and fibroblasts, resulting in a vascularized connective tissue that is not dissimilar in appearance or composition to tumor stroma.7,8 Other plasma proteins (e.g., plasma fibronectin), as well as locally synthesized structural proteins (e.g., cellular fibronectins, tenascin), hyaluronan, and at least two proteoglycans (chondroitin sulfate-rich proteoglycan and decorin) also contribute to this new tissue.6,11

The fibrin gel deposited by tumors is modulated by proteases (see Fig. 29.1) and is gradually replaced by the ingrowth of fibroblasts and new blood vessels which give rise to loose connective tissue, similar to the “granulation tissue” of healing wounds. After an additional period of time, this granulation tissue is further transformed into the poorly vascularized, densely collagenous scar-like connective tissue characteristic of tumor desmoplasia. Simultaneously, of course, other tumor cells have broken away from the original tumor site and have begun to recapitulate at nearby sites and particularly at the tumor’s growing edge in the same sequence of events—increased vascular permeability and new fibrin deposition. Thus, at any one time, growing desmoplastic tumors consist of older, generally more centrally placed portions comprising tumor cells that are encased in poorly vascularized, dense collagenous stroma and a more active, newer, fibrin-rich peripheral zone that interfaces with the surrounding host tissue.

VPF/VEGF: A Multifunctional Cytokine Critical to Tumor Angiogenesis and Stroma Formation

At this point, something more needs to be said about VPF/VEGF because of its central role in tumor angiogenesis and stroma generation (for more detailed accounts, see recent reviews11,12). VPF/VEGF is expressed in several different isoforms as the result of alternative splicing of a single, highly conserved gene. VPF/VEGF is the founding member of a family of proteins whose members include placenta growth factor and VEGF B, C, D, and E.12 At present, much more is known about VPF/VEGF (sometimes referred to as VEGF A) than about other, more recently discovered family members. In addition to its potent function as an effector of vascular hyperpermeability, VPF/VEGF has other important actions that contribute importantly to angiogenesis and stroma generation. Thus, VPF/VEGF stimulates endothelial cell division and migration. It also induces endothelial cells to express increased amounts of tissue factor, urokinase, tissue plasminogen activator and matrix metalloproteases.11,12,18,19 Collectively, these endothelial cell products induce clotting and initiate fibrinolysis and degradation of collagen and other elements of pre-exisiting matrix, all important steps in angiogenesis and stroma generation.

A number of factors serve to regulate VPF/VEGF expression in tumor cells. VPF/VEGF expression is greatly stimulated by tissue hypoxia and, perhaps independently, by low tissue pH, conditions that are commonly present in the tumor microenvironment.11,12 However, it would be a mistake to think that hypoxia is the only factor responsible for VPF/VEGF overexpression by tumor cells. Many tumor cells make substantial amounts of VPF/VEGF under normoxic or even hyperoxic conditions. Other factors that induce VPF/VEGF overexpression include cytokines (e.g., epidermal growth factor, basic fibroblast growth factor), certain hormones (e.g., thyroglobulin), and, perhaps of more general interest, various oncogenes (e.g., src, ras) and tumor suppressor genes including the von Hippel Lindau protein.11,12,20,21

Relation of Tumor Stroma Generation to Wound Healing and Other Examples of Pathologic and Physiologic Angiogenesis and Stroma Generation

The events of stroma generation in transplantable tumors closely mimic those of normal wound healing.4,22 As in tumors, VPF/VEGF expression is strikingly upregulated in wound healing as it occurs in several tissues22 as well as in a variety of analogous pathologic and physiologic processes that involve new blood vessel and stroma formation; these include rheumatoid arthritis, psoriasis, delayed hypersensitivity, diabetic retinopathy, and corpus luteum formation.11 In all these processes, the initial event is a local increase in vascular permeability, followed, in turn, by extravascular clotting, fibrin deposition, and infiltration of new blood vessels and connective tissue cells, leading to the development of granulation tissue and finally of dense fibrous connective tissue (termed “desmoplasia” in tumors and “scars” or fibroplasia in the other entities). It would seem, therefore, that tumors have preempted and subverted, for their own purposes, a fundamental host mechanism, the wound healing response, as the means to acquire the stroma they need to grow and spread.4 Of course, there are some differences. Platelets, which play several critical roles in wound healing, seem not to participate to any great extent in tumor stroma generation; however, many platelet functions can be subsumed by tumor cells, which express similar or analogous cytokines and growth factors. Tumors differ from healing wounds in another important respect. At wound sites, overexpression of VPF/VEGF and consequent vascular hyperpermeability are limited to a period of a few days, presumably until oxygen tension has returned to normal;22 by contrast, VPF/VEGF expression and vascular hyperpermeability are not limited in tumors and persist indefinitely. Thus, tumors behave in some sense as wounds that do not heal.4

The analogy between wound healing and tumor stroma generation may be taken one step further. Except in lower vertebrates that are capable of regenerating normal tissues, wound healing does not recapitulate ontogeny but, instead, replaces injured parenchyma and stroma with connective tissue whose functional capacities fall well short of the original normal tissue. In the same manner, tumor stroma, especially that of poorly differentiated malignant tumors, is generally a disorganized and poorly supportive parody of normal connective tissue. The vascular supply is often marginal. Tumor blood vessels are generally poorly differentiated, unevenly spaced, and often unequal to the task of supporting the growth and even the life of rapidly metabolizing tumor cells.23 The result is irregular bloodflow, uneven perfusion, shifting zones of anoxia, low pH, and, commonly, necrosis and apoptosis.24 In fact, the presence of necrosis is sometimes helpful to the pathologist in distinguishing malignant tumors from their benign counterparts and certain non-neoplastic processes.

Stroma Generation in Autochthonous Human Tumors

Detailed, interventional studies of the type required to elucidate the pathogenesis of tumor stroma generation in animal tumors are not ethically feasible in patients. Nonetheless, there is good reason to believe that similar mechanisms are involved in human malignancy. First, VPF/VEGF is overexpressed at both the mRNA and protein levels in the great majority of primary and metastatic human tumors that have been studied; these include carcinomas arising in the gastrointestinal tract, pancreas, stomach, breast, kidney and bladder, as well as glioblastomas.11 Second, both specific, high-affinity receptors for VPF/VEGF are also overexpressed in the microvascular endothelial cells that supply these tumors.11,25 Finally, many human tumors exhibit evidence of vascular hyperpermeability to plasma proteins, including spillage of fibrinogen with deposition of crosslinked extravascular fibrin as observed in animal tumors.4,26,27 Taken together, there is strong evidence that the pathogenesis of stroma formation in human tumors closely follows that in animal tumors, though allowance must be made for species differences and for the generally slower growth rate of autochthonous human tumors.

Role of the Surgical Pathologist in the Diagnosis and Management of the Cancer Patient

Surgical pathologists have the definitive role in tumor diagnosis. No matter how high the index of clinical suspicion, the diagnosis of cancer is not conclusively established nor safely assumed in the absence of a tissue diagnosis. With very few exceptions, definitive therapy for cancer should not be undertaken in the absence of a tissue diagnosis. Policies supporting this practice are written into the bylaws of most hospitals and are regularly monitored by hospital tissue committees and by accrediting agencies.

It is the task of the surgical pathologist to provide an accurate, specific, and sufficiently comprehensive diagnosis to enable the clinician to develop an optimal plan of treatment and, to the extent possible, estimate prognosis. There was a time not many years ago when the simple designation “benign” or “malignant” provided the clinician with all of the information necessary to provide appropriate care for the patient. This is no longer the case. Cancer is not a single disease. There are more than 300 distinct varieties of tumors, each with a characteristic biology. Moreover, tumors have a course of historical development and progression; in an individual patient, they may be first recognized at any stage along that course. The tremendous advances in all fields of oncology require a great deal of additional information, and nearly every case, in fact, requires a fuller understanding of the patient’s particular tumor to allow the most appropriate classification for research, for prognosis, and for therapeutic intervention. Details of the type and origin of the tumor, its differentiation, level of invasion, the numbers of lymph nodes with and without metastatic tumor, their architecture, the presence or absence of hormone receptors, the activity of specific enzymes, ploidy, frequency of mitosis, and percentage of cells in the S-phase may all be relevant in the pathologic assessment of neoplasia. Molecular pathology, for example, using nucleic acid probes with or without amplification by the polymerase chain reaction to detect expression of specific tumor genes or gene mutations, has not yet reached standard practice but promises a golden age for pathology in the next decade.

Surgical pathologists deal primarily with structure. Careful gross examination of excised tissue, first with the naked eye or with the help of a dissecting microscope, is followed by a more detailed examination of tissue sections in the compound light microscope. Intraoperative examination may make use of frozen tissue sections, but in most instances, pathologists rely on the better preservation of structure afforded by permanent tissue sections stained with hematoxylin and eosin (H & E) and occasionally other dyes. Histochemistry, immunohistochemistry, and electron microscopy are helpful or necessary supplements for diagnosis in 10 to 15% of solid tumors. In addition, surgical pathologists collaborate closely with cytopathologists in diagnoses involving exfoliated cells or needle aspirates and with clinical pathologists who make use of other techniques, such as culture for microorganisms, flow cytometry, and specialized laboratory tests of a biochemical, immunologic, or molecular nature. In order to perform most of these supplementary studies, the specimen must be specially processed while it is still fresh; that is, prior to tissue fixation. It is a responsibility of the surgical pathologist to coordinate these various activities and to synthesize the information provided by each into a comprehensive diagnosis that is maximally informative to the clinician caring for the patient.

Methods for Obtaining Specimens

Tissue may be obtained in a number of ways, each with its appropriate place and uses, depending on the clinical circumstances. Cytological examination of exfoliated, scraped, or brushed cells can be a rapid, efficient, and low-risk technique for establishing an accurate diagnosis. This approach, along with the related technique of fine-needle aspiration is discussed in greater detail later; for obvious reasons, these approaches do not always reveal the primary tumor site or the extent of disease. Cutting-needle biopsies, core- needle biopsies, or drill biopsies obtain tissue cores for histologic examinations or special studies that permit evaluation of architectural structure but have a greater risk of bleeding and patient discomfort than use of fine needles. Incisional biopsy (along with fine-needle aspiration) is often the method of choice for lesions that are inoperable, too large for ready excision, or when excision could lead to functional or cosmetic impairment. Care must be taken that incisional biopsies are performed in a fashion that will not compromise definitive therapy; that is, the tissue excised should be confined to an area which will be encompassed by subsequent treatment. Excisional biopsy is often favored because it provides generous amounts of tissue for diagnosis and may itself afford sufficient surgical therapy for some tumors, for example, small- to medium-sized breast cancers.

There are many potential pitfalls in biopsy interpretation. These include inadequate tissue sampling and artifacts induced by the procedure itself, such as thermal damage caused by an electrocautery or laser. Except for excisional biopsies, negative findings do not exclude the possibility that a tumor or any other significant pathologic condition is present but was not included in the tissue submitted for examination. Thus, for procedures short of complete excision, the clinician must be prepared to perform a second, often more extensive procedure if the first does not yield sufficient diagnostic information.

Gross Handling of Specimens

The pathologist must regard, and therefore properly triage biopsies, particularly excisional biopsies, as the definitive surgical specimen. To do this well, the pathologist must be informed about the clinical history, differential diagnosis, relevant laboratory results, gross tissue examination, and frozen section findings, if any, since they may individually or together dictate whether special studies are required. Specimens should be marked with clips, sutures, or ink to provide anatomic orientation, and these should be described in the pathology submission sheet. Often, tissue arrives in the pathology laboratory in formalin or other fixatives. At that stage, it is already too late to perform many special studies (e.g., microbiologic cultures, certain types of immunohistochemistry and optimal electron microscopy) that may prove to be critical for diagnosis. This fact emphasizes the importance of consulting the pathologist in advance in order to avoid the need for repeat biopsy. Frequently the goal of biopsy is to determine whether the lesion is benign or malignant, with the expectation of performing additional surgery if the lesion proves malignant. In this case, supplementary tests may properly be deferred to subsequent, more definitive surgery, at which time larger amounts of tissue become available.

The gross specimen should be described with regard to its appearance and characteristics, taking care to measure in three dimensions the size of the specimen and, if visible, the lesion itself along with the distances between the lesion edges and the excision (resection) margins. Excision margins should be identified and marked with ink prior to any dissection, thus permitting accurate measurement of these distances microscopically. Depending on the type of specimen and the clinical circumstances, margins can be evaluated by analysis of frozen sections. All lymph nodes associated with the specimen need to be dissected out, described along with their location, and processed for histology.

A still more careful examination is required for certain biopsies, for example, those of the breast, where no lesion may be visible to the naked eye. In addition, breast specimens with calcification often require specimen radiography. Ideally, therefore, a radiograph of the intact specimen should be obtained, following which the margins should be inked, the specimen “breadloafed” and radiographs taken of each slice. Sections should then be coded, processed individually for histology, and correlated with the corresponding radiographs.28,29

Preparation of Microscopic Sections

Microscopic examination requires that tissues be cut with a microtome into thin sections that can be stained with dyes such as hematoxylin and eosin (H & E), toluidine blue, and other special stains for specific tissue components, such as mucus, glycogen, cytoplasmic granules, collagen, bacteria, and fungi. Two types of sectioning methods are most commonly used: frozen sections and paraffin-embedded or permanent sections. Frozen sections can be prepared rapidly (within minutes) during the course of surgery while the patient is still under anesthesia and therefore are of the greatest practical value in situations where an immediate answer is required for an important clinical question. At one time, frozen sections were commonly performed intraoperatively in patients with suspected breast cancer, with the expectation that definitive radical surgery would follow immediately if cancer was found. With less aggressive surgical therapy now common for treatment of breast cancer, this practice has precipitously declined.

However, frozen sections continue to have many important applications. First, they are useful for determining whether a lesion is a neoplasm and, if so, whether it is benign or malignant. Second, they can provide information as to the extent of regional tumor metastases which may govern decisions concerning further surgery; for example, mediastinal lymph node involvement in primary carcinoma of the lung, peripancreatic lymph node involvement by carcinoma of the pancreas. Third, they allow the pathologist to determine whether the resection margins are adequate following definitive cancer surgery, such as resection of skin, gut, or pulmonary lesions. If resection margins are inadequate, additional tissue can be removed immediately, without the need for a subsequent operation. Some tumors, such as those arising in the soft tissues or breast, are best evaluated in permanent sections, however. Finally, perhaps the most common current use of frozen sections is to determine the appropriate additional workup necessary for a particular tissue specimen while it is still fresh; for example, if the metastatic tumor found in a lymph node is recognized as a poorly differentiated carcinoma, special fixation and electron microscopy may be required for proper diagnosis. On the other hand, if the tumor is a lymphoma, an entirely different set of studies may be required, such as those for cell surface antigen markers and gene rearrangement.

In contrast to frozen sections, permanent sections are prepared from tissues that have been fixed, dehydrated and embedded in paraffin wax as a supporting medium prior to sectioning. Though requiring more time for preparation (generally 12 to 24 hours), permanent sections offer a number of important advantages over frozen tissue sections. Sections are generally thinner (typically 5 μm) and, due to avoidance of freezing artifacts, are of better overall quality and therefore permit greater certainty of interpretation. A broader repertoire of stains is also available for permanent sections. Certain tissues, such as those containing fat or bone, cut poorly as frozen sections but may be satisfactorily studied in permanent sections. As a general rule, if insufficient tissue is available for both frozen and permanent sections, only permanent sections should be prepared. While the opinions just expressed certainly represent a majority view, some excellent pathology departments routinely diagnose tumors on the basis of frozen sections and prepare permanent sections primarily for archival purposes.

Microscopic Interpretation of Tissue Sections

In cases of suspected cancer, the first task of the surgical pathologist is to decide whether a neoplasm is present. As noted earlier in this chapter, the word “tumor” is Latin for “swelling,” and various types of “swelling” can masquerade as neoplasms. These include inflammatory lesions, repair, hypertrophy, hyperplasia (e.g., keloids), choristomas (ectopic rests), and hamartomas (masses of mature cells that are appropriate to a given site but are arranged in a disorganized fashion as the result of aberrant differentiation). This initial distinction is often made easily, for example, hyperplastic polyps of the colon, nasal polyps, and skin tags are not likely to be confused with true neoplasms. Sometimes, however, the task is less straightforward. Not infrequently, tumors generate an extensive inflammatory response, and it is not unusual, for example, in endoscopic biopsies of gastric carcinomas, to find only after a prolonged search rare individual cancer cells “buried” in an extensive inflammatory cell infiltrate. Healing ulcerations of the gastrointestinal or cervical mucosae may sometimes closely resemble the carcinomas or premalignant lesions (e.g., squamous intra-epithelial lesion, low- or high-grade) that arise in those tissues. Finally, atypical hyperplasia can be very difficult to distinguish from in situ carcinoma and, even when no evidence of tumor is found, may represent an important diagnostic finding. For example, patients whose breast biopsies show atypical hyperplasia have a four- to five-fold increased risk for developing breast cancer at a later time.30

Having decided that a neoplasm is present on the basis of criteria such as cellular abnormalities or invasion (see below), the pathologist’s next task is to classify it. A number of classification schemes are possible, but the most important of these is based on the tumor’s histogenetic or cytogenetic origin. Histogenetic/cytogenetic classification is often supplemented by other useful descriptors such as those provided by the tumor’s gross or microscopic appearance (e.g., polypoid, papillomatous), the degree of cellular differentiation (e.g., well- or poorly differentiated [Fig. 29.4]), and, perhaps most importantly, by the expected biologic behavior (benign versus malignant). Broadly speaking, tumors of epithelial cell origin are termed adenomas or papillomas when benign and carcinomas when malignant. Carcinomas account for ~80% of all malignant tumors. Their classification is often further qualified on the basis of the type of epithelium present, for example, glandular (adenocarcinoma [Fig. 29.5]), squamous (squamous cell carcinoma [Fig. 29.6]), transitional cell (transitional cell carcinoma). Addition of the suffix “-oma” to the cell of origin also describes benign tumors of mesenchymal origin (e.g., lipomas, fibromas, leiomyomas). Malignant tumors of mesenchymal origin are designated sarcomas (e.g., liposarcomas, fibrosarcomas, leiomyosarcomas). Most tumors comprise a single type of neoplastic cell. However, a few tumors contain neoplastic cells of more than a single type, for example, Wilms’ tumors. Even rarer are tumors containing neoplastic cells that derive from more than a single germ layer, for example, teratomas (dermoid cysts). Certain tumors have long been identified with trivial names that do not follow any well-ordered classification scheme. Examples include seminomas (for carcinomas of testicular epithelial cell origin), hypernephromas (for renal cell carcinomas), and melanomas (for melanocarcinomas) (Fig. 29.7). Other tumors, due to prolonged use, continue to bear eponyms (e.g., Hodgkin’s disease, Ewing’s sarcoma, Kaposi’s sarcoma).

Figure 29.4. Undifferentiated carcinoma of lung.

Figure 29.4

Undifferentiated carcinoma of lung. Tumor is composed of irregularly arranged pleomorphic tumor cells with large nuclei and prominent nucleoli. A central necrotic zone, typical of this type of tumor, is present. There is little stroma. H&E; magnification; (more...)

Figure 29.5. Adenocarcinoma of breast.

Figure 29.5

Adenocarcinoma of breast. Tumor is arranged in the form of fairly well-differentiated glands separated by a fibrous connective tissue stroma. Moderate numbers of inflammatory cells, mostly lymphocytes, are present in stroma. H&E; magnification: (more...)

Figure 29.6. Squamous cell carcinoma of the skin of the face.

Figure 29.6

Squamous cell carcinoma of the skin of the face. Tumor consists of island of well-differentiated squamous epithelium separated by fibrous connective tissue stroma. Note that epithelium forms “keratin pearls”. H&H; magnification: (more...)

Figure 29.7. Malignant melanoma arising in skin.

Figure 29.7

Malignant melanoma arising in skin. Tumor is composed of large, irregular cells with large nuclei, prominent nucleoli, and abundant, clear cytoplasm, peppered with dots and larger accumulations of melanin pigment. H&E; magnification: x100.

The pathologist must carry classification further still. Even within a single organ and within a single type of epithelium, several different types of tumors may arise, each with its own special characteristics, prognosis, and response to therapy. In the breast, for example, the two most common types of malignant tumor are infiltrating ductal carcinoma (sometimes designated as carcinoma “not otherwise specified” or NOS) which accounts for ~78% of infiltrating breast cancers, and infiltrating lobular carcinomas, which account for an additional ~9% of breast cancers. These two tumors, together accounting for nearly 90% of breast cancers, have similar prognoses that are less favorable than those of the other, less common types of breast carcinoma (i.e., tubular, mucinous or colloid, medullary, papillary, and adenoid cystic carcinomas).31

One of the most important distinctions the surgical pathologist can make is that between tumors that are benign or malignant. In general, benign tumors share certain properties. The neoplastic cells that make up the tumor are usually well differentiated, closely resembling the corresponding cells of normal tissue. Benign tumors tend to expand uniformly in all directions unless impeded from doing so by surrounding structures, for example, compression by the bony skull often causes meningiomas to take on a flattened appearance. As expansile masses, benign tumors cause compression atrophy of surrounding normal tissues that results in the formation of a thin rim of fibrous connective tissue; this enveloping connective tissue rim may serve as a “capsule” that renders benign tumors discrete, readily palpable, and easily movable. However, not all benign tumors have capsules, for example, leiomyomas of the uterus, hemangiomas, and tubular adenomas (adenomatous polyps) of the large intestine.

Malignant tumors, or cancers, are characterized primarily by the abnormality of their neoplastic cells. These cellular abnormalities are of two general types, those involving intercellular relationships and those affecting individual neoplastic cells. With regard to the former, malignant tumors commonly exhibit increased cell number and altered orientation of both neoplastic cells and stroma that may be best described as “helter-skelter” or disorganized. For example, carcinomas of the skin may comprise squamous cells that differentiate and mature fairly normally; however, the cells are commonly organized into nests, in which the least differentiated cells are situated peripherally and the most differentiated cells are positioned centrally where they form keratin pearls. Further, these tumor cell nests are surrounded by disorganized stroma. Disturbed intercellular arrangements such as these are of great help to the pathologist reading a tissue section; much of tumor diagnosis depends on the pathologist’s ability to recognize altered microscopic tissue patterns.

Abnormalities of individual neoplastic cells may also be helpful in diagnosis, particularly increased numbers of mitoses and cytological features relating to the state of tumor cell differentiation. Cytological features of malignancy include altered polarity, tumor cell enlargement, increased ratio of nuclear to cytoplasmic area (may approach 1:1 instead of the normal 1:4 or 1:6, though exceptions exist), pleomorphism (variation in size and shape) of tumor cells and their nuclei, clumping of nuclear chromatin and distribution of chromatin along the nuclear membrane, enlarged nucleoli, atypical or bizarre mitoses (e.g., tripolar), and tumor giant cells with one or more nuclei. Some malignant tumors, however, are well differentiated, so well differentiated, in fact, that their malignant cells cannot be distinguished from those of benign tumors or even from normal cells by any available diagnostic method. In such instances, the recognition of abnormal cellular relationships becomes especially important for correct diagnosis.

Anaplasia (Greek, “to form backwards”) is the term pathologists commonly use to describe the degree of tumor cell differentiation or, more correctly, the lack thereof. Though well entrenched, the term is an unfortunate one. It implies that tumors arise from mature, differentiated cells by a process of de-differentiation (i.e., differentiation in reverse). Few pathologists hold that view today. Mammalian cells, once differentiated, generally lack the capacity to reverse that process. Also, there is strong and growing evidence for the alternative explanation, namely, that tumors arise from populations of undifferentiated “stem” or “reserve” cells that are present in many, perhaps in all, organs capable of cell renewal.32–34 Stem cells comprise a minority cell population that lacks differentiation markers, making them difficult to identify. However, positive recognition of stem cells has been achieved in several organs; for example, bone marrow, epidermis, liver, and gastrointestinal tract mucosa.35 Stem cells have a high capacity for cell proliferation but, unless stimulated, may divide infrequently. Stem cells alone have the capacity to regenerate normal tissues and, by extension, tumor cell populations. Oncologists, of course, are well aware that stem cells are the critically important target of cancer therapy. Destruction of differentiated tumor cells, without simultaneous killing of tumor stem cells, will not lead to permanent tumor eradication.

Malignant tumors invariably lack a capsule. Instead, they extend crab-like projections into the surrounding host tissues without respect for normal anatomic boundaries. This behavior is referred to as invasion. Malignant tumor cells often invade lymphatics and veins and are transported by lymph or bloodflow to distant sites, opening the possibility of metastasis. Invasion is not a property confined to malignant tumor cells; many proliferations in fetal life, placental trophoblasts, and inflammatory cells also have the capacity to invade tissues. However, cancers need not be invasive at the time of removal, either because they have been “caught” before they had time to invade or because they have not yet progressed to the point where they have acquired the capacity to invade. Epithelial tumors with all other properties of malignancy that have not extended through the underlying basement membrane at the time of diagnosis are described as in situ carcinomas and can almost certainly be cured by complete excision.36

Ancillary Staining and Analytical Methods

Special stains are commonly employed to aid in tumor differential diagnosis and classification. Examples include the Van Gieson’s stain or the Masson trichrome method for distinguishing collagen and muscle, the Weigert’s stain for elastic tissue, silver stains for reticulin fibers, and special stains for mucins, amyloid, lipids, myelin and glycogen—all substances whose identification may aid in the diagnosis of one or another type of tumor. In other instances, enzyme histochemistry may be essential for defining cell lineage, as in certain types of leukemia; for example, chloroacetate esterase or endogenous peroxidase staining for cells of myelomonocytic lineage, alpha naphthyl butyrate esterase (so-called “nonspecific” esterase) staining for monocytes and macrophages.

Other techniques that may occasionally aid the surgical pathologist in tumor diagnosis are specimen radiography (for localizing and analyzing crystalline material, such as calcium, in breast biopsies) and morphometry.

Excision Margins

An important concern for the pathologist is the adequacy of tumor excision. Depending on the tissue, this decision can be made on either frozen or permanent sections. If the tumor forms a discrete mass and the margins of the specimen are clearly recognizable, determination of excision margins is usually straightforward. Examples of tumors whose excision is likely to give clearly defined margins include those arising in the gastrointestinal tract, lung, and skin. On the other hand, the margins of tumors arising in soft tissues (e.g., many sarcomas and breast carcinomas) and diffusely infiltrating tumors (e.g., infiltrating lobular carcinoma of the breast, signet ring tumors of the gastrointestinal tract, and nerve-invading tumors, such as adenoid cystic carcinomas of the salivary glands, gliomas, and glioblastomas) may be much more difficult to define. With at least certain histologic patterns of breast carcinoma, factors such as the extent of intraductal growth must be considered when evaluating resection margins.37,38 In patients treated with excision and radiotherapy for invasive breast cancer, the evaluation of excision margins in the context of an extensive intraductal component provides more prognostic information than the evaluation of margins alone.39,40

Tumor Grading, Staging, and Prognosis

Finally, pathologists are often called upon to grade tumors or to participate in their staging in order to estimate tumor prognosis. Tumor staging (e.g., the well-known TNM system) has proved to be of great value in estimating prognosis. Staging attempts to measure the extent of spread of a cancer within a patient on the basis of such parameters as the size of the primary tumor, the degree of lymph node involvement, and the presence of metastases. It is obvious that objective determinations made by the pathologist on resected tumor specimens have a critical impact on accurate tumor staging. With rare exceptions, such as papillary carcinoma of the thyroid, the single most important risk factor in determining tumor prognosis is the presence of metastases to regional lymph nodes. Therefore, the pathologist must search diligently to find, examine, and prepare histologic sections from all lymph nodes included in resected tissue.

Tumor grading has traditionally referred to a pathologist’s judgment as to a tumor’s degree of differentiation and growth rate, often on a scale of I to III where III represents the least differentiated, fastest dividing tumors (i.e., those tumors presumed to have the worst prognosis). Formal grading systems have improved in recent years with stricter standardization of criteria41 (Fig. 29.8). Tumor grading does, however, have shortcomings. First, a different scale is required for each type of tumor, and scoring is subjective and not always reproducible. Second, tumors are typically heterogeneous so that areas differing significantly in differentiation and mitotic activity exist side by side, with the attendant risk of sampling error. Because prognosis is invariably linked to the most malignant portions of a tumor, it follows that, for accurate diagnosis and grading, sufficient tissue and microscopic sections must be sampled so that the most malignant areas are found. Moreover, a regular (though not invariant) feature of malignant tumors is progression,42,43 the property by which tumors become more and more malignant over time. Tumor progression is thought to result from genomic instability44 that leads to specific mutations of oncogenes and tumor suppressor genes, from other genetic alterations, such as gene amplification, as well as from epigenetic changes that result in altered patterns of gene expression. Overt carcinogens, environmental promoters, and local factors, such as hypoxia and nutrient deprivation, may all contribute to these changes and therefore to tumor progression. Finally, the correlation between histologic appearance and biologic behavior is seldom perfect.

Figure 29.8. Actuarial rates of distant failure for 1,081 patients with invasive breast cancer related to histologic grade of tumor.

Figure 29.8

Actuarial rates of distant failure for 1,081 patients with invasive breast cancer related to histologic grade of tumor. Grading was performed using Elston’s modification of the Bloom and Richardson grading system, which takes into consideration (more...)

Of course, pathologists are continuously on the lookout for more useful tumor-specific features that may be important independent predictors of tumor prognosis. Recent attempts to identify such predictors have borne fruit in certain specific cancers. Thus, carcinomas of the prostate may be usefully graded on the basis of tissue architecture and neoplastic cell pattern.45 A number of different criteria including nuclear differentiation, degree of gland or tubule formation, and mitotic activity have been usefully combined to grade breast carcinomas.41,46,47 Carcinoma of the breast illustrates other tumor-specific factors that affect prognosis and will now be discussed in greater detail, as an important example.

Factors Important in Predicting Risk for Local Recurrence or Distant Metastases in Patients with Invasive Breast Cancer

Separate consideration must be given to the risks of local recurrence of cancer in the breast and distant metastases; the factors that affect each are not identical.

Local Recurrence

In patients treated for infiltrating ductal carcinoma with breast-conserving surgery and radiation therapy, the factors predictive of local recurrence are not clearly related to known factors that predict for the development of distant metastasis. A recent large study on a total of 584 patients with clinical stage I or II infiltrating ductal carcinomas of the breast makes this point.48 Treatment consisted of complete surgical excision of the primary tumor without microscopic margin evaluation, followed by radiation therapy totalling at least 60 Gray to the primary site. Of 34 separate tumor characteristics that were subjected to multivariate analysis, the only factor that was found to be associated with an increased risk of local breast recurrence was the presence of an extensive intraductal component (EIC-positive). EIC positivity was identified in two distinct groups of tumors: (1) tumors which were predominantly ductal carcinomas in situ with areas of focal invasion; and (2) primarily invasive tumors in which (a) the ducts and lobules were not obliterated, (b) virtually all preserved ducts were involved by ductal carcinoma in situ, and (c) tumors in which ductal carcinoma in situ was present adjacent to the invasive tumor.

Twenty-eight percent of all cases fell into the EIC-positive group, and 26% of such patients developed local tumor recurrence, compared with only 7% of patients with EIC-negative tumors (p = .001).49 A number of other investigators have now confirmed the finding that the presence of EIC is associated with a higher risk for breast cancer recurrence after local excision and radiation therapy.50,51

There seem to be two likely explanations for the increased risk of local recurrence in patients with EIC-positive tumors. One is that the residual tumor in this group of patients is less radiosensitive than that in patients with EIC-negative tumors. The second is that the subclinical residual tumor burden following excision is consistently larger in EIC-positive than in EIC-negative patients and may be too large to have been eradicated by the cosmetically acceptable doses of radiation therapy that were delivered. While both these explanations are possible, it has been found that the residual tumor burden is consistently larger in EIC-positive than in EIC-negative patients.37

The presence of EIC positivity is currently used to determine the extent of surgical excision. Patients with EIC-positive tumors frequently require a larger excision than patients with EIC-negative tumors. A minority of patients with EIC-positive tumors may require a mastectomy to obtain adequate margins. Patients with EIC-positive tumors with negative margins may be adequately treated with local excision “lumpectomy” and radiation therapy.40

Distant Metastases

As with many other tumors, the single most important factor predicting for the systemic spread of breast cancer is involvement of the regional lymph nodes. Unless treated, most patients with involved axillary lymph nodes will ultimately die from metastatic spread of their disease. Since the natural history of breast cancer is often protracted, clinically evident metastases may not appear for many years; for example, ≥10 to 20 years after the primary tumor and axillary lymph nodes have been removed. The greater the number of involved axillary lymph nodes, the greater is the likelihood that tumor cells have spread elsewhere in the body to indeterminate locations only to become clinically manifest at a later time. Thus, in one large series, 87% of the patients with ≥13 involved axillary lymph nodes developed metastatic spread within 10 years, whereas only 20% of node-negative patients did so.52

Given the important prognostic significance of positive lymph nodes and recent evidence that patient survival improves with adjuvant therapy, criteria are now urgently needed for identifying the 20 to 30% of lymph node–negative patients who will nonetheless develop metastatic disease after primary breast treatment. Factors worthy of consideration include the degree of tumor differentiation and the mitotic index as discussed above. In addition, other more specific factors may play a role. One likely candidate is the capacity of tumor cells to find their way into vascular spaces, such as lymphatics or small blood vessels.53–56

Newer Criteria for Assessing Breast Carcinomas

Several scoring systems have been used to grade breast cancers. Generally speaking, most include the degree of nuclear differentiation, the degree to which the tumor is able to form glands, and some attempt to estimate tumor growth rate by measuring mitotic activity. Using such criteria, the survival rates at 5 years in one series of node-negative patients with grade 1 through grade 3 tumors varied from 86% through 64%, and at 15 years from 49% through 25%.57

One approach for assessing tumor differentiation involves the use of flow cytometry. Tumors with normal diploid or tetraploid DNA content have been repeatedly shown to have a better prognosis than aneuploid tumors.58–60 However, not all workers agree with this conclusion as it applies to node-negative patients.61,62

Tumor growth rate may be measured in a number of ways, including standard counts of mitotic figures per 10 high power fields, the percentage of cells in the S-phase as determined by flow cytometry and 3H thymidine labeling index.41,46,47 In a large study of DNA ploidy and S-phase fraction in lymph node–negative patients with breast cancer, no correlation was found between S-phase fraction and survival among patients with aneuploid tumors.58 These aneuploid tumors constituted two-thirds of cases studied. Among diploid tumors, however, the S-phase fraction was highly predictive for the risk of recurrence.58

One of the most intensively studied and widely used measures of evaluating breast cancers is their expression of estrogen receptor (ER) and progesterone receptor (PR). These are, in fact, the only measures for which standardized quality control is currently available. Most studies have shown that lymph node–negative patients with ER-positive tumors have a significantly better disease-free survival and, in some cases, better overall survival than patients whose tumors are ER negative.63 The difference between the survival rates of patients with ER-positive tumors and ER-negative tumors decreases with time, and some studies have suggested that the survival curves will eventually merge.64 Taken together, these observations suggest that tumors expressing estrogen and progesterone receptors tend to proliferate more slowly than tumors lacking such receptors. Thus, ER measurements may not represent an independent prognostic factor per se but may instead provide yet another method of assessing tumor growth rate or differentiation.

Another criterion for assessing breast cancer prognosis has come into prominence recently, that of measuring microvascular density. As noted earlier, tumors must induce new blood vessels if they are to grow beyond the minimal size. Therefore, intratumor microvascularity, taken as a measure of tumor angiogenesis, might be expected to provide a useful index of tumor aggressiveness. Investigating this possibility, Weidner and colleagues65,66 performed counts of blood vessel frequency on breast cancer tissue sections. Immunostaining sections with anti–factor VIII–related antigen to ensure detection of all microvessels, they found that, as with other tumor properties such as mitotic index, individual breast cancers were heterogeneous with respect to microvessel density. They, therefore, selected zones that exhibited the highest blood vessel density (“hot spots”) and found that hot spots in cancers of patients with metastases had mean vessel counts of 101 ± 49 per 200 × field, significantly higher than the value of 45 ± 21 for patients without metastases. Multivariate analysis of the data revealed that intratumor microvascular density provided an independent prognostic indicator of lymph node metastasis and of both relapse-free and overall patient survival. The majority of follow-up studies have confirmed these findings.67 Unfortunately, measurements of tumor microvascular density are laborious and subject to interobserver variability and for these reasons are unlikely to be used routinely outside of research settings. Nonetheless, Weidner’s studies emphasize the importance of angiogenesis in human tumor biology and support the principle that angiogenesis may be a worthy target in biologic approaches to breast cancer therapy. Recent studies suggest that measurements of microvessel density may also provide useful prognostic information for carcinomas arising in organs besides breast, such as head and neck,68 lung,69,70 and prostate.71

A large number of studies are now in progress attempting to identify other factors which may predict for risk in node-negative patients. These include evaluation of Ki-67,72 epidermal growth factor receptors,73–75 insulin-like growth factors,76,77 transforming growth factor-α,78,79 cathepsin D,80 and various oncogenes and their products. A recent review of prognostic factors in breast cancer catalogs and evaluates them.81

Oncologists have long sought therapeutic agents that would spare normal tissue and specifically target tumor cells. The use of antiestrogenic agents against estrogen receptor–positive breast cancers has long been an established practice. A newer approach has been to target the HER2/neu antigen, which is overexpressed in certain breast and ovarian cancers. The antigen is targeted by a therapeutic agent, trastuzumab (Herceptin, Genentech) consisting of a humanized monoclonal antibody that binds to the HER2/neu protein.82 Initial clinical trials have demonstrated that this agent specifically targets tumor cells and has a significant impact on patient survival.83

In the past, pathologists and oncologists have sought markers that predict tumor prognosis. We are now entering an era in which it will be more important to identify markers that help to predict the response to new and different therapeutic modalities.

Surgical Pathologist’s Report

The findings should be presented descriptively and comprehensively in terms that are understandable to both the pathologist and the clinicians caring for the patient. The report should provide enough information so that the clinician caring for the patient can follow the thought processes of the pathologist, much as if he were viewing the case with the pathologist at a double-headed microscope. The report should contain all the information to which the pathologist has access (i.e., tumor size, grade, and nodal status) that is necessary to stage a patient with cancer. This information varies with tumor origin, type, and staging system employed. The report should include the results of all specialized tests performed, their interpretation, and the synthesis and coordination of all clinically useful information available to the pathologist that may be of aid in diagnosis and management. Finally, reports should be issued in a timely manner so that they are available to the clinician within a few days of tissue submission. Failure to report results promptly may delay patient care (thus uselessly adding to the cost of medical care), lead to error and confusion, and at the very least prolong anxiety in patients who are often already distraught.

Role of the Cytopathologist

Cytology is used for both screening and diagnosing of lesions that may represent cancer or its precursors. Specific benefits include cost-effectiveness, rapid turnaround time, and tissue diagnosis with minimal patient risk. Since cytologic specimens usually consist of a very small amount of cells or tissue fragments, optimal technique for both sample collection and slide preparation is crucial. Moreover, as in all areas of pathology, cytologic diagnosis should never be made “in a vacuum;” pertinent clinical data and communication between the cytopathologist and clinician are essential and will facilitate rapid, accurate, and definitive cytologic diagnoses.

Methods for Obtaining Specimens

Two categories of methods are involved in obtaining cells for microscopic examination. The first is to obtain the medium that contains naturally exfoliated cells, such as urine, sputum, and body cavity fluids. The second is to specifically obtain with an instrument, such as a brush or a needle, the cells of interest for examination.

Preparation of Specimens

The preparation of cytologic specimens depends on the type of specimens. When the cells are collected with an instrument, the cells can be either rinsed into a preservative solution, such as Saccomanno’s, 50% ethanol, a balanced electrolyte solution, or other preservatives, or simply directly spread onto slides.84–87 When the cells are collected in a medium, whether natural or artificial, the specimen needs to go through a process in order to separate the medium from the cells. This can be done with centrifugation, cytocentrifugation, filtering, or processing through a machine that spreads a thin layer of cells on slides. The optimal final product is a slide with a thin layer of evenly dispersed cells on it. Cells thinly spread on a slide dry out very easily; therefore, the slides with unpreserved cells directly spread onto them need to be fixed (either in 95% ethanol or with commercially available spray fixatives) immediately and then stained with Papanicolaou’s or Hematoxylin and Eosin stain. Slides with unfixed cells can also be left to air dry intentionally and then stained with a Romanowsky-type stain.

Microscopic Interpretation of Cytologic Specimens

In contrast to surgical pathologists, cytopathologists deal primarily with cells without regard to stroma. Although some architectural features are maintained in cytologic specimens, many are lost in the process of specimen collection and preparation. Therefore, cytopathologists rely mainly on the cytologic features of malignancy described earlier and residual structural features, such as cohesion versus dyshesion, to determine the benign versus malignant nature of the lesion. Cytopathologists usually report the result in one of four categories—“positive,” “suspicious,” “atypical,” or “negative.”88–90 A positive diagnosis indicates that the pathologist is sufficiently confident of the malignant nature of the lesion to recommend that the patient undergo definitive treatment, such as surgical resection or chemotherapy, on the basis of that diagnosis alone. Where there is any doubt, the report should be less definitive and in the suspicious category. Other diagnostic tests, such as a repeat cytology sample, or biopsy should be done to determine with certainty the nature of the lesion before the patient undergoes definitive therapy. Occasionally, other (e.g., clinical, radiologic), evidence of malignancy is so strong that clinicians feel confident to implement definitive therapy with a suspicious diagnosis. That should be the decision of the responsible clinician. When there are cellular abnormalities whose clinical significance is not known, the report should be in the atypical category. Other diagnostic tests may be in order, depending on the clinical situation. Definitive therapy should never be initiated solely on the basis of atypia. A negative cytology means that no abnormal cells were found in the sample examined. It is important for all to realize that this does not necessarily indicate absence of malignancy in the patient. False-negative cytologies are often the result of sampling error. However, laboratory error may result in both false-negative and false-positive results. Not all types of cytologic samples receive diagnoses using these four categories, however. For example, Pap test results are described according to The Bethesda System for Cervical and Vaginal Cytology. Whenever possible, general diagnostic categories (atypical, positive, etc.) should be followed by more precise diagnoses, indicating specific neoplasms, infections, or other processes as is done in surgical pathology reports.

Exfoliative Cytology

This involves microscopic examination of cells exfoliated from the female genital (“Pap” smear), respiratory (sputum), and urinary (urine) tracts. The use of the Pap smear to screen for cervical cancer and its precursors in the general asymptomatic population has been instrumental in lowering the mortality rate from cervical cancer over the last four decades.91–94 A problem inherent to all screening tests is the need to balance sensitivity and specificity. Lowering the threshold for diagnosis of atypia means that fewer cases of neoplasia will be missed. However, the trade-off is that more patients without neoplastic disease will require additional, expensive studies, such as colposcopy, to rule out the presence of cancer or its precursors. The importance of proper sample collection and preparation needs to be emphasized. Failure to fix samples immediately, thick smears, and the presence of significant amounts of blood may all result in specimens that are unsatisfactory or suboptimal for diagnosis. To put the sampled materials in a preservative medium and to use a machine, such as a ThinPrep processor, to make slides with a thin layer of cells can obviate most of the above pitfalls in specimen preparation, but the cost of preparation would be significantly increased.95 Sensitivity can be increased by using a cytobrush because of its ability to sample a broad area.96,97

Cytologic examinations of sputum and urine are not currently used to screen the general population, but instead to detect cancers in high-risk patients who either have had exposures that increase their risk of developing cancers or already have symptoms that may be caused by cancers in the lung or urinary bladder. Urinary cytology is also used for detecting local recurrence or second primary tumors in patients with previously resected urethelial carcinoma. For patients with symptoms or other clinical evidence of disease, more invasive procedures, such as bronchoscopy, cystoscopy, and colposcopy, are called for to collect cells or tissue for cytologic and/or histologic examination if a diagnosis cannot be made on exfoliative cytology.

Endoscopic Cytology

In areas amenable to endoscopy, such as the bronchial tree and gastrointestinal and urinary tracts, cytologic specimens obtained with brushing and washing techniques may serve a diagnostic function. The brush can be rolled on slides to make direct smears or can be immersed in a preservative for subsequent preparation of slides by a machine. Specimens collected by washing are processed like those collected with a medium. Paired cytology and biopsy can improve the likelihood of diagnosing malignancy in a single procedure.98,99 Because brush samples cover a wide area, they provide greater diagnostic sensitivity, particularly for the diagnosis of early lesions that are not grossly obvious. However, endoscopic biopsies generally provide more information, particularly in determining tumor type and presence of invasion.

Cytology of Body Cavity and Cerebrospinal Fluids

Fluids are removed from body cavities not only for the purpose of therapy (e.g., to relieve pressure on vital organs) but also as a form of diagnosis. Although immediate fixation is not necessary, fluids do need to be refrigerated promptly and may also require anticoagulation (heparin, 1 unit per 100 mL) if the fluid is bloody. The presence of tumor cells in body cavity implies metastasis, with the exception of mesotheliomas (Fig. 29.9). It is usually difficult, if not impossible, to determine the site of primary tumor, although certain morphologic clues may occasionally allow the cytopathologist to suggest a site of origin. If consistent with the clinical findings and results from other studies, a positive cytologic diagnosis can lead directly to treatment. As always, current cytology specimens should be compared with previous cytology or histology specimens, if available.

Figure 29.9. Ovarian papillary serous adenocarcinoma in ascitic fluid.

Figure 29.9

Ovarian papillary serous adenocarcinoma in ascitic fluid. Cells are present in three-dimensional clusters with nuclear molding and irregular hyperchromatic nuclei. Papanicolaou; magnification: x250.

Reactive mesothelial cells share certain characteristics with carcinoma cells, including large nuclei with nucleoli and even mitotic figures, and should always be considered in the differential diagnosis. Mesothelial cells survive and multiply when exfoliated into effusions. Panels of immunocytochemical stains may also prove useful (see below). Unfortunately, there are, at present, no available antibodies that distinguish benign from malignant mesothelial cells and very few that reliably identify the site of origin of metastatic cancers.100,101

Sometimes (e.g., mesotheliomas), tumor growth may be confined to the surfaces lining body cavities, without significant exfoliation of malignant cells. Under these circumstances, examination of effusions for exfoliated cells may be fruitless, requiring the need for a biopsy of the cavity wall. An alternative approach for future investigation may be to analyze tumor cell–free effusions for secreted products of malignant cells or other tumor cell markers. One successful though preliminary application of this approach has been the finding of high levels of the angiogenic factor VPF/VEGF in effusions by immunoassay.102

Aspiration Cytology

The earliest work on aspiration cytology was reported from the Memorial Hospital in New York in the 1930s.103 Subsequently, the impetus for this technique shifted to Europe and was not “rediscovered” in the United States until the 1970s.104 Considerable controversy exists over who is best qualified to perform fine-needle aspiration (FNA). At present, cytopathologists, surgeons, and other clinicians successfully perform such aspirations.105 In fact, the most critical and technically demanding step in aspiration cytology is generally not the aspiration itself but rather the preparation of adequate slides after the sample has been obtained.106–108 To obviate the artifacts associated with poor preparation, the needle can be rinsed with a preservative solution and the specimen processed with a machine as described above.85

In the case of nonpalpable lesions, aspiration is performed by a radiologist under computed tomography (CT), ultrasound, or fluoroscopic guidance. Such deep aspiration procedures are expensive, time consuming, and invasive. For these reasons, it is desirable that a cytotechnologist or cytopathologist attend the procedure to ensure specimen adequacy and optimal slide preparation. A further advantage of this attendance is the ability of experienced personnel to triage material effectively for special studies, such as immunocytochemistry, electron microscopy, flow cytometry, and tissue culture.

Definitive aspiration cytology diagnoses, rendered by an experienced cytopathologist, can provide the basis for definitive therapy. However, such diagnoses need to be viewed in the context of all other laboratory studies and clinical findings. Specific problems and pitfalls that attend aspirations of various sites will now be briefly discussed.


Fine-needle aspiration permits the accurate diagnosis of papillary, medullary, and anaplastic carcinomas but is less useful in the diagnosis of follicular nodules. There are cytologic features which help distinguish among the various types of follicular lesions,109 but a definitive diagnosis may not be possible by FNA, particularly the distinction between follicular adenomas and carcinoma. Thus, in some instances, FNA will serve only to distinguish patients needing immediate surgery for thyroid disease from those who may be safely monitored, with or without hormonal suppression.106,110,111 However, even this limited information can eliminate much unnecessary surgery.


The diagnostic specificity of breast aspiration is very high in the hands of an experienced cytopathologist, and a positive diagnosis may safely lead to mastectomy or other definitive treatment.106,112,113 Of course, atypical and suspicious cases will require further workup. Aspiration cytology of the breast may also be performed on nonpalpable lesions under the guidance of conventional or stereotactic mammography or ultrasonography,114–118 but the ultimate value of this new technique is not yet clear. Inherent problems for breast cytology include the inability to distinguish infiltrating from in situ carcinoma and the difficulty in rendering a specific benign diagnosis, when compared with core biopsies.119–121 However, needles used in aspiration cytology cost much less than the core needles and are more readily tolerated by patients.


Aspiration cytology of the lung may lead to the diagnosis of both primary and metastatic tumors and non-neoplastic lesions, such as tuberculosis and fungal infections.106,108,122 As with histologic tissue sections, it is not always possible to distinguish primary from metastatic carcinomas.


Aspiration cytology is particularly useful in diagnosing malignancies in the liver, pancreas, kidney, and retroperitoneum prior to treatment.108 A diagnosis of metastatic tumor or lymphoma spares the patient from major surgery, and provides the basis for definitive therapy.123 Poorly differentiated tumors may be difficult to type, but the use of adjunct techniques is often helpful in establishing a definitive diagnosis (see Fig. 29.10).

Figure 29.10. Metastatic neuroendocrine tumor as seen in a fine-needle aspiration of liver.

Figure 29.10

Metastatic neuroendocrine tumor as seen in a fine-needle aspiration of liver. Note eccentric nuclei with “salt and pepper” chromatin and abundant granular cytoplasm. Immunocytochemical stains confirmed the diagnosis. Papanicolaou; magnification: (more...)

Lymph Nodes

Many cytopathologists believe that aspiration cytology has only a limited role in the diagnosis of lymph node lesions. However, FNA can provide useful information to obviate the need for surgery in cases of suspected carcinoma metastatic to palpable lymph nodes with a known primary. Lymph node aspirates may also be useful for diagnosing lymphoproliferative diseases and infection.124,125

Application of Ancillary Studies on Cytologic Materials

Virtually all ancillary studies, such as those involving immunohistochemistry, electron microscopy, flow cytometry, and molecular biology, can be applied on cytologic materials (see below for details).

There have been few diagnostic tests introduced into medicine which have actually lowered the cost of high-quality patient care. Cytopathology is such a test. It offers the advantages of low morbidity, rapid turnaround time, and outstanding cost-effectiveness. The problems and pitfalls of cytology should not detract from its usefulness. All procedures have limitations, and the oncologist needs to be informed as to both the benefits and the pitfalls of this approach.

Role of the Immunohistochemist

Immunohistochemistry has become an important adjunct in the evaluation of human neoplasms. A detailed discussion of the technical aspects of immunohistochemistry is beyond the scope of this chapter, and the interested reader is referred to several review articles and monographs.126–130 The commercial availability of a broad range of reagents (including prediluted reagents in kit form) has made it possible for high-quality immunohistochemistry to be performed in most pathology laboratories. The most commonly employed immunohistochemical techniques are those in which enzymes, such as horseradish peroxidase or alkaline phosphatase, are used in conjunction with specific antibodies (to provide color reactions at sites of antigen-antibody interactions). Variations of the avidin-biotin complex (ABC) technique are currently the most widely utilized in current practice. The ABC procedure generally requires three sequential steps: an unlabeled primary antibody, a biotin-labeled anti-immunoglobulin secondary antibody, and, finally, preformed avidin-biotin-peroxidase complexes. One variation of the ABC method employs streptavidin, which has greater sensitivity than avidin and exhibits less nonspecific binding.131 It should be noted that the sensitivity of any immunohistochemical procedure is, in large part, related to the reagents and detailed procedures employed. As a consequence, it is difficult to compare the results of immunohistochemical studies from different institutions which employ different reagents and methods.

Virtually any type of pathologic specimen may be suitable for immunohistochemical staining, including fresh frozen tissue, fixed tissue, and cytologic preparations. Unfortunately, however, not all antigens are equally well preserved after these various treatments, and the approach taken for immunohistochemical staining must depend on the antigen(s) of interest. For example, while a large number of cytoplasmic antigens are detectable in fixed, paraffin-embedded tissue, other antigens, such as many cell surface–associated antigens, are destroyed or masked by common fixatives and may be demonstrable only in fresh frozen tissue or in cytologic preparations. Antigen retrieval methods, such as pretreatment with proteolytic enzymes or heating (using a microwave oven, steamer, pressure cooker, or autoclave), may permit the identification of otherwise undemonstrable antigens in fixed, paraffin-embedded tissue sections.132,133 Finally, not all fixatives are equivalent with regard to antigen preservation. While crosslinking fixatives, such as formaldehyde, are often suitable, they are suboptimal for detecting certain antigens of diagnostic importance, such as those located on intermediate filaments, which are best demonstrated in fresh-frozen or alcohol-fixed tissue.134–136


Immunohistochemistry has widespread applicability in the evaluation of human tumors. Some of the more common applications are listed in Table 29.1 and are discussed below.

Table 29.1. Common Applications of Immunohistochemistry in the Evaluation of Human Tumors.

Table 29.1

Common Applications of Immunohistochemistry in the Evaluation of Human Tumors.

Categorization of “Undifferentiated” Malignant Tumors

Not infrequently, a pathologist examining routine H & E-stained paraffin sections recognizes the presence of a malignant tumor but is unable to characterize the tumor further. This is understandable in that “undifferentiated” tumors often lack characteristics that would permit more accurate classification. Yet further classification is often important in making clinical decisions related to appropriate therapy and prognosis. Immunohistochemistry may be helpful in such situations (Fig. 29.11). Before performing immunohistochemistry, however, the pathologist must first develop a differential diagnosis, and this will depend on the tumor’s histologic appearance, anatomic location, and the clinical setting. Only then is he or she in a position to select antibodies that will permit a more definitive diagnosis.

Figure 29.11. Immunoperoxidase staining of a monoclonal antibody specific for keratin in a poorly differentiated squamous cell carcinoma of skin.

Figure 29.11

Immunoperoxidase staining of a monoclonal antibody specific for keratin in a poorly differentiated squamous cell carcinoma of skin. This “spindle cell” form of the tumor mimics tumors of connective tissue origin, and its true nature can (more...)

One common problem in tumor diagnosis, that of undifferentiated tumors composed of large cells with an epithelioid appearance, will serve as an example. The differential diagnosis in such cases typically includes undifferentiated carcinoma, lymphoma, and melanoma. Distinction among these tumor types can often be made using a panel of antibodies as illustrated in Table 29.2. Unfortunately, this table presents an ideal result that is not always achieved in practice. Some carcinomas show staining for vimentin137,138 or S100 protein,139 some lymphomas express epithelial membrane antigen,140 some melanomas show immunoreactivity for keratin,141 and some neoplasms other than melanomas express HMB-45.142 Such results emphasize the need to use a panel of antibodies, rather than a single antibody, when evaluating tumors.

Table 29.2. Idealized Imunohistochemical Evaluation of the“Undifferentiated” Malignant Tumor*.

Table 29.2

Idealized Imunohistochemical Evaluation of the“Undifferentiated” Malignant Tumor*.

Determination of Site of Origin of Metastatic Tumors

On routine microscopic examination, tumors may be classifiable with regard to general type (e.g., carcinoma), but not with regard to site of origin. It would be highly desirable to have available antibodies specific for tumors arising in different sites. At present, however, very few organ- or tissue-specific antigens have been identified, thus limiting the ability of immunohistochemistry to resolve such problems in every instance. A number of useful antigens are listed in Table 29.3. It should be noted that antigens specific for some of the more common tumors, such as carcinomas of the lung, colon, endometrium and pancreas, are not currently available. Furthermore, some of the antigens listed in Table 29.3 have now been demonstrated in neoplasms other than those for which they were initially thought to be “specific.” For example, the melanoma-associated antigen detected by one widely used antibody (HMB-45) has been found in some nonmelanocytic tumors.142 A more recent approach for the subclassification of metastatic carcinomas exploits differences in cytokeratin profiles in tumors from different primary sites.143

Table 29.3. Antigens with Highly Restricted Specificity.

Table 29.3

Antigens with Highly Restricted Specificity.

Subclassification of Tumors in Various Organ Systems

In some organs and tissue compartments, it may be difficult to subclassify certain tumors solely on the basis of histologic grounds because of overlapping features. Some of these distinctions are (at least at present) only of academic interest (e.g., determining whether a high-grade spindle cell sarcoma shows neural, myogenous, or fibrohistiocytic differentiation), but others have therapeutic and prognostic significance. For example, in some cases, it may be difficult or impossible to distinguish with certainty an anaplastic seminoma from an embryonal carcinoma of the testis by routine microscopic examination, a distinction with both therapeutic and prognostic implications. However, immunostaining for the intermediate filament keratin is often useful in making this distinction because seminomas are typically keratin negative, whereas embryonal carcinomas are usually keratin positive.144,145 Similar situations are encountered in other organ systems and tissue compartments.

Distinction between Carcinomas and Malignant Mesotheliomas

A common problem encountered by the surgical pathologist is the distinction between metastatic adenocarcinoma and malignant mesothelioma involving the pleura or peritoneum.146–150 Immunohistochemical staining using a panel of antibodies may be useful in assisting in this distinction (Table 29.4).

Table 29.4. Immunohistochemical Distinction between Metastatic Adenocarcinoma and Malignant Mesothelioma Involving the Pleura or Peritoneum.

Table 29.4

Immunohistochemical Distinction between Metastatic Adenocarcinoma and Malignant Mesothelioma Involving the Pleura or Peritoneum.

Categorization of Leukemias and Lymphomas

One of the most common uses for immunohistochemistry is the correct diagnosis and classification of leukemias and lymphomas. A detailed discussion of this subject is beyond the scope of this chapter, and the interested reader is referred to several recent articles and reviews.151–155 In brief, immunohistochemistry, in conjunction with morphology and histochemistry, is a useful adjunct in making the distinction between acute leukemias of lymphoid and nonlymphoid types and in distinguishing hairy-cell leukemia from other types of leukemic infiltrates in the bone marrow and at other sites. In addition, this technique is useful for subclassifying non–Hodgkin’s lymphomas and Hodgkin’s disease and in distinguishing them from each other in problematic cases.

Detection of Antigens of Potential Prognostic or Therapeutic Significance

A variety of antigens of possible prognostic and therapeutic importance can be detected using immunohistochemistry, including estrogen and progesterone receptors in breast cancers,156–158 protein products of oncogenes (such as HER-2/neu in breast cancers,159–161) antigens associated with tumor cell proliferation such as Ki-67 and PCNA/cyclin,162–163 and the P-glycoprotein product of the multiple drug resistance (MDR) gene.164 Ki-67 is of particular interest.165 It is a nuclear antigen present in all proliferating cells; that is, present in the G1, S, G2 and M phases of the cell cycle but absent in Go cells. Therefore, by staining for this antigen, it is possible to measure the tumor growth fraction directly and in a simpler manner, which is more readily applicable to clinical specimens than are radioactive labeling methods using [3H]-thymidine. Ki-67 staining also yields results which are more reproducible than those obtained by counting mitotic figures. Recently, a number of antibodies have become available to identify the Ki-67 antigen in formalin-fixed, paraffin-embedded tissue sections.166


An appreciation of the limitations of immunohistochemistry in tumor diagnosis is as important as an understanding of its many useful applications. Potential limitations in the immunohistochemical evaluation of solid tumors can be broadly characterized as technical and interpretive.

Technical Limitations

Because demonstration of different types of antigens by immunostaining requires appropriate tissue preparation, advance planning for immunohistochemistry is essential so that the specimen may be handled appropriately. For example, if an excised lymph node on clinical grounds or at intraoperative examination (i.e., frozen section or tissue imprint) conveys features suspicious for a lymphoma, a portion of the specimen should be snap-frozen to permit reliable demonstration of lymphocyte surface markers, since these are not well demonstrated in fixed, paraffin-embedded tissue. In cases of suspected carcinoma, in which the demonstration of intermediate filament proteins is likely to be important, fixation of a portion of the tumor in an alcohol-based fixative is advisable.

As with any laboratory procedure, the use of appropriate positive and negative controls is mandatory in immunohistochemistry and serves as a check on the technical adequacy of the procedure. Results of immunostaining must always be viewed with caution, if the appropriate controls are omitted or suboptimal.

Interpretive Limitations

Correct interpretation of immunohistochemical stains performed on tumor specimens is dependent not only on the technical adequacy of the procedure but on interpretive factors as well. In most situations, it is more useful to employ a panel of antibodies than a single antibody. The antibodies that make up the panel must be selected thoughtfully, on the basis of a carefully prepared differential diagnosis. A “shotgun” approach to immunostaining is strongly discouraged, as it will only serve to compound diagnostic confusion.

Accurate interpretation of staining also requires familiarity with the characteristics of “true-positive,” “false-positive,” “true-negative” and “false-negative” staining. Negative reactions are more difficult to interpret than are positive reactions. Even with the use of other controls, it is difficult to be certain that a reaction is a true negative, unless the section in question stains positively for a complementary antigen. For example, in the analysis of an undifferentiated malignant tumor in which the differential diagnosis includes lymphoma and carcinoma, a negative reaction for keratin (the intermediate filament characteristic of many carcinomas) does not by itself rule out the possibility of carcinoma. However, if a negative keratin stain is accompanied by positive staining for the leukocyte common antigen (a marker present in most lymphomas), the likelihood of lymphoma is greatly enhanced.

Some antibodies are of great diagnostic value in terms of both sensitivity and specificity (e.g., antibodies to prostate-specific antigen), whereas others are of limited diagnostic value even when used as part of a panel (e.g., antibodies to the intermediate filament vimentin). Pathologists who use immunohistochemistry must be experienced and aware of the limitations of a methodology which is evolving at a rapid pace. An antigenic profile suitable today for diagnosing a particular type of tumor may tomorrow be shown to be suboptimal or less specific than was originally thought. Immunohistochemistry is a valuable tool for aiding in the diagnosis of difficult tumors. However, it is only an adjunct to diagnosis, and the results must be interpreted in the context of other findings, particularly routine histologic sections and the clinical setting.

Role of the Electron Microscopist

Though making use of radically different technology, electron microscopy seeks the same type of information as that gleaned from immunohistochemistry, that is, detection of differentiated organelles (or markers, in the case of immunohistochemistry) that permit more accurate tumor identification and classification. Electron microscopy (EM) is not useful in determining whether individual cells are malignant or benign. It is a powerful tool for recognizing subcellular structures that are not detectable by light microscopy but which, when present, allow confident identification of cells as, for example, of epithelial or melanocyte origin. Although advances in immunohistochemistry have somewhat reduced the need for EM in tumor diagnosis, they have by no means eliminated this need altogether, and EM remains, at present, a powerful but generally underutilized approach to tumor diagnosis.167–171 Moreover, validation of new immunohistochemical reagents is often best accomplished by ultrastructural study of replicate tissue samples.

Technical Considerations

Appropriate tissue handling, fixation, and processing are of even greater importance in EM than they are for immunohistochemistry.172 Advanced planning and consultation between the clinician, surgical pathologist, and electron microscopist, are, therefore, important. In many cases, it is advantageous to have a pathologist or knowledgeable technician in the operating room or at the bedside at the time of biopsy, in order that tissue may be fixed immediately and trimmed appropriately. Tissues must be cut into small pieces because chemical fixatives penetrate tissues slowly (over minutes to hours), and the electron microscope glaringly exposes artifacts in poorly fixed tissues that are not detectable at the lower resolution afforded by light microscopy. In at least one dimension, tissues must be no thicker than 1 mm, and to achieve this small size, further trimming may be necessary after brief preliminary fixation. Mixtures of glutaraldehyde and paraformaldehyde (e.g., Karnovsky’s fixative) provide optimal fixation.172 Although these reagents are best when freshly prepared, it is also possible to freeze vials of fixative beforehand so that may be thawed immediately prior to use. Tissues fixed in formalin or in other “routine” fixatives designed for light microscopy give inadequate tissue preservation for electron microscopy. Once tissues are fixed inappropriately, they generally cannot be recovered for adequate electron microscopy, and repeat biopsy becomes the best option. Peripheral blood, bone marrow, and cell-containing fluids (e.g., pleural effusions, spinal or synovial fluids) are handled somewhat differently from samples of solid tissue and require the presence of a member of the electron microscopy staff when the sample is obtained.172


The great strength of EM lies in its exquisite resolution, which permits the recognition of intracellular structures, organelles, or products that are undetectable by light microscopy.

EM is often helpful in the diagnosis of “undifferentiated” malignant tumors and in determining the origin of metastatic tumors of unknown primary site (Figs. 29.12 and 29.13). The recognition of cytoplasmic premelanosomes within tumor cells permits the distinction of amelanotic malignant melanomas from undifferentiated carcinomas and lymphomas with which they can be confused. Other ultrastructural features whose recognition may permit definitive diagnosis are the cytoplasmic granules characteristic of carcinoid tumors; the norepinephrine- and epinephrine-containing granules found in pheochromocytomas; “terminal webs” characteristic of primary gastrointestinal carcinomas of absorptive epithelial cell origin;174 lamellar (surfactant) bodies found only in type II pneumocytes and therefore diagnostic of alveolar cell carcinomas of the lung;175 tonofilaments and desmosomes found in mesothelial cells and squamous cells; and cytoplasmic glycogen aggregates and calligraphic nucleoli typical of germinomas. The presence of intercellular junctions permits the distinction of carcinomas from lymphomas, even if the carcinoma’s primary site cannot be determined. Other examples include some thyroid carcinomas that may be identified by the polarized nature of their cells, which often contain small apical vesicles filled with colloid at one pole and a basal lamina underlying the opposite pole. Large numbers of mitochondria characterize oncocytomas, whether originating in the thyroid or elsewhere. EM combined with morphometric analysis to calculate a nuclear contour index may be required for the diagnosis of mycosis fungoides.176 EM may also, at times, correct faulty impressions derived from light microscopy. For example, a mistaken diagnosis of poorly differentiated adenocarcinoma may result from the misinterpretation of vascular spaces as tumor cell acini.

Figure 29.12. A.

Figure 29.12

A. Electron micrograph from lung mass shows typical surfactant-containing lamellar bodies (surfactant bodies) (arrowhead) that fill the cytoplasm of a tumor cell, allowing the specific diagnosis of primary alveolar cell carcinoma of the lung to be made. (more...)

Figure 29.13. High-magnification electron micrograph of pleural tumor shows three typical features of mesothelial cells: numerous elongated, thick surface microvilli that do not display evidence of terminal web differentiation; desmosomes that connect individual cells surrounding the extracellular acinar space and basal lamina (arrowhead).

Figure 29.13

High-magnification electron micrograph of pleural tumor shows three typical features of mesothelial cells: numerous elongated, thick surface microvilli that do not display evidence of terminal web differentiation; desmosomes that connect individual cells (more...)

EM is also helpful in subclassifying tumors, an exercise that may have important therapeutic implications. The use of ultrastructural cytochemistry for endogenous peroxidase may allow the important distinction of acute myeloblastic leukemia from acute lymphoblastic leukemia. Histiocytosis X may be diagnosed by identification of Birbeck bodies characteristic of Langerhans’ cells. EM may also permit accurate diagnosis of lysosomal storage diseases and of bacterial, fungal, and viral infections.

Finally, EM is important in identifying the histogenesis of newly recognized neoplasms. A recent example (Fig. 29.14) is the recognition that certain spindle cell tumors of the gastrointestinal tract, previously thought to be of smooth muscle origin, in fact arise from autonomic neurons (gastrointestinal tract autonomic nerve tumors or GAN tumors).177

Figure 29.14. This high-magnification micrograph of a gastric tumor shows tumor cells diagnostic for the newly delineated entity, the gastrointestinal autonomic nerve tumor (or GAN tumor).

Figure 29.14

This high-magnification micrograph of a gastric tumor shows tumor cells diagnostic for the newly delineated entity, the gastrointestinal autonomic nerve tumor (or GAN tumor). Elongated tumor cells are neurites which contain numerous mitochondria and neurofilaments; (more...)


As with immunohistochemistry, the limitations are both technical and interpretive. We have already alluded to certain technical limitations (those involving tissue handling, prompt and appropriate fixation, and suitable processing). Another is sampling error, attributable to the very small size of a specimen that can be studied on an EM grid. One other limitation that deserves mention is expense. Whereas the availability of commercial reagents, defined protocols, and relatively simple interpretation have permitted immunohistochemistry to be established in almost any hospital pathology laboratory, the same cannot be said for diagnostic electron microscopy. The costly electron microscope and fairly elaborate support equipment, and in particular the need for experienced technical and professional personnel have limited the application of this methodology to large secondary- and tertiary-care centers, mostly at academic institutions. Of particular importance is the need for a pathologist who is well trained in both surgical pathology and electron microscopy. The widely employed practice of asking a basic science-oriented electron microscopist or an electron microscopy technician to take a few pictures of a tumor specimen is a prescription for almost certain failure and must never be allowed.

Role of the Clinical Pathologist

The role of the clinical pathologist or “laboratorian” is obvious and familiar to the oncologist and requires only brief mention here. Clinical pathologists direct hospital laboratories and, thus, in addition to routine laboratory testing of cancer patients, are interested in measurements of body fluids that could lead to the early detection and monitoring of cancer. There has been a great deal of interest in the field of tumor antigens and tumor markers, and some of these are discussed elsewhere in this volume. At least in theory, tumor-specific antigens circulating in the plasma could be of utility in tumor diagnosis and prognosis, assessment of tumor burden, prediction of recurrence, and guidance for treatment. Properties of an ideal tumor marker include great sensitivity, specificity, and accuracy in reflecting total tumor burden. A tumor marker should also be prognostic of outcome and predictive of tumor recurrence. Unfortunately, none of the tumor markers discovered to date fulfills all of these criteria. In fact, none is uniquely produced by tumor cells. Normal cells of one sort or another make all of the tumor markers thus far recognized, and plasma or serum levels in tumor patients differ only quantitatively, not qualitatively, from those of normal controls or patients with other diseases.

The role of the clinical pathologist, however, has recently expanded into other areas of oncology. Particularly important is the molecular biologic diagnosis of tumors, for example, T-cell lymphomas, by the detection of gene rearrangements.178,179 Demonstration of such rearrangements may be especially important as a supplement to the work of the surgical pathologist and immunohistochemist in distinguishing “clonal” and, therefore, presumably malignant lesions from benign but highly reactive lymph nodes. There is increasing interest in the clinical laboratory in the assessment of solid tumor clonality in understanding tumorigenesis and as an aid to diagnosis and estimation of prognosis. In recent years, the cytogenetics laboratory has enjoyed a renaissance of activity in recognizing consistent chromosome abnormalities in a growing list of leukemias, lymphomas, and solid tumors. At present, specific chromosomal abnormalities are of greatest clinical importance in only a few tumors, primarily lymphomas and leukemias;180,181 for example, in acute and chronic myeloid leukemia and in acute lymphocytic leukemia. However, it is now clear that nonrandom chromosomal changes are to be found in a variety of solid tumors, and it is likely that cytogenetic information will become increasingly useful in defining tumor progression and prognosis.33,182–185 Along a similar vein, there has been increasing interest in recent years in the use of flow cytometric analysis of tumor cell suspensions for the determination of tumor ploidy and the fraction of replicating cells (see above). Early results indicate that such information is of use in defining the prognosis of renal, breast, and certain other types of tumors.33,58,59,61186

Role of the Autopsy Pathologist

Autopsies do not receive the respect that was at one time accorded them, and the autopsy rate has declined precipitously throughout the United States from ~90% of hospital deaths in some teaching hospitals in the late 1960s to the current average rate of only 15%.178,187–191 One widely voiced but erroneous reason for this change in attitude and practice is the belief that autopsies no longer yield much in the way of useful information because they have been preempted by new technologies, such as magnetic resonance imaging and other pathologic and biochemical tests performed on the patient during life. In a series of cases from 1986 to 1995, clinically undiagnosed or misdiagnosed malignancies were found in 44% of autopsies—a rate similar to that reported in studies from 1923 and 1972. Among the undiagnosed cancers, 57% were felt to be directly related to the patient’s death.192 Another concern is the clinicians’ fear of malpractice suits resulting from new findings revealed at autopsy that had not been diagnosed during life. Still another is the fact that the Joint Commission for the Accreditation of Health Care Organizations has greatly reduced its emphasis on the hospital autopsy rate for accreditation purposes. Psychological factors may also play a negative role. Because of the significant side effects that accompany the longer survivals achieved with modern cancer therapy, the family of the deceased may feel that the patient had “suffered enough.” Also, the autopsy serves as a symbol of failure, reminding the clinician that he was unable to cure the patient. Finally, there is a negative economic incentive for performing autopsies. Neither the pathologist, the hospital, nor the oncologist is reimbursed the cost in time, effort, and materials involved in performing an autopsy or in persuading a reluctant family to permit an autopsy.

Despite these objections, the autopsy continues to have an important role in patient care. In fact, advances in technology have done little to change the incidence of unexpected, clinically significant findings at autopsy. In our experience, it is most unusual for an unexpected autopsy finding to lead to litigation, and if an egregious error in patient management has occurred, is it not the responsibility of the medical profession to discover this?

The autopsy has an important role in evaluating the care of the cancer patient who has succumbed to his illness. While the autopsy will obviously not offer direct benefit to the dead patient, it may be essential for supporting or refuting clinical impressions, determining the extent of residual disease and the adequacy of therapy, evaluating new therapies, identifying the ultimate and proximate causes of death, and revealing unexpected findings that affected patient care. As a means of advancing knowledge, clinicians should regard autopsy as the final contribution they and the deceased patient can make to science and to an understanding of disease. The usefulness of the autopsy is greatly enhanced when the clinician takes the time to address the pathologist with specific questions that he would like answered at postmortem examination, and makes it a point to view the dissected organs, and if the pathologist issues a timely and “clinician-friendly” report that contains a minimum of jargon and attempts to integrate the anatomic findings with the clinical picture.

Summary and Conclusions

Perhaps the most important theme of this chapter has been its emphasis on the role of the pathologist as a member of the medical team caring for the patient with cancer. The importance of close communication between the oncologist, surgeon, radiotherapist, other clinicians, and the pathologist cannot be overemphasized. Patient care will be optimized if the pathologist is consulted in advance of procedures designed to obtain tissue samples for definitive diagnosis. There may be only a single opportunity to obtain tissue that will make a complete diagnosis possible, and it would be unfortunate if that opportunity was lost because portions of the specimen were not appropriately triaged for immunohistochemistry, electron microscopy, flow cytometry, culture, or other special procedures. Implementation of a high level of communication and cooperation between pathologists and clinicians has led to important contributions in the treatment and care of patients with malignant melanoma and breast cancer, among other examples. These examples should serve as useful models for the study of other types of cancers.

The authors thank Mr. Peter K. Gardner for his expertise in compiling this manuscript.


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Leukemias and ascites tumors are also embedded in stroma, that are provided by blood plasma and peritoneal exudate, respectively. Also, leukemias commonlyinduce stroma analgous to that of solid tumors in the bone marrow, as do ascites tumors in tissues lining the peritoneal cavity.

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