Prognostic Markers and Trials in Penile Cancer

New tumor biomarkers open the potential for designing personalized therapy for penile squamous cell carcinoma. Despite the initial promising results of some biomarkers, controversy remains due to contradictory studies. Further robust research work is required before incorporating biomarkers in the personalized management of penile cancer. This narrative review aims to highlight some of the most commonly and recently investigated biomarkers of penile cancer and to summarize the ongoing registered clinical trials for the management of penile cancer patients.


Introduction
The incidence of penile cancer (PC) is rare, 1 with approximately 26 000 new cases diagnosed annually worldwide.The incidence rate varies depending on the geographic location, with higher incidence rates in some regions of South America, Asia, and Africa than in Western Europe and North America. 2 A meta-analysis reported the age-standardized incidence of PC (expressed as cases per 100 000 person-years) to be highest in Latin America (1.40 cases), followed by Africa (0.99 cases), North America (0.91 cases), Europe (0.90 cases), then Asia (0.44 cases), while the least incidence was reported in Oceania (0.42 cases). 3Studies have demonstrated the highest rate in Brazil where the incidence ranged between 2.9 and 6.8 cases per 100 000. 4,5The second-highest rate of PC is reported in Uganda, where 3-4 new cases per 100 000 men are diagnosed annually. 4,6fortunately, the clinical presentations of invasive PC vary, leading to delayed diagnosis, with poor patient survival in some cases. 7One of the most significant prognostic factors in PC is the presence of lymph node (LN) metastases. 80][11][12] Table 1 demonstrates the reported survival rates according to LN involvement.
Inguinal LN dissection (ILND) is indicated if positive inguinal LNs are detected to limit the spread of the disease.Nevertheless, approximately one-quarter of clinically node-negative patients have microscopic LN metastases 13 that are not feasibly detected using conventional imaging techniques 14 but can be detected using sentinel node biopsy techniques based on protocols related to the stage of the disease. 15In some regions, prophylactic ILND in cN0 patients is undertaken, resulting in the unnecessary risk of procedure morbidities such as wound breakdown, lymphoceles, and lymphedema. 8,16There is no current reliable prognostic biomarker which can be used in primary cancers to predict LN metastasis.
Investigating the association between different biomarkers and relevant variables in PC patients improves our understanding of the pathways of tumor development and progression.This, in turn, has provided new insights into identifying new therapeutic targets. 17owever, to date, no biomarker has reliably provided an aid to diagnosis which is utilized in mainstream practice.

Methods
For this narrative review, a literature search was conducted on the databases of PubMed/MEDLINE, Cochrane library, and ClinicalTrials.gov.The search used the keywords "Penile cancer," "lymph node metastasis," "survival," and "biomarkers" to identify relevant articles.The search was limited to English-published articles.The search yielded 69 results, out of which 25 were used.As a large number of biomarkers were evaluated in the retrieved studies, the review focused on the most commonly evaluated or discussed biomarkers.The studies were included if they assessed the prediction of LN metastasis, overall survival (OS), or disease-specific survival (DSS) in patients with PC.In addition, articles that were case reports or did not include data on the prognostic value of the biomarker for lymphatic metastasis or survival were excluded (Figure 1).The reference lists of the retrieved articles were searched for further related studies and previous reviews.

Squamous Cell Carcinoma Antigen
The squamous cell carcinoma antigen (SCCAg) is a tumor-associated glycoprotein.Seven studies [18][19][20][21][22][23][24] assessed the potential association between elevated serum levels of SCCAg and LN metastasis in penile squamous cell carcinoma (SCC) patients.A statistically significant association with nodal disease was reported by 4 studies: 2 in univariate analysis 19,22 and 2 studies in multivariate analysis. 18,20Zhu et al 22 reported that at a cut-off level > 1500 ng/L, the sensitivity and specificity of SCCAg were 34.3% and 89.3%, respectively, for predicting LN metastasis (P = .005).However, they found that SCCAg poorly predicted occult inguinal metastasis in clinically node-negative patients (Table 2).
Two studies 19,23 examined the association between elevated SCCAg levels and survival in patients with penile SCC after surgery (Table 2).Li et al 19 found that DSS was significantly lower by 28% in patients with elevated SCCAg levels compared to those with normal levels in univariate analysis, but the association was not significant in multivariate analysis (hazard ratio (HR): 4.564, 95% CI: 0.583-35.7,P = .148).Liu et al 23 found a non-significant impact on OS (HR: 1.285, 95% CI: 0.632-2.616,P = .489).
The results for SCCAg are not reliable enough for including the biomarker in routine practice to identify LN metastasis as the results are controversial, though there seems a tendency for higher levels in patients with LN metastases.The 2 studies that investigated the impact of SCCAg levels on patients' survival reported non-promising results.

C-Reactive Protein
C-reactive protein (CRP) is synthesized by the liver in response to an inflammatory stimulus and was recorded in several conditions including infection, trauma, and malignant tumors. 25Elevated CRP levels in malignancies could be explained by the inflammatory state induced by tumor growth or the immune reaction triggered by tumor antigens. 26High plasma CRP levels were associated with poor prognosis in some cancers such as renal cell carcinoma. 27effens et al 28 reported a significant association between high preoperative serum CRP levels above 15 mg/L and nodal disease (P = .007).In addition, patients with high CRP levels had a worse 5-year cancer-specific survival (CSS, P = .001).Multivariate analysis identified CRP as an independent prognostic factor for CSS (HR: 3.34, 95% CI: 1.04-10.72,P = .04).The study by Al Ghazal et al 29 linked also high CRP serum levels above 20 mg/L with nodal metastasis.Li et al 19 demonstrated that CRP levels ≥ 4.5 mg/L were significantly associated with extranodal extension (P < .001),pelvic LN metastases (P = .007),and 3-year CSS (P < .001)(Table 2).Multivariate regression analysis showed that the combined use of high CRP and SCCAg levels was significantly associated with 3-year CSS (HR: 3.39, 95% CI: 1.104-10.411;P = .033).Although the findings of the mentioned studies show promising results for the association of CRP with LN metastasis and patients' survival, these results are too heterogeneous to accept CRP as a routine marker.

MAIN POINTS
The results concerning the use of Ki-67 as a marker for LN metastasis appear heterogeneous and thus the marker cannot currently be recommended to be used in clinical practice.As for the prediction of survival, the results indicate a poor impact on patients' survival, with no potential for clinical use.

Human Papillomavirus Status
Human papillomavirus (HPV) is a double-stranded DNA virus.Human papillomavirus is sexually transmitted and has been linked with PC, particularly the HPV 16 and 18 subtypes. 39,40The HPV infection binds-through the HPVE7 and E6 oncoproteins-to the host retinoblastoma and p53 proteins, disrupting apoptosis and leading to abnormal cellular proliferation. 41The HPV is detected in up to 50% of PC.Basaloid and warty PC show a high prevalence of HPV while verrucous and papillary PC exhibited HPV in approximately one-third of patients. 42 a marker for LN metastasis, 4 studies [43][44][45][46] reported the lack of significant association between HPV positivity and nodal disease (Table 2).
Six studies evaluated the impact of HPV infection on survival in PC.Four studies stated the lack of significant association, 43,[45][46][47] while 2 studies 44,48 reported that HPV infection was significantly associated with good outcomes and improved DSS (Table 2).

P16INK4a
P16INK4a is a tumor-suppressor gene that prevents cell division.Immunohistochemical staining in high-risk HPV genotypes demonstrated the overexpression of P16INK4a. 49Therefore, P16INK4a expression can be used as a surrogate biomarker for HPV infection. 50igh sensitivity (100%) and relatively low specificity (57%) were reported for P16INK4a immunostaining when used as a predictor for high-risk HPV DNA. 51sitive P16INK4a immunoreaction was reported to have an inverse relationship with occult LN metastasis, 52 but its performance as a predictor was poor and lacked statistical significance.Ferrándiz-Pulido et al. 46 Tang et al. 53 and Mohanty et al 54 reported the lack of significant association with positive LNs (Table 2).
Positive P16INK4a immunoreaction has been associated with a tendency toward improved survival on univariate analysis in 4 studies 38,[54][55][56] and multivariate analysis in 3 studies. 38,55,56On the other hand, other studies showed a lack of significant association between P16INK4a immunoreaction and OS 46,53 or CSS 51 in univariate analysis.Steinestel et al 51 stated that P16INK4a positivity and high-risk HPV status suggested a less aggressive behavior of PC, but no significant association was found with CSS.The multivariate analysis by Bethune et al 38 showed that lacking p16 expression predicted worse OS (HR: 0.54, 95% CI: 0.31-0.93,P = .026),but not CSS (HR: 0.53, 95% CI: 0.26-1.06,P = .073;Table 2).

P53
TP53 is a tumor suppressor gene that exhibits mutations in approximately two-thirds of adult solid tumors. 57A mutated TP53 gene results either in producing an anomalous p53 protein (in 90% of cases) or its absence (in 10% of cases).The anomalous protein accumulates in the nucleus of cancer cells which can be demonstrated by immunohistochemical staining. 58The prevalence of p53 overexpression ranges from 26% to 91% in PC. 59,60 Eight studies 23,36,54,56,59,[61][62][63] reported the relationship between p53 overexpression and positive LN in PC.Six studies found a significant association, 23,36,56,59,61,62 with increased risk on univariate analysis ranging between 1.04 and 266.4.Multivariate analysis was conducted by Lopes et al. 59 Zhu et al. 36 and Liu et al 23 who found p53 to be an independent predictor of LN metastasis, whereas Zhu et al 62 found a non-significant association on multivariate regression (OR: 3.22, 95% CI: 0.96-10.86,P = .058;Table 2).

Cytogenetics in Penile Cancer
Advances in genome studying techniques enable sequencing of the entire genome of a tumor cell, which opens the potential of identifying new biomarkers in cancer patients.Whole exome sequencing is used to identify genetic imbalance which is defined as "a genome showing any loss or gain of DNA sequences compared with the reference DNA whole sequence of the genome of interest." 71The epigenetic analysis identifies potentially reversible alterations of the genome-such as methylation and histone modification-which are known as epigenetic modifications and may induce genetic instability. 72genetic imbalance was reported in PC patients, as comparative genomic hybridization enabled the identification of DNA copy-number alterations.73,74 Copy-number alterations of 3p, 3q, and 8p were associated with reduced CSS and DSS.Several studies reported on the association between the amplification of the MYC gene with CSS in PC. 75,76 The MYC gene is a proto-oncogene in the 8q24 chromosome that encodes a transcription factor responsible for regulating cellular proliferation, differentiation, and apoptosis.77 Busso-Lopes et al 74 demonstrated the presence of MYC gene amplification in PC patients, but no prognostic significance was detected.
Another genetic imbalance in PC is the loss of heterozygosity on chromosomes 6, 9, and 12, which correlated with metastasis and advanced stage. 78These findings suggest a promising potential prognostic role of cytogenetic markers in PC patients.
There is a paucity of data on epigenetic modifications and their prognostic value in PC.One study 72 identified methylation epi-signatures that were associated with HPV status and LN metastasis, with a sensitivity and specificity of 93% and 80%, respectively.Another study 79 found an association of low brain-derived neurotrophic factor gene methylation with LN metastasis and a shorter DSS using univariate analysis, but this significance was not detected in multivariate analysis.

Current Ongoing Clinical Trials on the Management of Penile Cancer
The search continues to find new treatment lines for PC that can improve patient outcomes.Current clinical trials that are registered on Clinicaltrials.govare listed in Table 3.
The sequencing of surgery and chemotherapy or radiotherapy is assessed in the AFU-GETUG 25 trial and the InPACT trial.
The AFU-GETUG 25 trial (NCT02817958) compares LN dissection (LND) and adjuvant chemotherapy to neoadjuvant chemotherapy followed by bilateral LND.The chemotherapy regimen (TIP) includes paclitaxel, ifosfamide, and cisplatin for 4 cycles every 3 weeks.The estimated study completion date is in September 2024.This non-randomized, open-label trial targets the enrolment of 37 participants.The first arm will be subjected to adjuvant chemotherapy TIP after modified bilateral LND (4 cycles every 21 days).The second arm will undergo fine needle biopsy or sentinel node + neoadjuvant chemotherapy TIP followed by modified bilateral LND.The primary outcome is survival without locoregional LN recurrence.The eligibility criteria included adult men with histologically proven PC, stage cN1 and cN2 or nodes involvement risk ≥ pT1b and/or grade 2. The secondary outcomes include a complete response rate to neoadjuvant chemotherapy, survival, toxicity, and quality of life.
The InPACT trial (NCT02305654) is a phase III, open-label, randomized trial that assesses the sequencing of surgery, chemotherapy, and chemoradiotherapy.The study is still recruiting, and the estimated sample size is 400 participants.The study enrolls adult men with histologically proven SCC of the penis, any T stage, N1 to N3 nodal stages, and no metastasis.The primary outcome is OS, while secondary outcomes include DSS, grade 3 or 4 toxicity, disease-free survival, surgical complication, quality of life, and pathological complete remission.
Several studies are evaluating the use of new drugs in PC patients.The ORPHEUS phase II trial (NCT04231981) evaluates INCMGA00012, a new drug acting on PI3K and indoleamine 2,3-dioxygenase in patients with advanced stages.The study recruited 18 patients and its completion date is estimated to be in December 2022.Eligible patients are those above 18 years old, with histologically proven penile SCC, locally advanced unresectable or metastatic stage 4 cancer.The primary outcome is the objective response rate, and the secondary outcomes include clinical benefit rate, progression-free survival (PFS), duration of response, OS, maximum tumor shrinkage, and adverse events.The open-label, single-arm, phase II ALPACA trial (NCT03391479) assesses avelumab in patients with advanced PC (locally advanced or metastatic) who are either unfit for or progressing on platinumbased chemotherapy.The study aims to enroll 24 patients.The studied outcomes include objective response rate (primary) as well as PFS and OS (secondary outcomes).Patients are eligible for enrolment if they are above 18 years, with histologically proven SCC of the penis, unres ectab le/me tasta tic stage, and unfit for platinum-based chemotherapy or progressed on/after treatment with platinum-based chemotherapy.
The PULSE trial (open-label, single-arm, NCT03774901) uses avelumab as maintenance therapy in stable diseases with first-line chemotherapy and will enroll 32 participants.The eligibility criteria include age above 18 years and histologically confirmed unresectable locally advanced or metastatic penile SCC.The studied outcomes are PFS (primary) as well as OS, quality of life, and adverse events (secondary outcomes).
The LATENT study (NCT03357757, open-label, single-arm) evaluates the combined use of avelumab and valproic acid in SCC patients with advanced p16-positive tumors.The study estimates to enroll 39 participants.The study will include patients above 18 years old of either sex and with confirmed SCC of the penis, cervix, vulva, vagina, or anus.The primary outcomes are the efficacy of the intervention and the proportion of patients completing 4 doses of the treatment.Secondary outcomes are OS, PFS, adverse events, and immunoscore.
Atezolizumab is under evaluation in an open-label, phase-II study (PERICLES; NCT03686332) with or without radiotherapy in advanced penile SCC.The study enrolled 32 men ≥18 years of age, with advanced histologically documented SCC of the penis or distal urethra.The primary endpoint is PFS, and the secondary endpoints are OS and the percentage of patients completing the full course of radiotherapy.The role of gene-modified HPV virus (MEDI0457) is evaluated in an open-label, single-arm, phase II clinical trial (NCT03439085), along with durvalumab for recurrent or metastatic HPV-related cancers, including PC.The study enrolls 77 adult participants of either sex with recurrent/ metastatic HPV-associated cancers and cancers refractory to standard therapy.The primary outcome is the objective response rate.The secondary outcomes comprise the disease control rate, PFS, and OS.

Conclusion
New tumor biomarkers may allow for tailoring personalized therapy in cancer patients by identifying those at high risk of LN metastasis who will benefit from pelvic LND, radiotherapy, chemotherapy, or combination treatments.Several biomarkers for PC have been identified, but heterogeneity in outcomes and no improvement beyond current normal practice means the non-feasibility of incorporating these biomarkers in patient management currently.The lack of standard definitions of the markers' positivity or the optimal cut-off values may contribute to this.In addition, studies were not powered for conducting regression analysis to adjust for confounders due to their relatively small sample sizes.
Further collaborative research is necessary to validate the incorporation of current and new biomarkers in the management of PC.
The new therapeutic agents being investigated, such as checkpoint inhibitors, may enhance the response and therefore reduce the administered dose of cytotoxic drugs or radiation which will decrease the adverse effects of therapy or may allow treatment to be directed toward those who will benefit most.

Declaration of Interests:
The authors declare that they have no competing interest.

Funding:
The authors declare that this study had received no financial support.

Figure 1 .
Figure 1.Flow chart of the search results and article selection.
Several clinical trials are assessing anti-PD-L1 monotherapy in PC patients.Three clinical trials are evaluating avelumab.

Table 1 .
Survival Rates in Penile Cancer Patients According to the Stage of Nodal Involvement

Table 2 .
Summary of the Reported Biomarkers' Association with Lymph Node Metastasis and Survival in Penile Cancer Patients

Table 3 .
Ongoing Studies Investigating New Treatment Lines for Penile Cancer (from clinicaltrials.gov)Pembrolizumab is another drug under assessment in 2 trials.An open-label, single-arm, phase-II trial (HERCULES; NCT04224740) assesses the drug in advanced PC along with the standard-of-care chemotherapy.The study targets to enroll 33 adult men with penile SCC and metastatic disease or recurrent locally advanced disease not amenable to curative therapy.The primary outcome is the overall response rate.The secondary outcomes are PFS, OS, clinical benefit rate, and quality of life.The open-label, single-arm PEVOsq trial (NCT04357873) uses pembrolizumab in association with vorinostat in 111 patients with recurrent and/or metastatic SCC of different body regions.Patients must have histologically confirmed recurrent and/ or metastatic SCC of the head and neck, cervix, lung, anus, vulva, or penis and radiologically confirmed progressive recurrent and/or metastatic disease.The studied outcomes include the objective response rate (primary) as well as the duration of response, PFS, and OS.The human anti-PD-L1 monoclonal antibody is being assessed in an open-label single-arm, phase II trial (NCT04718584) enrolling 127 patients with tumors of the urinary and genital systems (muscularinfiltrating bladder cancer suitable for surgery, advanced opaque cell renal carcinoma, and advanced PC).The primary outcome is a complete response, whereas the secondary outcomes are recurrencefree survival, PFS, disease control rate, duration of response, OS, and adverse events.