Immunotherapy in Genitourinary Cancers: Role of Surgical Pathologist for Detection of Immunooncologic Predictive Factors : Advances in Anatomic Pathology

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Immunotherapy in Genitourinary Cancers: Role of Surgical Pathologist for Detection of Immunooncologic Predictive Factors

Bahlinger, Veronika MD*,†; Hartmann, Arndt MD*,†; Eckstein, Markus MD*,†

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Advances In Anatomic Pathology 30(3):p 203-210, May 2023. | DOI: 10.1097/PAP.0000000000000383
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Genitourinary (GU) malignancies include a broad spectrum of distinct entities affecting kidneys, bladder, prostate, penile, and testicles. Prostate adenocarcinoma is the most common GU malignancy in men with over 1,400,000 new diagnosis per year, followed by almost 950,000 new cases of bladder and kidney cancer in men and women combined.1 Treatment options significantly increased in past years, and the current standard of care armamentarium for GU malignancies includes antihormonal therapy, chemotherapy, and PARP inhibition for prostate cancer patients; chemotherapy, immunotherapy, and anti–fibroblast growth factor receptor–targeted treatment for urothelial cancer patients; chemotherapy for testicular cancer patients; and a broad spectrum of multikinase inhibitors, immunotherapy, and targeted therapy against the vascular endothelial growth factor pathway for kidney cancer patients.2–4

Immunotherapeutic approaches base on a long-lasting era of research. For example, systemic cytokine therapy with interleukin-2 and interferon α were applied for the treatment of metastatic kidney cancer.5 Moreover, Bacillus Calmette-Gúerin instillation therapy for high-risk non–muscle-invasive bladder cancer represents one of the most successful and oldest immunotherapy.6 In the recent years with a steadily growing understanding of immunologic determinants and escape mechanisms, the treatment paradigms in some GU entities changed. For example, urothelial and kidney carcinomas are supposed to represent highly immunogenic tumors,7–9 while other GU malignancies such as testicular germ cell tumors often present with a strongly immunosuppressed microenvironment which seems not to be suitable for immune checkpoint inhibition.10 Matching with these considerations single-arm phase 2 clinical trials studying the efficacy of anti–programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors in testicular germ cell tumors did not demonstrate meaningful responses.11,12

Penile carcinomas, however, show promising features indicating high immunogenicity and thus approachability for immune therapy including human papillomavirus association and high prevalence of antitumoral immune infiltrates with marked immune checkpoint expression levels,13 but immunotherapy trials are still in their beginnings for this entity.

Based on these considerations, in this review, we will mostly focus on urothelial and kidney carcinoma. Several immune checkpoint inhibitors are currently approved for the treatment of metastatic urothelial cancer, for example, frontline monotherapy with atezolizumab or pembrolizumab in platinum-ineligible patients, avelumab maintenance after response to frontline platinum-based chemotherapy, and second-line pembrolizumab following disease progression to frontline platinum-based chemotherapy.14–17 Importantly, only 2 approvals for the metastatic setting base on successful pivotal trials (avelumab maintenance and second-line pembrolizumab treatment). Just recently, adjuvant nivolumab treatment following radical cystectomy has been approved in addition by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) based on impressive results from CheckMate 274.18

For the treatment of locally advanced and metastatic clear cell renal cell carcinoma several combination therapies or monotherapies have been approved: Dependent on clinical risk stratification several immune checkpoint inhibitor (+/− multikinase inhibitor) combinations have been approved for the frontline treatment of locally advanced (irresectable) and metastatic clear cell renal cell carcinoma.19–21 Following disease progression to multikinase/vascular endothelial growth factor inhibitors nivolumab has been approved in the metastatic second-line setting.22 Moreover, recently FDA and EMA also approved adjuvant pembrolizumab therapy for patients with intermediate and high-risk kidney cancer or oligometastatic disease following complete metastasis and primary tumor resection.23 Of note, these indications are mostly accounting for clear cell renal cell carcinoma, whereas clinical trials studying immune checkpoint inhibitors in patients with other kidney carcinoma entities (eg, SUNNIFORECAST study, NCT03075423) are still running without having reported final results so far.

Besides these entity-specific approvals, there are tissue agnostic approvals for microsatellite instability-high (MSI-H)/mismatch repair–deficient (MMRd) carcinomas (excluding MSI-H colorectal carcinoma) and for those with high tumor mutational burden (TMB; excluding urothelial carcinoma)24,25; however, it is important to note, that these 2 options have only been approved by the FDA but not the EMA. Table 1 summarizes the different therapy options among urothelial and clear cell renal cell carcinoma.

TABLE 1 - Overview of Genitourinary Cancers and the Approved Use of Immunotherapy
Malignancy Clinical Setting Approved Immunmodulative Drugs Condition Biomarker
Bladder cancer
 Noninvasive urothelial bladder cancer Bacillus Calmette-Guerin unresponsive Pembrolizumab26
Cave: phase III data not reported yet
High risk, with carcinoma in situ
 Adjuvant, muscle-invasive bladder cancer Nivolumab18
 Locally advanced or metastatic First line Atezolizumab15 Cisplatin-ineligible urothelial carcinoma or not eligible for any platinum-containing chemotherapy PD-L1 assessment necessary: PD-L1 stained tumor infiltrating immune cells covering ≥5% of the tumor area
First line Pembrolizumab14 Cisplatin-ineligible urothelial carcinoma or not eligible for any platinum-containing chemotherapy PD-L1 assessment necessary: whose tumors express PD-L1 Combined Positive Score ≥10
Second line Nivolumab27 Disease progression during or following platinum-containing chemotherapy
Second line Avelumab28 Disease progression during or following platinum-containing chemotherapy
Maintenance Avelumab17 Maintenance treatment of patients with locally advanced or metastatic urothelial carcinoma
Kidney cancer
 Locally advanced, metastatic Adjuvant Pembrolizumab23 High risk for recurrence: pT2, grad 4 or sarcomatoid N0, M0, pT3, and pT4 any grade and N-positive or M1 with no evidence of disease after surgery
 Metastatic First line Avelumab21 In combination with axitinib
First line Nivolumab19 In combination with ipilimumab or cabozantinib
Second line Nivolumab22 Patients who have received prior antiangiogenic therapy
First line Pembrolizumab20 In combination with axitinib or lenvatinib
Locally advanced, metastatic tissue agnostic approval Second line Pembrolizumab25 Adult and pediatric patients with unresectable or metastatic, microsatellite instability (MSI)-high or mismatch repair (MMR) deficient solid tumors that have progressed following prior treatment and who have no satisfactory alternative treatment options MSI or MMR analysis
Second line Pembrolizumab24 Adult and pediatric patients with unresectable or metastatic tumor mutational burden–high solid tumors, that have progressed following prior treatment and who have no satisfactory alternative treatment options Tumor mutational burden–high (TMB-H) ≥10 mutation/Mb, as determined by an FDA-approved test
FDA indicates Food and Drug Administraion; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1.

In the following, this review provides a streamlined overview on the role of a surgical pathologist to assess biomarkers and current indications for PD-L1 assessment for immune therapy response prediction. In addition, this review explores potentially emerging future biomarkers for disease treatment, which will probably improve patient's selection.


Interfering pathways within the tumor immune microenvironment, for example, therapeutic inhibition of crucial immune checkpoints such as cytotoxic T-lymphocyte–associated antigen 4, PD-1, or PD-L1 allows reactivation or release of antitumoral immune responses, which then can effectively target and clear tumor cells. PD-1 which binds to PD-L1 suppresses the immune system through many mechanisms including suppressing T-cell–mediated immune responses, decreasing the production of inflammatory cytokines, and increasing the activity of regulatory T cells.29 Inhibition of negative regulatory immune checkpoints expressed or induced by malignant cells can reinduce the restrained capability of immune cells to sufficiently destroy tumor cells.29 As surrogate predictive biomarkers for an immune checkpoint protein targeted therapy approach multiple markers such as expression of PD-L1 on immune and tumor cells within the tumor microenvironment have been discussed. So far, PD-L1 is established as valid predictive marker for immune checkpoint inhibitor success in lung cancer or head and neck squamous cell carcinoma, but its role in GU malignancies, especially in clear cell renal cell carcinoma or urothelial carcinoma is controversially discussed.30

Programmed Death-Ligand 1 Assessment in Urothelial Carcinoma

PD-L1 assessment for patient selection for anti-PD-1 and anti-PD-L1 agents is currently prescribed by the FDA and EMA in 2 indications: platinum-ineligible patients in the first-line metastatic setting need to exceed either the cutoff of immune cells 5% or Combined Positive Score 10 to receive monotherapy with atezolizumab or pembrolizumab, respectively.31 Recently, FDA and EMA also approved adjuvant treatment with nivolumab following radical cystectomy for patients with locally advanced high-risk bladder cancer based on results from CheckMate 274.18 The EMA, however, only approved the treatment for patients with tumors exceeding a tumor cell/Tumor Proportion Score cutoff of 1% although benefits were shown for the whole intention to treat population. These indications include 3 different scoring algorithms, which have been established based on 3 different FDA-approved and EMA-approved closed companion diagnostic assays (CDAs):

  • Algorithm: VENTANA IC-Score; cutoff 5%; drug: atezolizumab; CDA: VENTANA SP142.
  • Algorithm: Combined Positive Score; cutoff 10; drug: pembrolizumab; CDA: pharmDX 22c3.
  • Algorithm: Tumor cell/Tumor Proportion Score; cutoff 1%; drug: nivolumab; CDA: pharmDX 28-8.

A more detailed overview with description of the respective scoring algorithms is summarized in Table 2. Since scoring algorithms and CDAs are bound to specific therapeutic agents, surgical pathologists must consider, which therapy the clinician intends to initiate and which CDA, scoring algorithm and respective cutoff is required. Since critical clinical information, however, is often lacking in routine pathology request forms, several best practice recommendations in the contemporary literature suggested to report results for all relevant scoring algorithms.30,32,33 Although CDAs differ to some extent in staining intensity and patterns, multiple studies demonstrated that all commonly used CDAs (including pharmDX 22c3 and 28-8 and VENTANA SP142 and SP263) demonstrate an exchangeable performance in detecting PD-L1-positive immune cells.34–36 Importantly, the VENTANA SP142 CDA shows significantly reduced sensitivity to detect PD-L1-positive tumor cells compared with the other 3 assays why usage of the VENTANA SP142 is not recommended to detect PD-L1 tumor cell expression.33 Thus, except for the VENTANA SP142 assay (not suitable for tumor cell-dependent scoring algorithms) all approved CDAs can be considered exchangeable for PD-L1 assessment allowing to assess multiple scoring algorithms with one specific CDA in daily routine PD-L1 assessment. In addition, reporting all relevant scores is particularly important in the metastatic first-line setting where 2 different drugs with 2 different algorithms and cutoffs are approved. Two recent studies demonstrated, that interalgorithm variability is much bigger than interassay variability; thus, patients can be eligible for one drug, but not for the other one based on differential PD-L1 expression of immune or tumor cells, different consideration of PD-L1-positive staining cells and different cutoffs.34,37

TABLE 2 - Different Algorithms and Cutoffs for PD-L1 Assessment in Urothelial Carcinoma
Anti-PD-1/PD-L1 Therapeutics Companion Diagnostic Assay Algorithm Used Cutoff for PD-L1 Positivity Considerations for Evaluation
Atezolizumab VENTANA PD-L1 (SP142) VENTANA Immune Cell (IC)-Score: number of PD-L1-positive tumor-infiltrating ICs as a proportion of the total TC and IC area IC ≥5% Plasma cells have to be excluded from scoring All immune cells are included (incl. neutrophil granulocytes
Nivolumab pharmDX 28-8 Tumor cell/Tumor Proportion (TC/TPS) Score: number of PD-L1-positive TCs as a proportion of the total TC area TC/TPS ≥1%
Pembrolizumab pharmDX 22c3 Combined Positive Score (CPS): Number (Count) of PD-L1-positive TCs and number of PD-L1-positive ICs as a proportion of the total TC area CPS ≥10 Plasma cells have to be excluded from scoring
Neutrophil granulocytes not included
PD-1 indicates programmed cell death protein 1; PD-L1, programmed death-ligand 1.

Furthermore, PD-L1 assessment is not trivial due to the huge differences of the specific scoring algorithms. Thus, pathologists performing PD-L1 assessment on routine bases should ideally participate in dedicated PD-L1 assessment trainings on regular base which are usually hosted by academic institutions or CDA manufacturers. Systematic trainings programs have proven to be critical to achieve high interobsrver and intraobserver concordance, thus leading to high-quality PD-L1 assessment in daily practice.36 In addition, PD-L1 testing laboratories should participate in external quality assurance programs on regular base even if closed CDAs are used to assess PD-L1.30 Further considerations on optimal routine PD-L1 testing include the correct choice of representative tumor tissue (most recent tissue, ideally metastatic biopsy) for the current disease setting and optimal fixation of tissue samples (eg, in 10% neutral buffered formalin in a quantity that covers the tissue volume in a ratio of at least 1:10, and an adequate fixation period between 12 and 24 h) are preferable. Moreover, scoring should be performed on whole slide sections, and highly necrotic tumor samples should be avoided if possible. On-slide positive controls such as tonsil tissue should also be used for every PD-L1 staining. In addition, it must be mentioned that approved CDAs are usually not approved for usage in cytologic specimens, cytoblocks, or decalcified (bone) samples. However, if those samples represent the most recent and representative tumor sample, they can be used with certain limitations, although assessment of the VENTANA IC-Score is not possible in tissue samples without preserved spatial tissue integrity.30,34,36 Multiple best practice recommendations, reviews, and workshops are available and highlight the further need of harmonization for PD-L1 testing.30,32,33Figure 1 demonstrates PD-L1 assessment in upper tract urothelial carcinoma.

Programmed death-ligand 1 assessment in upper tract urothelial carcinoma: Representative images of the VENTANA PD-L1 (SP263) assay on tumor cells A, and predominantly on immune cells B. All images showed with ×400 magnification.

Kidney Tumors and the Role of Programmed Death-Ligand 1 Assessment for Immunotherapy Stratification

As mentioned above, immunotherapy has significantly improved the standard of care for clear cell renal cell carcinoma patients and is now commonly used in daily practice.38 The data landscape regarding the predictive value of PD-L1, however, is inconsistent; for example, the CheckMate 025 trial reported a slightly worse outcome for patients with PD-L1 tumor cell expression >1%.22 However, other above-mentioned pivotal trials could neither validate a positive or negative influence for immune checkpoint inhibitor response clear cell renal cell carcinoma.20,39,40 Thus, PD-L1 assessment is currently not prescribed for renal cell carcinoma.

Programmed Death-Ligand 1 Expression in Low Immunogenic Prostate Cancer

Due to its biological behavior, prostate cancer progresses more slowly and is usually less lethal than other cancers. In contrast, in case of systemic dissemination, the 5-year survival rate drops significantly.41 Although immunomodulatory therapeutics such as immune checkpoint inhibitors and other compounds have been extensively studied in metastatic disease, no regimen demonstrated superiority above comparator treatments; thus, no immune checkpoint inhibitor is specifically approved for the treatment of prostate cancer so far.42 In general, studies showed a low immunogenic microenvironment in prostate cancer, which generally presents with low overall immune infiltration and immune checkpoint protein expression like PD-L1.42–44 However, ∼8% of patients with metastatic prostate cancer who have MSI-H/DNA-MMRd tumors can receive pembrolizumab based on the tissue agnostic FDA-only approval for microsatellite instable/MMRd tumors.25,45 In addition, patients with metastatic prostate cancer and high TMB are also falling within the FDA-only tissue agnostic approval of pembrolizumab for TMB-high tumors.24

Emerging Biomarkers for Immunomodulative Therapeutic Options in Genitourinary Malignancies

Histologic Characteristics as Predictive Marker

In November 2021, adjuvant Pembrolizumab treatment for locally advanced renal cell carcinoma has been approved by the FDA. Among the KEYNOTE-564, a double-blind, phase 3 trial, Pembrolizumab treatment led to a significant improvement in disease-free survival as compared with placebo after surgery among patients with kidney cancer who were at high risk for recurrence. Interestingly, high risk of recurrence includes tumors with pT2 tumor stage and present sarcomatoid histology or nuclear grade 4 (Fuhrman nuclear grading, not WHO recommended grading) histology.46Figure 2 demonstrates these 2 histologic features and pathologists should be aware of these 2 specific high-risk features in pT2 clear renal cell carcinomas. Of note, this represents the first immunotherapy indication which is dependent on classic histopathology. Thus, standard pathologic gross dissection of kidney tumor specimens should ensure a representative coverage of the whole tumor (especially in pT2 tumors) to avoid missing sarcomatoid tumor areas which approximately occur in 1% to 5% of kidney carcinomas.47 However, it has to be mentioned that the use of the Fuhrman grading system within the pivotal trial appears to be bizarre since the WHO nucleolar grading system has been shown to be superior to the outdated Fuhrman grading system as a prognostic predictor, and has been proposed as official grading system >6 years ago with the 2016 issue of the WHO classification system.48 Nevertheless, for pT2 renal cell carcinomas, the Fuhrmann grade should be reported in the future until comparative studies might have proven the WHO grading system to be equivalent for identifying high-risk pT2 renal cell carcinoma patients. Other high-risk features include stages pT3 and pT4, pathologic confirmed lymph node metastases, and/or patients with completely resected distant metastases without evidence of further disease manifestations.

A, Clear cell renal carcinoma with sarcomatoid variant. B, Clear cell renal cell carcinoma with Fuhrmann nuclear grade 4 histology. All images showed with ×400 magnification.

Microsatellite Instability/DNA-Mismatch Repair Deficiency and Prevalence in Genitourinary Malignancies

Classic microsatellite instability is considered to be a strong predictive biomarker for immunotherapy response and is mainly caused by pathogenic hereditary (mutations) or sporadic alterations (mutations, promoter methylation in case of MLH1) of DNA-mismatch repair enzymes including MSH2, MSH6, PMS2, and MLH1 which is a status defined as MMRd.49 A MMRd status leads to a nucleotide length variation of DNA repeat regions called microsatellites.50 More specifically, MMRd leads to a strong accumulation of mutations due to an impaired detection and repair of nucleotide mismatches, eventually leading to the formation of nonsynonymous mutations which have the potential to be presented and recognized as neoantigens; thus, strong neoantigens can lead to the induction of strong antitumor immune responses.51 In 2017, pembrolizumab was approved by the FDA for the treatment of metastatic MMRd/MSI-H-positive solid tumors (excluding MMRd/MSI-H colorectal carcinoma) which had progressed on prior therapy and did not have alternative treatment options based on a nonrandomized single-arm study of around 200 patients of different tumor entities.45 MMRd/MSI-H status can roughly find in 3% of prostate carcinomas, 1% to 2% of urothelial carcinomas of the bladder tumors, 3% of urothelial carcinoma of the upper urinary tract, and 1% of renal cell carcinoma.52,53 Although these rates are overall low, patients without treatment options might benefit from a MMRd/MSI-H status assessment. Assessment of MMRd via immunohistochemistry or MSI-H via different molecular pathologic approaches to detect microsatellite instability are standard techniques in surgical and molecular pathology for decades.54 Detection of MSI-H status is most commonly performed using the Bethesda consensus panel consisting of the well-known 3 dinucleotides (D2S123, D17S250, and D5S346) and 2 mononucleotides (BAT25 and BAT26).55 Based on the approval, both the immunohistochemical detection of an MMRd and the molecular pathologic detection of an MSI-H status are sufficient for pembrolizumab therapy.45 Due to the significantly better cost and time efficiency, immunohistochemistry MMRd assessment seems to be the first choice for an initial screening.

Tumor Mutational Burden

Besides PD-L1 expression, TMB has been associated with responses to immunomodulatory therapies in several tumor types. Like MMRd, tumors with a hypermutated/TMB-high phenotype have a higher likelihood to present strong neoantigens thus offering a target structure for strong antitumoral immune responses. Despite promising results from post hoc analyses in the past, prospective organ-specific (controlled phase III) studies were not able to validate the predictive potential for immune checkpoint inhibition so far.30,43 TMB is commonly defined as the overall number of somatic nonsynonymous mutations per megabase (mut/Mb), including frame-shift mutations, insertions, point mutations, and deletions.24 Compared with the other GU malignancies, bladder tumor is a prime example of a long-term carcinogenic exposure-related tumor, which harbors relatively high TMB.43 Therefore, among phase 2 IMvigor 210 trial using atezolizumab in advanced urothelial carcinoma TMB was assessed in 150 patients using a 315-gene next-generation sequencing panel. The median mutational load was significantly higher in responders to atezolizumab than nonresponders (12.4 vs. 6.4 mut/Mb).56 Compared with this and although kidney tumors are also good responders to immunotherapeutic agents, these tumors demonstrate also relatively low TMB compared with bladder cancer and other cancer types traditionally associated with immunotherapy response with approximately TMA of 2.7 Mut/Mb.43,57 In prostate cancer, the TMB is also relatively low with nonsynonymous mutational load ∼1.5 mut/Mb in primary tumors and 2.9 in metastatic tumors.58 Future clinical trials must show whether the necessary associations are presented in distinct cancer entities and whether summarizing of nonsynonymous mutations as a marker for high neoantigen load are sufficient in some GU entities to cover the complex interplay of the tumor immune microenvironment.43 Moreover, as surgical pathologists, there is a need of harmonization of TMB measurement and to address this using a cost-effective procedure to include more patients. Despite a lack of prospective phase III data, pembrolizumab monotherapy is FDA-only approved for patients with refractory solid tumors with TMB ≥10 mut/Mb, based on results from the phase 2 KEYNOTE-158 trial.25 The mutational number defining TMB “high” appears to vary across cancer types, and it is unlikely that there will be a universal number for all cancer types that defines a TMB-high status and thus a high likelihood of benefiting from immune checkpoint inhibition.30 FDA-approved or authorized diagnostic assays for TMB assessment include the MSK-IMPACT and FoundationOne CDx panels. However, these panels are very cost consuming and whether small, targeted panels could also provide a sufficient TMB estimation must be tested in future studies.30,43 Importantly, within KEYNOTE-158 only a small fraction of GU malignancies was included, for example, prostate, renal, and urothelial.

Gene Expression Signatures

RNA-based gene expression signatures have been utilized to understand responses to immunomodulatory therapy, elucidate mechanisms of resistance, and provide subclassification of urothelial carcinoma.59,60 Analyses of gene expression profiles in the T-cell–inflamed microenvironment have shown pan-tumor immune-related signatures that correlate with anti-PD-1 benefit, including interferon-responsive genes related to antigen presentation, chemokines, cytolytic activity, and adaptive immune resistance.61 Recently, for example, for muscle-invasive bladder cancer in KEYNOTE-052, stromal signatures were significantly associated with worse outcomes; however, in the KEYNOTE-045 trial, this association could not be confirmed.62 In kidney tumors, certain gene expression signatures designed to assess the activity of a specific biologic or signaling pathway showed that higher-than-median expression of a myeloid inflammation gene signature comprising cytokines and chemokines associated with recruitment of myeloid-derived suppressor cells associating with worse outcomes to immune checkpoint inhibitor monotherapy in kidney carcinomas.63 Although positive prospective data are missing yet—mostly due to lacking prospective implementation—implementation into clinical trials might lead to novel therapy stratification tools.


In summary, until today, the role of surgical pathologists to predict immunotherapy in GU malignancies lies mainly in PD-L1 assessment of urothelial carcinoma and assessment of MMRd/MSI-H statuses in multiple cancer entities. However, with the recent approval of adjuvant pembrolizumab treatment in renal cell carcinoma, well-established histologic characteristics are gaining significant importance in terms of being predictive markers for immunotherapy benefits. The emerging role of new biomarkers such as MSI-H/MMRd status, TMB, or gene expression signatures has to be validated in entity-specific trials and pathologists should be aware of these new advances.


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immunotherapy; genitourinary tumors; PD-1; PD-L1; tumor mutational burden; microsatellite instability

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