Low co-expression of PD-L1 and oncogenic receptor tyrosine kinases HER2 and cMET in urothelial carcinoma is associated with discordant expression between primary and metastatic sites.

OBJECTIVES: Novel regimens targeting immune checkpoints and the cMET or HER2 pathways are under investigation in metastatic urothelial carcinoma (mUC) though co-expression of these molecular targets has not been defined. We sought to characterize the protein co-expression rates of PD-L1, cMET and HER2 in primary and metastatic mUC lesions and agreement rates in paired biopsies. MATERIALS AND METHODS: We assessed PD-L1, cMET and HER2 protein expression by immunohistochemistry (IHC) in archival mUC samples identified from an institutional database (n = 143). Correlation of expression between primary and metastatic biopsies was performed in patients with available paired biopsies (n = 79). Protein expression levels by predefined thresholds were measured, and Cohen's kappa statistics (κ) were utilized to assess the agreement in expression between paired primary and metastatic samples. RESULTS: In primary tumors (n = 85), high expression of PD-L1, cMET, and HER2 was observed in 14.1%, 34.1%, and 12.9%, respectively. In metastatic samples (n = 143), high expression of PD-L1, cMET and HER2 was detected in 9.8%, 41.3%, and 9.8%, respectively. Expression agreement rates between paired specimens (n = 79) were PD-L1: 79.7% (κ = 0.09), cMET: 69.6% (κ = 0.35), HER2: 84.8% (κ = 0.17). High PD-L1/cMET co-expression was observed in only 5.1% (n = 4) of primary and 4.9% (n = 7) of metastatic specimens. High co-expression of PD-L1/HER2 occurred in 3.8% (n = 3) of primary samples and no metastatic samples. The overall co-expression agreement between paired samples was 55.7% (κ = 0.22) for PD-L1/cMET and 67.1% (κ = 0.06) for PD-L1/HER2, but agreement for high co-expression between paired samples was very low (2.5% for PD-L1/cMET and 0% for PD-L1/HER2). CONCLUSIONS: Tumor co-expression of high cMET or HER2 and PD-L1 is low in this cohort. Agreement of high co-expression between primary and metastatic sites is rare. Biomarker-based strategies used in selection of patients for contemporary trials testing combinations of immune checkpoint inhibitors with either cMET or HER2-targeted agents should account for discordant biomarker expression between primary and metastatic sites.

Prognostic value of galectin-1 and galectin-3 expression in localized urothelial bladder cancer.

Background: Galectin-1 (Gal-1) and Galectin-3 (Gal-3) are carbohydrate binding proteins with a wide range of biological activity, including regulation of cellular adhesion, proliferation, and apoptosis in solid tumors. Prior small studies have reported that Gal-3 expression is associated with progression of disease in urothelial carcinoma (UC), from non-muscle invasive UC progression to muscle invasive UC. We assessed Gal-1 and Gal-3 protein expression H-score utilizing a tissue microarray (TMA) created from 301 cystectomy specimens. Methods: Immunohistochemistry for Gal-1 and Gal-3 was performed on TMA generated from tumor blocks from chemotherapy naïve cystectomy specimens. The variable of interest, H-score, was defined as the product of the percentage of cells staining positive (0-100) and intensity score (0-3) scored by a single pathologist. Survival end points were analyzed using Kaplan-Meier and Cox Proportional Hazards methods. Clinical data including Charlson Comorbidity Index (CCI), pathologic tumor (T) stage, tumor size, node stage, and surgical margins, were included in multivariable analysis. Results: We found that Gal-1 and Gal-3 expression correlated with intratumoral T stage (median Gal-1 H-score was 0 across non-invasive tissue types and 200 in invasive, P<0.01 and median Gal-3 score was 270 across non-invasive tissue types and 70 in invasive, P<0.01). However, the highest intratumoral H-score per cystectomy core did not independently predict for recurrence-free survival (RFS) (Gal-1: HR =1.02, P=0.44, Gal-3: HR =1.01, P=0.65) or OS (Gal-1: HR =1.02, P=0.44, Gal-3: HR =1.01, P=0.72) in this cohort. Significant intratumoral heterogeneity was present for both Gal-1 and Gal-3, with an average difference between the highest and lowest H score was 95 for Gal-1 and 109 for Gal-3 for cystectomy specimens with more than one biopsy. Conclusions: Gal-1 and Gal-3 H-score per bladder did not independently predict for RFS or OS. Intra-tumoral Gal-1/Gal-3 heterogeneity complicates the use of Gal-1 and Gal-3 expression as a prognostic biomarker. Future studies should consider the evaluation of serum and urinary galectins as an approach to mitigate tumor heterogeneity.

Identification of potential biomarkers and novel therapeutic targets through genomic analysis of small cell bladder carcinoma and associated clinical outcomes.

OBJECTIVES: Small cell bladder carcinoma (SCBC) represents a rare histologic variant with a poor prognosis and for which no routine biomarkers exist. Limited reports of genomic sequencing in SCBC have demonstrated a high prevalence of TP53 and RB1 gene mutations, though the prognostic value of these and other gene variants in SCBC remains undefined. In this study, we performed targeted genomic sequencing on a cohort of SCBC patients and correlated genomic findings with clinical outcomes to identify potential novel biomarkers. MATERIALS AND METHODS: Thirty-one patients with SCBC and available treatment-naïve tumor specimens were identified from an institutional database (23 limited stage [LS], 8 extensive stage [ES]). Small cell carcinoma specimens were microdissected and subjected to tumor next-generation whole-exon sequencing with a 592 gene panel. Kaplan-Meier techniques and Cox proportional hazards models were used to evaluate genomic aberration association with relapse-free survival (RFS) and overall survival (OS) in the limited stage cohort. RESULTS: The most common pathogenic gene variants included ARID1A (48%), TP53 (48%) and RB1 (48%). Mutations in genes with potential therapeutic targets not routinely evaluated in SCBC included BRCA1/2 (16%), POLE (13%), JAK2 (13%), PDGFB (13%) and FGFR3 (3%). Multiple novel biomarker candidates showed trends for improvements in OS in the LS subset including ERCC2 (HR 0.322, P = 0.122) and RB1 (HR 0.481, P = 0.182), while LS patients with TP53 mutations (HR 2.730, P = 0.056), and MCL1 gene amplification (HR 4.183, P = 0.018) suggested inferior OS. Additionally, gene or copy number variants with potential prognostic benefit included UBR5 and DAXX (P = 0.02, [hazard ratios nonestimable due to zero events in biomarker positive groups]). CONCLUSIONS: These results support the role for tumor genomic profiling in SCBC and identify multiple potential novel biomarkers and therapeutic targets in this rare disease. Efforts to validate these findings should lead to improved decision-making and treatment outcomes in SCBC.

Racial and Socioeconomic Disparities in MRI-Fusion Biopsy Utilization to Assess for Prostate Cancer.

OBJECTIVE: To evaluate whether racial disparities in MRI-Bx usage persisted after correction for socioeconomic, demographic, and clinical factors. METHODS: This is a retrospective cohort study of patients who received either MRI-Bx or systematic biopsy (SB) within a single academic medical center between January 2018 - June 2020. For each patient, socioeconomic variables including household income, education, percent below poverty, and unemployment were estimated using 2015 American Community Survey census-tract level data. Chi-square analysis was used to examine differences in clinical and demographic characteristics between the two groups. The Benjamini-Hochberg procedure was used to control false discovery rate (FDR) for multiple testing. RESULTS: Eighteen percent of Black men (53/295) received MRI-Bx while 41% (228/561) of white men received MRI-Bx. Patients coming from highly impoverished areas were less likely to receive MRI-Bx, 25% vs 75%, respectively. In multivariate analysis, race remained significantly different across MRI-Bx and SB groups. Clinical factors including family history, DRE, BMI, and prostate volume were not significantly different between patients receiving MRI-Bx and SB. CONCLUSION: Black men are less likely to receive MRI-Bx than white men, even after adjusting for clinical and socioeconomic characteristics. Further work is necessary to identify and study methods to increase equity in PCa diagnostic testing.

Genomic analysis of response to bacillus Calmette-Guérin (BCG) treatment in high-grade stage 1 bladder cancer patients.

BACKGROUND: Intravesical bacillus Calmette-Guérin (BCG) therapy is standard treatment for high-risk non-muscle invasive bladder cancer (NMIBC) but overall efficacy is low, and no reliable predictive biomarkers currently exist to refine patient selection. We performed genomic analysis on high-grade (HG) T1 NMIBCs to determine if response to therapy is predicted by certain mutational and/or expressional changes. METHODS: Patients with HG T1 NMIBC treated with induction BCG were stratified by response into durable and non-durable responders. Baseline tumor samples were subjected to targeted DNA sequencing and whole-exome RNAseq. Genomic variants differing significantly between response groups were analyzed using Ingenuity Pathway Analysis (IPA) software. Variant selection was refined to target potential biomarker candidates for responsiveness to BCG. RESULTS: Among 42 patients, the median follow-up was 51.7 months and 40.5% (n=17) were durable BCG responders. Deleterious mutations in the RNA sequence of JCHAIN, S100A7, CLEC2B, and ANXA10 were more common in non-durable responders. Mutations in MCL1 and MSH6 detected on targeted sequencing were more commonly found in durable responders. Of all deleterious DNA and RNA mutations identified, only MCL1 was significantly associated with longer recurrence free survival (RFS) (P=0.031). CONCLUSIONS: Differences in the genomic profiles of HG T1 NMIBC tumors exist between those who show durable response to BCG and those who do not. Using pathway analysis, those differences imply upregulation of several interconnected inflammatory pathways among responders. Specific variants identified here, namely MCL1, are candidates for further study and, if clinically validated, may serve as useful biomarkers in the future.

Racial Disparities in Prostate Specific Antigen Screening and Referral to Urology in a Large, Integrated Health Care System: A Retrospective Cohort Study.

PURPOSE: Contemporary trends and racial disparities in prostate cancer screening and referral to urology for prostate cancer risk are not well characterized, despite consensus that Black men are at higher risk for poor prostate cancer outcomes. The objective of this study was to characterize current racial disparities in prostate cancer screening and referral from primary care to urology for prostate cancer concern within our large, integrated health care system. MATERIALS AND METHODS: This retrospective cohort study used data from Atrium Health's enterprise data warehouse, which includes patient information from more than 900 care locations across North Carolina, South Carolina and Georgia. We included all men seen in the ambulatory or outpatient setting between 2014 and 2019 who were ≥40 years old. Clinical and demographic data were collected for all men, including age and race. Racial outcomes were reported for all groups with >2% representation in the population. Between-group comparisons were determined using chi-squared analysis, Wilcoxon rank sum testing and multivariable logistic regression, with significance defined as p <0.05. RESULTS: We observed a significant decrease in prostate specific antigen testing across all age and racial groups in a cohort of 606,985 men at Atrium Health, including 87,189 Black men, with an overall relative decline of 56%. As compared to White men, Black men were more likely to undergo prostate specific antigen testing (adjusted OR 1.24, 95% CI 1.22-1.26) and be referred to urology for prostate cancer (adjusted OR 1.94, 95% CI 1.75-2.16). CONCLUSIONS: There was a continued significant decline in prostate cancer screening between 2014 and 2019. Despite having modestly elevated odds of being screened for prostate cancer compared to White men, Black men are relatively underscreened when considering that those who undergo prostate specific antigen screening are more likely to be referred by primary care to urology for additional prostate cancer diagnostic evaluation.

Association of 5-Alpha Reductase Inhibitor Use with Prostate Specific Antigen Level at the Time of Urology Referral in a Retrospective Cohort at a Large, Integrated Health Care System.

INTRODUCTION: 5-Alpha reductase inhibitor (5-ARI) use leads to a 50% decline in serum prostate specific antigen (PSA) without a concomitant decrease in prostate cancer (PCa) risk. We hypothesize that failure to account for the effect of 5-ARI use on serum PSA leads to increased PCa risk at urology referral among 5-ARI users. METHODS: This is a retrospective cohort study for the years 2018-2019. Atrium Health is a large, vertically integrated health system with over 900 care locations in North Carolina and South Carolina. Men ≥40 years old during 2018-2019 who had a PSA test performed were included. We determined differences in corrected serum PSA level at the time of referral to urology. 5-ARI users and nonusers were compared using the chi-square test, Student's t-test and gamma regression. RESULTS: From 2018-2019, there were 91,368 men who underwent PSA testing, including 2,939 5-ARI users. At referral, 5-ARI users had similar uncorrected median PSA (5.8 vs 5.6 ng/ml, p=0.05). After correcting for the effect of 5-ARIs on PSA, 5-ARI users had a median PSA of 11.6 ng/ml at urology referral, compared to 5.6 ng/ml in nonusers. CONCLUSIONS: Men taking 5-ARIs have higher corrected serum PSA at time of referral to urology. As the unadjusted PSA at referral to urology for PCa risk was similar between 5-ARI users and nonusers, this indicates that the effect of 5-ARI use on serum PSA levels is not routinely accounted for when assessing PCa risk.

Human epidermal growth factor receptor 2 overexpression is frequently discordant between primary and metastatic urothelial carcinoma and is associated with intratumoral human epidermal growth factor receptor 2 heterogeneity.

Human epidermal growth factor receptor 2 (HER2) overexpression occurs in 5-10% of primary urothelial carcinomas (UCs) but has not reliably predicted benefit from HER2-targeted agents in the metastatic setting. HER2 testing of primary tumors may not reflect the HER2 status of distant metastases. We assessed the concordance of HER2 expression in paired primary and distant metastatic UC lesions. Specimens from 149 patients with metastatic UC underwent immunohistochemical staining for HER2, including 79 paired primary and distant metastatic tumors. HER2 status was defined using 2018 ASCO/CAP guidelines. HER2 intratumoral heterogeneity (ITH) was defined as HER2 3+ expression in 5-50% of tumor cells. The HER2-positive, -equivocal, and -negative rates observed were 10.6%, 24.7%, and 64.7% for primary tumors and 9.8%, 12.6%, and 77.6% for metastatic tumors, respectively. HER2 ITH occurred in 44% of HER2-positive primary tumors. Low agreement of HER2-positive status between primary and metastatic tumors was observed (к = 0.193, P = 0.079). Loss of HER2 overexpression in the metastatic lesion was observed in 55% (5 of 9 cases) of HER2-positive primary cases and was associated with the presence of HER2 ITH in the primary tumor (Fisher's exact P = 0.048). Change from negative primary to positive metastasis was seen in 2% (1 of 50) of cases. No differences in metastasis-free survival or overall survival were observed in accordance with HER2 status defined by either the primary or metastatic lesion. These findings are likely to impact patient selection for HER2 targeted therapies in UC. Confirmation and evaluation of the clinical significance of HER2 discordance is warranted, preferably in the context of a clinical trial.

Neoadjuvant atezolizumab and chemotherapy in patients with resectable non-small-cell lung cancer: an open-label, multicentre, single-arm, phase 2 trial.

BACKGROUND: Approximately 25% of all patients with non-small-cell lung cancer present with resectable stage IB-IIIA disease, and although perioperative chemotherapy is the standard of care, this treatment strategy provides only modest survival benefits. On the basis of the activity of immune checkpoint inhibitors in metastatic non-small-cell lung cancer, we designed a trial to test the activity of the PD-L1 inhibitor, atezolizumab, with carboplatin and nab-paclitaxel given as neoadjuvant treatment before surgical resection. METHODS: This open-label, multicentre, single-arm, phase 2 trial was done at three hospitals in the USA. Eligible patients were aged 18 years or older and had resectable American Joint Committee on Cancer-defined stage IB-IIIA non-small-cell lung cancer, an Eastern Cooperative Oncology Group performance status of 0-1, and a history of smoking exposure. Patients received neoadjuvant treatment with intravenous atezolizumab (1200 mg) on day 1, nab-paclitaxel (100 mg/m2) on days 1, 8, and 15, and carboplatin (area under the curve 5; 5 mg/mL per min) on day 1, of each 21-day cycle. Patients without disease progression after two cycles proceeded to receive two further cycles, which were then followed by surgical resection. The primary endpoint was major pathological response, defined as the presence of 10% or less residual viable tumour at the time of surgery. All analyses were intention to treat. This study is registered with ClinicalTrials.gov, NCT02716038, and is ongoing but no longer recruiting participants. FINDINGS: Between May 26, 2016, and March 1, 2019, we assessed 39 patients for eligibility, of whom 30 patients were enrolled. 23 (77%) of these patients had stage IIIA disease. 29 (97%) patients were taken into the operating theatre, and 26 (87%) underwent successful R0 resection. At the data cutoff (Aug 7, 2019), the median follow-up period was 12·9 months (IQR 6·2-22·9). 17 (57%; 95% CI 37-75) of 30 patients had a major pathological response. The most common treatment-related grade 3-4 adverse events were neutropenia (15 [50%] of 30 patients), increased alanine aminotransferase concentrations (two [7%] patients), increased aspartate aminotransferase concentration (two [7%] patients), and thrombocytopenia (two [7%] patients). Serious treatment-related adverse events included one (3%) patient with grade 3 febrile neutropenia, one (3%) patient with grade 4 hyperglycaemia, and one (3%) patient with grade 2 bronchopulmonary haemorrhage. There were no treatment-related deaths. INTERPRETATION: Atezolizumab plus carboplatin and nab-paclitaxel could be a potential neoadjuvant regimen for resectable non-small-cell lung cancer, with a high proportion of patients achieving a major pathological response, and manageable treatment-related toxic effects, which did not compromise surgical resection. FUNDING: Genentech and Celgene.

Discordance of high PD-L1 expression in primary and metastatic urothelial carcinoma lesions.

Immune checkpoint inhibitors (ICI) targeting PD-(L)1 are effective in select patients with advanced urothelial carcinoma (UC). High PD-L1 expression enriches for response to ICIs; however, the predictive value of PD-L1 expression is limited, which may be due in part to dynamic expression of PD-L1 in the tumor environment. We sought to characterize PD-L1 expression in primary UC and paired metastatic lesions to gain insight into the potential discordance of tumor PD-L1 expression during the metastatic process. MATERIALS AND METHODS: Immunohistochemical staining for PD-L1 using the SP-142 antibody was performed on primary tumors and matched metastatic specimens in 77 evaluable subjects with advanced UC. Immunohistochemical staining was scored for the percentage of cells positive (<5%, ≥5%) in tumor cell (TC) and immune cell (IC) compartments. Correlation of PD-L1 expression in TCs and ICs was estimated using Spearman's correlation coefficients (rho, ρ). Cohen's kappa statistics (κ) were utilized to assess the agreement in PD-L1 expression between groups. RESULTS: High (≥5%) PD-L1 expression in primary and metastatic biopsies, respectively, was observed in 6.0% and 7.7% of TCs and in 14.5% and 11.5% of ICs. IC PD-L1 expression in primary tumors was not correlated with IC PD-L1 expression in paired metastatic lesions (ρ = 0.05, P = 0.67) and there was poor agreement in high expression rates between primary and metastatic lesions in the IC compartment (κ= 0.086). CONCLUSION: High PD-L1 IC expression is temporally and spatially discordant between primary and metastatic UC lesions. Future studies of PD-(L)1 targeted therapies in patients with metastatic UC may benefit from use of fresh biopsies of metastatic lesions to define PD-L1 expression when feasible.

Patient HLA class I genotype influences cancer response to checkpoint blockade immunotherapy.

CD8+ T cell-dependent killing of cancer cells requires efficient presentation of tumor antigens by human leukocyte antigen class I (HLA-I) molecules. However, the extent to which patient-specific HLA-I genotype influences response to anti-programmed cell death protein 1 or anti-cytotoxic T lymphocyte-associated protein 4 is currently unknown. We determined the HLA-I genotype of 1535 advanced cancer patients treated with immune checkpoint blockade (ICB). Maximal heterozygosity at HLA-I loci ("A," "B," and "C") improved overall survival after ICB compared with patients who were homozygous for at least one HLA locus. In two independent melanoma cohorts, patients with the HLA-B44 supertype had extended survival, whereas the HLA-B62 supertype (including HLA-B*15:01) or somatic loss of heterozygosity at HLA-I was associated with poor outcome. Molecular dynamics simulations of HLA-B*15:01 revealed different elements that may impair CD8+ T cell recognition of neoantigens. Our results have important implications for predicting response to ICB and for the design of neoantigen-based therapeutic vaccines.