Eugene J. Pietzak

Next-generation Sequencing of Nonmuscle Invasive Bladder Cancer Reveals Potential Biomarkers and Rational Therapeutic Targets

BACKGROUND Molecular characterization of nonmuscle invasive bladder cancer (NMIBC) may provide a biologic rationale for treatment response and novel therapeutic strategies. OBJECTIVE To identify genetic alterations with potential clinical implications in NMIBC. DESIGN, SETTING, AND PARTICIPANTS Pretreatment index tumors and matched germline DNA from 105 patients with NMIBC on a prospective Institutional Review Board-approved protocol underwent targeted exon sequencing analysis in a Clinical Laboratory Improvement Amendments-certified clinical laboratory. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Comutation patterns and copy number alterations were compared across stage and grade. Associations between genomic alterations and recurrence after intravesical bacillus Calmette-Guérin (BCG) were estimated using Kaplan-Meier and Cox regression analyses. RESULTS AND LIMITATIONS TERT promoter mutations (73%) and chromatin-modifying gene alterations (69%) were highly prevalent across grade and stage, suggesting these events occur early in tumorigenesis. ERBB2 or FGFR3 alterations were present in 57% of high-grade NMIBC tumors in a mutually exclusive pattern. DNA damage repair (DDR) gene alterations were seen in 30% (25/82) of high-grade NMIBC tumors, a rate similar to MIBC, and were associated with a higher mutational burden compared with tumors with intact DDR genes (p<0.001). ARID1A mutations were associated with an increased risk of recurrence after BCG (hazard ratio=3.14, 95% confidence interval: 1.51-6.51, p=0.002). CONCLUSIONS Next-generation sequencing of treatment-naive index NMIBC tumors demonstrated that the majority of NMIBC tumors had at least one potentially actionable alteration that could serve as a target in rationally designed trials of intravesical or systemic therapy. DDR gene alterations were frequent in high-grade NMIBC and were associated with increased mutational load, which may have therapeutic implications for BCG immunotherapy and ongoing trials of systemic checkpoint inhibitors. ARID1A mutations were associated with an increased risk of recurrence after BCG therapy. Whether ARID1A mutations represent a predictive biomarker of BCG response or are prognostic in NMIBC patients warrants further investigation. PATIENT SUMMARY Analysis of frequently mutated genes in superficial bladder cancer suggests potential targets for personalized treatment and predictors of treatment response, and also may help develop noninvasive tumor detection tests.

Genetic Determinants of Cisplatin Resistance in Patients With Advanced Germ Cell Tumors

Purpose Owing to its exquisite chemotherapy sensitivity, most patients with metastatic germ cell tumors (GCTs) are cured with cisplatin-based chemotherapy. However, up to 30% of patients with advanced GCT exhibit cisplatin resistance, which requires intensive salvage treatment, and have a 50% risk of cancer-related death. To identify a genetic basis for cisplatin resistance, we performed whole-exome and targeted sequencing of cisplatin-sensitive and cisplatin-resistant GCTs. Methods Men with GCT who received a cisplatin-containing chemotherapy regimen and had available tumor tissue were eligible to participate in this study. Whole-exome sequencing or targeted exon-capture-based sequencing was performed on 180 tumors. Patients were categorized as cisplatin sensitive or cisplatin resistant by using a combination of postchemotherapy parameters, including serum tumor marker levels, radiology, and pathology at surgical resection of residual disease. Results TP53 alterations were present exclusively in cisplatin-resistant tumors and were particularly prevalent among primary mediastinal nonseminomas (72%). TP53 pathway alterations including MDM2 amplifications were more common among patients with adverse clinical features, categorized as poor risk according to the International Germ Cell Cancer Collaborative Group (IGCCCG) model. Despite this association, TP53 and MDM2 alterations predicted adverse prognosis independent of the IGCCCG model. Actionable alterations, including novel RAC1 mutations, were detected in 55% of cisplatin-resistant GCTs. Conclusion In GCT, TP53 and MDM2 alterations were associated with cisplatin resistance and inferior outcomes, independent of the IGCCCG model. The finding of frequent TP53 alterations among mediastinal primary nonseminomas may explain the more frequent chemoresistance observed with this tumor subtype. A substantial portion of cisplatin-resistant GCTs harbor actionable alterations, which might respond to targeted therapies. Genomic profiling of patients with advanced GCT could improve current risk stratification and identify novel therapeutic approaches for patients with cisplatin-resistant disease.

Neoadjuvant Treatment of High-Risk, Clinically Localized Prostate Cancer Prior to Radical Prostatectomy

Multimodal strategies combining local and systemic therapy offer the greatest chance of cure for many with men with high-risk prostate cancer who may harbor occult metastatic disease. However, no systemic therapy combined with radical prostatectomy has proven beneficial. This was in part due to a lack of effective systemic agents; however, there have been several advancements in the metastatic and castrate-resistant prostate cancer that might prove beneficial if given earlier in the natural history of the disease. For example, novel hormonal agents have recently been approved for castration-resistant prostate cancer with some early phase II neoadjuvant showing promise. Additionally, combination therapy with docetaxel-based chemohormonal has demonstrated a profound survival benefit in metastatic hormone-naïve patients and might have a role in eliminating pre-existing ADT-resistant tumor cells in the neoadjuvant setting. The Cancer and Leukemia Group B (CALGB)/Alliance 90203 trial has finished accrual and should answer the question as to whether neoadjuvant docetaxel-based chemohormonal therapy provides an advantage over prostatectomy alone. There are also several promising targeted agents and immunotherapies under investigation in phase I/II trials with the potential to provide benefit in the neoadjuvant setting.

Prognostic Value of TERT Alterations, Mutational and Copy Number Alterations Burden in Urothelial Carcinoma

Point mutations in the TERT gene promoter occur at high frequency in multiple cancers, including urothelial carcinoma (UC). However, the relationship between TERT promoter mutations and UC patient outcomes is unclear due to conflicting reports in the literature. In this study, we examined the association of TERT alterations, tumor mutational burden per megabase (Mb), and copy number alteration (CNA) burden with clinical parameters and their prognostic value in a cohort of 398 urothelial tumors. The majority of TERT mutations were located at two promoter region hotspots (chromosome 5, 1 295 228 C>T and 1 295 250 C>T). TERT alterations were more frequently present in bladder tumors than in upper tract tumors (73% vs 53%; p=0.001). ARID1A, PIK3CA, RB1, ERCC2, ERBB2, TSC1, CDKN1A, CDKN2A, CDKN2B, and PTPRD alterations showed significant co-occurrence with TERT alterations (all p<0.0025). TERT alterations and the mutational burden/Mb were independently associated with overall survival (hazard ratio[HR] 2.31, 95% confidence interval [CI] 1.46-3.65; p<0.001; and HR 0.96, 95% CI 0.93-0.99; p=0.002), disease-specific survival (HR 2.23, 95% CI 1.41-3.53; p<0.001; and HR 0.96, 95% CI 0.93-0.99; p=0.002), and metastasis-free survival (HR 1.63, 95% CI 1.05-2.53; p=0.029; and HR 0.98, 95% CI 0.96-1.00; p=0.063) in multivariate models. PATIENT SUMMARY: The majority of TERT gene mutations that we detected in urothelial carcinoma are located at two promoter hotspots. Urothelial tumors with TERT alterations had worse prognosis compared to tumors without TERT alterations, whereas tumors with a higher mutational burden had more favorable outcome compared to tumors with low mutational burden.

Incidence and Effect of Thromboembolic Events in Radical Cystectomy Patients Undergoing Preoperative Chemotherapy for Muscle-invasive Bladder Cancer.

Background We evaluated the incidence and effect of thromboembolic events (TEEs) in patients with muscle‐invasive bladder cancer treated with preoperative chemotherapy (POC) and radical cystectomy (RC) with pelvic lymph node dissection (PLND). Patients and Methods We performed a retrospective review of all patients who had undergone POC followed by RC plus PLND for muscle‐invasive bladder cancer from June 2000 to January 2013 (n = 357). The chemotherapy type (neoadjuvant vs. induction), incidence and timing of TEE diagnosis (preoperatively vs. ≤ 90 days postoperatively), and effect of TEEs on clinical outcomes were recorded. Results Overall, 79 patients (22%; 95% confidence interval [CI], 18%‐27%) experienced a TEE: 57 (16%) occurred during POC and 22 (6.2%) were diagnosed postoperatively. Forty patients (11%; 95% CI, 8.1%‐15%) required an inferior vena cava filter. We found no significant differences in neoadjuvant versus induction chemotherapy and the risk of TEEs (difference, 3.3%; 95% CI, −5% to 12%; P = .5). No significant difference were found in the rates of POC completion according to the presence of a TEE (difference, 1.0%; 95% CI, −11% to 13%; P = .9). The occurrence of TEE did not significantly affect other perioperative outcomes. The risk of recurrence and overall survival were not associated with TEE on multivariable analysis. Conclusion We found a high incidence of TEEs (22%) in patients undergoing POC before RC plus PLND, with a 16% incidence in the preoperative period. TEEs in the POC setting leads to invasive procedures; however, we did not find a significant effect on POC completion or postoperative complication risk. Further research is required to determine whether preventative TEE measures during POC can improve clinical outcomes. Micro‐Abstract We hypothesized that the incidence of thromboembolic events (TEEs) in patients receiving preoperative chemotherapy (POC) before radical cystectomy and pelvic lymph node dissection might be severely underappreciated given the association between cisplatin and TEEs. We conducted a retrospective review of 357 consecutive patients who had received POC at our institution and provide a detailed review of the incidence and timing of the TEEs. The overall TEE rate was 22%, with a 16% incidence in the preoperative setting. Forty patients (11.2%) required an inferior vena cava filter. The occurrence of TEEs did not significantly affect other perioperative outcomes, including the risk of recurrence and overall survival.

Genomic Profile of Urothelial Carcinoma of the Upper Tract from Ureteroscopic Biopsy: Feasibility and Validation Using Matched Radical Nephroureterectomy Specimens

Urothelial carcinoma of the upper tract (UTUC) presents specific challenges regarding accurate staging and tumor sampling. We aimed to assess the feasibility of applying next-generation sequencing to biopsy specimens and gauged the concordance of their genetic profiles with matched radical nephroureterectomy (RNU) specimens. Of the 39 biopsy specimens collected, 36 (92%) had adequate material for sequencing using a hybridization-based exon capture assay (MSK-IMPACT). The most frequently altered genes across the patient cohort were consistent with the urothelial carcinoma-associated alterations identified in a cohort of 130 RNU specimens previously sequenced at our center, including mutations in the TERT promoter (64%), hotspot activating mutations in FGFR3 (64%), and frequent mutations in chromatin remodeling genes. For 12 patients, a matching tumor sample from a subsequent RNU was sequenced. We found a high level of concordance between matched biopsy and RNU specimens, up to 92% for the likely pathogenic alterations. PATIENT SUMMARY We evaluated the feasibility of genomic characterization of tumor tissue collected at the time of ureteroscopic biopsy and found high concordance with subsequent radical nephroureterectomy specimens. Molecular characterization of urothelial carcinoma of the upper tract biopsies could guide treatment decision-making and identify high-risk patients who could benefit from neoadjuvant chemotherapy and low-risk patients who could benefit from conservative or organ-sparing strategies.

The Impact of Plasmacytoid Variant Histology on the Survival of Patients with Urothelial Carcinoma of Bladder after Radical Cystectomy

BACKGROUND The clinical significance of the plasmacytoid variant (PCV) in urothelial carcinoma (UC) is currently lacking. OBJECTIVE To compare clinical outcomes of patients with any PCV with that of patients with pure UC treated with radical cystectomy (RC). DESIGN, SETTING, AND PARTICIPANTS We identified 98 patients who had pathologically confirmed PCV UC and 1312 patients with pure UC and no variant history who underwent RC at our institution between 1995 and 2014. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Univariable and multivariable Cox regression and Cox proportional hazards regression to determine if PCV was associated with overall survival (OS). RESULTS AND LIMITATIONS Patients with PCV UC were more likely to have advanced tumor stage (p=0.001), positive lymph nodes (p=0.038), and receive neoadjuvant chemotherapy than those with pure UC (46% vs 22%, p<0.0001). The rate of positive soft tissue surgical margins was over five times greater in the PCV UC group compared with the pure UC group (21% vs 4.1%, respectively, p<0.0001). Median OS for the pure UC versus the PCV patients were 8 yr and 3.8 yr, respectively. On univariable analysis, PCV was associated with an increased risk of overall mortality (hazard ratio=1.34, 95% confidence interval: 1.02-1.78, p=0.039). However, on multivariable analysis adjusted for age, sex, neoadjuvant chemotherapy received, lymph node status, pathologic stage, and soft margin status, the association between PCV and OS was no longer significant (hazard ratio=1.06, 95% confidence interval: 0.78, 1.43, p=0.7). This retrospective study is limited by the lack of pathological reanalysis, and the impact of other concurrent mixed histology cannot be determined in this study. CONCLUSIONS Patients with PCV features have a higher disease burden at RC compared with those with pure UC. However, PCV was not an independent predictor of survival after RC on multivariable analysis, suggesting that PCV histology should not be used as an independent prognostic factor. PATIENT SUMMARY Plasmacytoid urothelial carcinoma is a rare and aggressive form of bladder cancer. Patients with plasmacytoid urothelial carcinoma had worse adverse pathologic features, but this was not associated with worse overall mortality when compared with patients with pure urothelial carcinoma.

Histologic and Oncologic Outcomes Following Liver Mass Resection With Retroperitoneal Lymph Node Dissection in Patients With Nonseminomatous Germ Cell Tumor

OBJECTIVE To evaluate the oncologic outcomes and histologic concordance of postchemotherapy residual liver mass resection with postchemotherapy retroperitoneal lymph node dissection (PC-RPLND). METHODS Retrospective review of our prospectively maintained germ cell tumor (GCT) surgical database identified patients with nonseminomatous GCT who underwent both postchemotherapy residual liver mass resection and PC-RPLND between 1990 and 2015. RESULTS A total of 36 patients were identified, of whom 29 (81%) presented with a liver mass at initial diagnosis and 17 (47%) received second-line chemotherapy before liver resection. Teratoma was found in 8 (22%) and 5 (14%) of PC-RPLND and liver resection specimens, respectively. Viable GCT was found in 5 (14%) and 4 (11%) of PC-RPLND and liver resection specimens, respectively. Histologic discordance was observed in 4 of 19 patients (21%; 95% confidence interval [CI] 6.1%-46%); in all cases, liver resection specimens contained teratoma or viable GCT while PC-RPLND revealed only fibrosis or necrosis. At 3 years after surgical intervention, the Kaplan-Meier estimated probability of cancer-specific survival was 75% (95% CI 55%-85%) and the probability of progression-free survival was 75% (95% CI 56%-87%). CONCLUSION In this contemporary cohort, clinically significant discordance was observed between the histology of metastatic liver masses and that of retroperitoneal lymph nodes. The benefit of postchemotherapy liver mass resection for patients with advanced nonseminomatous GCT is supported by favorable survival outcomes. Until more reliable predictors of postchemotherapy histology exist, complete surgical resection of all sites of residual disease should be performed whenever feasible.

Clinical Outcomes in Patients with Panurothelial Carcinoma Treated with Radical Nephroureterectomy Following Cystectomy for Metachronous Recurrence

Purpose: We report pathological, functional and oncologic outcomes in patients treated with radical nephroureterectomy following radical cystectomy. Materials and Methods: We identified patients who underwent radical cystectomy and then radical nephroureterectomy for metachronous urothelial recurrence at our institution between January 1995 and December 2014. Univariable Cox regression was used to assess the association between overall survival and age, grade, stage, lymph node metastasis and radiographic findings. Results: Of the 3,173 patients treated with radical cystectomy 64 underwent subsequent radical nephroureterectomy for metachronous urothelial recurrence. Median age at radical cystectomy was 66 years (IQR 61–74). In the 64 patients who underwent radical nephroureterectomy median time from radical cystectomy to radical nephroureterectomy was 2.7 years (IQR 1.4–4.6). Among 37 patients who underwent ureteroscopy prior to radical nephroureterectomy 29 (78%) had a positive biopsy. Radical nephroureterectomy pathology findings revealed locally advanced disease (pT3/pT4) in 39% of cases and positive node status in 11% compared with locally advanced disease in 17% and positive node status in 6% on radical cystectomy pathology findings. The post‐radical nephroureterectomy estimated glomerular filtration rate was less than 60 and less than 30 ml/minute/1.73 m2 in 96% and 40% of patients, respectively. Median overall survival after radical nephroureterectomy was 3.1 years (95% CI 2.4–4.3). Only lymph node involvement at radical nephroureterectomy was significantly associated with worse overall mortality (HR 2.73, 95% CI 1.04–7.15, p = 0.041). Conclusions: The prognosis is poor in patients with panurothelial carcinoma treated with nephroureterectomy following cystectomy with locally advanced disease in a large proportion. Renal function after these procedures diminished and almost all patients were ineligible for cisplatin based chemotherapy.