Ajjai Alva

Integrative clinical genomics of metastatic cancer

Metastasis is the primary cause of cancer-related deaths. Although The Cancer Genome Atlas has sequenced primary tumour types obtained from surgical resections, much less comprehensive molecular analysis is available from clinically acquired metastatic cancers. Here we perform whole-exome and -transcriptome sequencing of 500 adult patients with metastatic solid tumours of diverse lineage and biopsy site. The most prevalent genes somatically altered in metastatic cancer included TP53, CDKN2A, PTEN, PIK3CA, and RB1. Putative pathogenic germline variants were present in 12.2% of cases of which 75% were related to defects in DNA repair. RNA sequencing complemented DNA sequencing to identify gene fusions, pathway activation, and immune profiling. Our results show that integrative sequence analysis provides a clinically relevant, multi-dimensional view of the complex molecular landscape and microenvironment of metastatic cancers.

Circulating Tumor Cells as Potential Biomarkers in Bladder Cancer.

PURPOSE We explored the diagnostic use of circulating tumor cells in patients with neoadjuvant bladder cancer using enumeration and next generation sequencing. MATERIALS AND METHODS A total of 20 patients with bladder cancer who were eligible for cisplatin based neoadjuvant chemotherapy were enrolled in an institutional review board approved study. Subjects underwent blood draws at baseline and after 1 cycle of chemotherapy. A total of 11 patients with metastatic bladder cancer and 13 healthy donors were analyzed for comparison. Samples were enriched for circulating tumor cells using the novel IsoFlux™ System microfluidic collection device. Circulating tumor cell counts were analyzed for repeatability and compared with Food and Drug Administration cleared circulating tumor cells. Circulating tumor cells were also analyzed for mutational status using next generation sequencing. RESULTS Median circulating tumor cell counts were 13 at baseline and 5 at followup in the neoadjuvant group, 29 in the metastatic group and 2 in the healthy group. The concordance of circulating tumor cell levels, defined as low-fewer than 10, medium-11 to 30 and high-greater than 30, across replicate tubes was 100% in 15 preparations. In matched samples the IsoFlux test showed 10 or more circulating tumor cells in 4 of 9 samples (44%) while CellSearch® showed 0 of 9 (0%). At cystectomy 4 months after baseline all 3 patients (100%) with medium/high circulating tumor cell levels at baseline and followup had unfavorable pathological stage disease (T1-T4 or N+). Next generation sequencing analysis showed somatic variant detection in 4 of 8 patients using a targeted cancer panel. All 8 cases (100%) had a medium/high circulating tumor cell level with a circulating tumor cell fraction of greater than 5% purity. CONCLUSIONS This study demonstrates a potential role for circulating tumor cell assays in the management of bladder cancer. The IsoFlux method of circulating tumor cell detection shows increased sensitivity compared with CellSearch. A next generation sequencing assay is presented with sufficient sensitivity to detect genomic alterations in circulating tumor cells.

Epigenetics in Prostate Cancer

Prostate cancer (PC) is the most commonly diagnosed nonskin malignancy and the second most common cause of cancer death among men in the United States. Epigenetics is the study of heritable changes in gene expression caused by mechanisms other than changes in the underlying DNA sequences. Two common epigenetic mechanisms, DNA methylation and histone modification, have demonstrated critical roles in prostate cancer growth and metastasis. DNA hypermethylation of cytosine-guanine (CpG) rich sequence islands within gene promoter regions is widespread during neoplastic transformation of prostate cells, suggesting that treatment-induced restoration of a “normal” epigenome could be clinically beneficial. Histone modification leads to altered tumor gene function by changing chromosome structure and the level of gene transcription. The reversibility of epigenetic aberrations and restoration of tumor suppression gene function have made them attractive targets for prostate cancer treatment with modulators that demethylate DNA and inhibit histone deacetylases.

IL-4 induces proliferation in prostate cancer PC3 cells under nutrient-depletion stress through the activation of the JNK-pathway and survivin up-regulation

Interleukin (IL)‐4 plays a critical role in the regulation of immune responses and has been detected at high levels in the tumor microenvironment of cancer patients where it correlates with the grade of malignancy. The direct effect of IL‐4 on cancer cells has been associated with increased cell survival; however, its role in cancer cell proliferation and related mechanisms is still unclear. Here it was shown that in a nutrient‐depleted environment, IL‐4 induces proliferation in prostate cancer PC3 cells. In these cells, under nutrient‐depletion stress, IL‐4 activates mitogen‐activated protein kinases (MAPKs), including Erk, p38, and JNK. Using MAP‐signaling‐specific inhibitors, it was shown that IL‐4‐induced proliferation is mediated by JNK activation. In fact, JNK‐inhibitor‐V (JNKi‐V) stunted IL‐4‐mediated cell proliferation. Furthermore, it was found that IL‐4 induces survivin up‐regulation in nutrient‐depleted cancer cells. Using survivin‐short‐hairpin‐RNAs (shRNAs), it was demonstrated that in this milieu survivin expression above a threshold limit is critical to the mechanism of IL‐4‐mediated proliferation. In addition, the significance of survivin up‐regulation in a stressed environment was assessed in prostate cancer mouse xenografts. It was found that survivin knockdown decreases tumor progression in correlation with cancer cell proliferation. Furthermore, under nutrient depletion stress, IL ‐4 could induce proliferation in cancer cells from multiple origins: MDA‐MB‐231 (breast), A253 (head and neck), and SKOV‐3 (ovarian). Overall, these findings suggest that in a tumor microenvironment under stress conditions, IL‐4 triggers a simultaneous activation of the JNK‐pathway and the up‐regulation of survivin turning on a cancer proliferation mechanism. J. Cell. Biochem. 113: 1569–1580, 2012.

Genomic Profiling of Penile Squamous Cell Carcinoma Reveals New Opportunities for Targeted Therapy

Penile squamous cell carcinoma (PeSCCA) is a rare malignancy for which there are limited treatment options due to a poor understanding of the molecular alterations underlying disease development and progression. Therefore, we performed comprehensive, targeted next-generation sequencing to identify relevant somatic genomic alterations in a retrospective cohort of 60 fixed tumor samples from 43 PeSCCA cases (including 14 matched primary/metastasis pairs). We identified a median of two relevant somatic mutations and one high-level copy-number alteration per sample (range, 0-5 and 0-6, respectively). Expression of HPV and p16 was detectable in 12% and 28% of patients, respectively. Furthermore, advanced clinical stage, lack of p16 expression, and MYC and CCND1 amplifications were significantly associated with shorter time to progression or PeSCCA-specific survival. Notably, four cases harbored EGFR amplifications and one demonstrated CDK4 amplification, genes for which approved and investigational targeted therapies are available. Importantly, although paired primary tumors and lymph node metastases were largely homogeneous for relevant somatic mutations, we identified heterogeneous EGFR amplification in primary tumor/lymph node metastases in 4 of 14 cases, despite uniform EGFR protein overexpression. Likewise, activating HRAS mutations occurred in 8 of 43 cases. Taken together, we provide the first comprehensive molecular PeSCCA analysis, which offers new insight into potential precision medicine approaches for this disease, including strategies targeting EGFR.

The changing natural history of metastatic prostate cancer.

Since 1941, the understanding of prostate cancer pathogenesis and therapy has undergone a significant transformation. Rigorous translational research has identified multiple mechanisms underlying castration resistance, the fatal clinical state of the disease. Therapeutic approaches targeting these mechanisms in metastatic castration-resistant prostate cancer have now been clinically validated in the clinic including high-potency androgen signaling inhibition, novel cytotoxic chemotherapy, and bone-targeted therapies. Despite these advances, cure remains an elusive goal. The natural history of metastatic prostate cancer has evolved particularly in the last 2 decades in step with improved management of age-associated comorbidities, improved imaging, and the expansion of novel therapies, thus providing new opportunities and challenges. It is also important to note that the advent of prostate-specific antigen testing caused a stage shift in the disease spectrum, thus leading to earlier interventions and potentially positively impacting survival. The optimal sequencing and combinations of available therapies, predictive biomarkers, and better understanding of mechanisms of resistance remain high priority. Further refinement of the clinical niche for novel therapies in hormone-sensitive and castration-resistant disease through rationally designed clinical trials incorporating molecular, clinical, and imaging biomarkers and quality-of-life correlatives is of paramount importance.

SWOG S0925: A Randomized Phase II Study of Androgen Deprivation Combined With Cixutumumab Versus Androgen Deprivation Alone in Patients With New Metastatic Hormone-Sensitive Prostate Cancer

PURPOSE Cixutumumab, formerly IMC-A12, is a recombinant human monoclonal immunoglobulin G1 antibody that targets insulin-like growth factor I receptor (IGF-IR). Cixutumumab was synergistic with castration in a hormone-sensitive prostate cancer xenograft model. PATIENTS AND METHODS Patients with new metastatic prostate cancer were randomly assigned within 30 days of initiating androgen deprivation (AD) to cixutumumab added to a luteinizing hormone-releasing hormone agonist with bicalutamide versus AD alone. With 180 patients and one-sided alpha of 0.10, there would be 90% power to detect an absolute 20% difference in undetectable prostate-specific antigen (PSA; ≤ 0.2 ng/mL) rate at 28 weeks (relative risk, 1.44); this end point was previously strongly correlated with survival. Secondary end points included the proportion of patients with PSA > 4.0 ng/mL, safety and tolerability, circulating tumor cell (CTC) levels, and seven plasma IGF-IR biomarkers. Fishers exact test was used for the primary end point, and extended Mantel-Haenszel χ(2) test was used for three PSA response categories. RESULTS The trial accrued 210 eligible patients (105 randomly assigned to each arm). Patient characteristics were similar in both arms. Undetectable PSA rate was 42 (40.0%) of 105 for cixutumumab plus AD and 34 (32.3%) of 105 for AD alone (relative risk, 1.24; one-sided P = .16). Lower baseline CTCs (0 v 1 to 4 v ≥ 5/7.5 mL whole blood) were associated with higher rate of PSA response (three categories; P = .036) in 39 evaluable patients. IGF-IR biomarkers were not correlated with PSA outcome, and cixutumumab did not significantly change these biomarker levels. CONCLUSION Cixutumumab plus AD did not significantly increase the undetectable PSA rate in men with new metastatic hormone-sensitive prostate cancer. CTCs at baseline may carry prognostic value.

Efficient delivery of radical cystectomy after neoadjuvant chemotherapy for muscle-invasive bladder cancer: a multidisciplinary approach.

Cystectomy delay >90 days after a diagnosis of muscle‐invasive bladder cancer (MIBC) adversely affects pathologic stage and survival outcomes in patients who undergo primary surgery. After neoadjuvant chemotherapy (NAC), the impact of the timing of cystectomy delivery on these outcomes is uncertain. Poor communication between urologic and medical oncologists can result in cystectomy delay after systemic treatment. The authors of this report hypothesized that a delay in cystectomy delivery after NAC is associated with adverse survival outcomes.

Frequent PD-L1 expression in primary and metastatic penile squamous cell carcinoma: potential opportunities for immunotherapeutic approaches

BACKGROUND Despite aggressive multimodal therapy, locally advanced and/or metastatic penile squamous cell carcinoma (SqCC) is associated with significant morbidity and mortality, indicating a need for new therapeutic options. Given the emerging clinical utility of immunotherapeutics, we sought to assess the incidence and potential clinical significance of PD-L1 expression in penile SqCC. PATIENTS AND METHODS Using an anti-PD-L1 primary antibody (clone 5H1), immunohistochemistry was carried out on whole tumor sections from 37 patients with penile SqCC treated at our institution between 2005 and 2013. PD-L1-positive tumors were defined as those with membranous staining in ≥5% of tumor cells. Association between PD-L1 expression and clinicopathologic parameters was examined using Fishers exact test. Correlation between PD-L1 expression in primary tumors and matched metastases was assessed using the Spearman rank correlation coefficient (ρ). The difference in cancer-specific mortality between PD-L1-positive and -negative groups was examined using the log-rank test. RESULTS Twenty-three (62.2%) of 37 primary tumors were positive for PD-L1 expression, and there was strong positive correlation of PD-L1 expression in primary and metastatic samples (ρ = 0.72; 0.032 < P < 0.036). Primary tumor PD-L1 expression was significantly associated with usual type histology (P = 0.040) and regional lymph node metastasis (P = 0.024), as well as decreased cancer-specific survival (P = 0.011). CONCLUSIONS The majority of primary penile SqCC tumors express PD-L1, which is associated with high-risk clinicopathologic features and poor clinical outcome. These data provide a rational basis for further investigation of anti-PD-1 and anti-PD-L1 immunotherapeutics in patients with advanced penile SqCC.

A Phase II Trial of Neoadjuvant nab-paclitaxel, Carboplatin, and Gemcitabine (ACaG) in Patients With Locally Advanced Carcinoma of the Bladder

OBJECTIVE To assess the activity of neoadjuvant nab-paclitaxel, carboplatin, gemcitabine (ACaG) followed by cystectomy in patients with muscle-invasive urothelial carcinoma of the bladder. METHODS Patients who were candidates for cystectomy received nab-paclitaxel 260 mg/m(2) on day 1, carboplatin area under the curve 5 on day 1, and gemcitabine 800 mg/m(2) on days 1 and 8, every 21 days for 3 cycles. The first 3 patients received nab-paclitaxel 100 mg/m(2) weekly and were not included in the efficacy analysis of evaluable patients. Efficacy was assessed by the percentage of patients with pathologic complete response (pT0) at cystectomy. Progression-free and overall survival was estimated using the Kaplan-Meier methods. RESULTS Of 29 patients enrolled, 26 received the planned 3 cycles with 82 cycles overall; doses were reduced in 16 patients. Of 29 patients, nearly all patients experienced grade 3-4 neutropenia; 17 patients (58.6%) required growth factor, and 16 patients (55.2%) experienced grade 3-4 thrombocytopenia; there was 1 toxicity-related death. Nonhematological toxicity was generally tolerable. Twenty-two of 26 patients were evaluable for the primary endpoint: 6 patients (27.3%, 95% confidence interval [CI] 10.7-50.2) had pT0, 6 pTis, 1 pT1, 54.5% of patients had no residual muscle-invasive disease (<pT2N0), and 81.8% had pN0 at cystectomy. By intent-to-treat (ITT) analysis, the pT0 rate was 27.6% (95% CI 12.7-47.2). CONCLUSION Neoadjuvant nab-paclitaxel, carboplatin, gemcitabine is feasible but grade 3-4 myelotoxicity is common. Although the regimen has activity, the pT0 rate is lower than those reported with cisplatin-based regimens and did not meet the predefined threshold to support further investigation. Taxane-based regimens remain investigational for neoadjuvant therapy of bladder cancer.