Hikmat Al-Ahmadie

Genome Sequencing Identifies a Basis for Everolimus Sensitivity

Tumor genome sequencing reveals the molecular basis of a patient’s unexpected and dramatic response to a cancer drug. Cancer drugs often induce dramatic responses in a small minority of patients. We used whole-genome sequencing to investigate the genetic basis of a durable remission of metastatic bladder cancer in a patient treated with everolimus, a drug that inhibits the mTOR (mammalian target of rapamycin) signaling pathway. Among the somatic mutations was a loss-of-function mutation in TSC1 (tuberous sclerosis complex 1), a regulator of mTOR pathway activation. Targeted sequencing revealed TSC1 mutations in about 8% of 109 additional bladder cancers examined, and TSC1 mutation correlated with everolimus sensitivity. These results demonstrate the feasibility of using whole-genome sequencing in the clinical setting to identify previously occult biomarkers of drug sensitivity that can aid in the identification of patients most likely to respond to targeted anticancer drugs.

The role of SPINK1 in ETS rearrangement-negative prostate cancers

ETS gene fusions have been characterized in a majority of prostate cancers; however, the key molecular alterations in ETS-negative cancers are unclear. Here we used an outlier meta-analysis (meta-COPA) to identify SPINK1 outlier expression exclusively in a subset of ETS rearrangement-negative cancers ( approximately 10% of total cases). We validated the mutual exclusivity of SPINK1 expression and ETS fusion status, demonstrated that SPINK1 outlier expression can be detected noninvasively in urine, and observed that SPINK1 outlier expression is an independent predictor of biochemical recurrence after resection. We identified the aggressive 22RV1 cell line as a SPINK1 outlier expression model and demonstrate that SPINK1 knockdown in 22RV1 attenuates invasion, suggesting a functional role in ETS rearrangement-negative prostate cancers.

B7-H3 and B7x are highly expressed in human prostate cancer and associated with disease spread and poor outcome

B7-H3 and B7x are recently discovered members of the B7-CD28 family thought to dampen peripheral immune responses via negative costimulation. We evaluated their potential expression in human prostate cancer using a large cohort of patients with 7 years of follow-up. We identified 823 patients with tissue available treated with radical prostatectomy between 1985 and 2003. Immunohistochemistry was performed on tissue microarray sections using anti-B7-H3 and -B7x. The percentage and intensity of immunoreactivity by tumor cells were blindly evaluated by two urological pathologists, and outcome analyses were conducted. Both B7-H3 and B7x were highly expressed; 93% and 99% of tumors had aberrant expression, respectively. The median percentage of tumor cells staining positive was 80% for each molecule. Strong intensity for B7-H3 and B7x was noted in 212 (26%) and 120 (15%) patients, respectively. Patients with strong intensity for B7-H3 and B7x were significantly more likely to have disease spread at time of surgery (P < 0.001 and P = 0.005, respectively). Additionally, patients with strong intensity for B7-H3 and B7x were at significantly increased risk of clinical cancer recurrence (P < 0.001 and P = 0.005) and cancer-specific death (P = 0.004 and P = 0.04, respectively). To our knowledge, we present the largest investigation of B7 family molecules in a human malignancy and a previously undescribed evaluation of B7x in prostate cancer. B7-H3 and B7x are abundantly expressed in prostate cancer and associated with disease spread and poor outcome. Given the proposed immune-inhibitory mechanisms of B7-H3 and B7x, these molecules represent attractive targets for therapeutic manipulation in prostate cancer.

TMPRSS2-ERG Gene Fusion Is Not Associated with Outcome in Patients Treated by Prostatectomy

A significant number of prostate cancers have been shown to have recurrent chromosomal rearrangements resulting in the fusion of the androgen-regulated TMPRSS2 promoter to a member of the ETS transcription factor family, most commonly ERG. This results in ERG overexpression, which may have a direct causal role in prostate tumorigenesis or progression. However, the clinical significance of the rearrangement is unclear, and in particular, relationship to outcome has been inconsistent in recent reports. We analyzed TMPRSS2-ERG gene rearrangement status by fluorescence in situ hybridization in 521 cases of clinically localized surgically treated prostate cancer with 95 months of median follow-up and also in 40 unmatched metastases. Forty-two percent of primary tumors and 40% of metastases had rearrangements. Eleven percent had copy number increase (CNI) of the TMPRRS2-ERG region. Rearrangement alone was associated with lower grade, but not with stage, biochemical recurrence, metastases, or death. CNI with and without rearrangement was associated with high grade and advanced stage. Further, a subgroup of cancers with CNI and rearrangement by deletion, with two or more copies of the deleted locus, tended to be more clinically aggressive. DNA index assessment revealed that the majority of tumors with CNI of TMPRSS2-ERG had generalized aneuploidy/tetraploidy in contrast to tumors without TMPRSS2-ERG CNI, which were predominantly diploid. We therefore conclude that translocation of TMPRSS2-ERG is not associated with outcome, and the aggressive clinical features associated with CNI of chromosome 21 reflect generalized aneuploidy and are not due to CNI specifically of rearranged TMPRSS2-ERG.

Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients

Tumor molecular profiling is a fundamental component of precision oncology, enabling the identification of genomic alterations in genes and pathways that can be targeted therapeutically. The existence of recurrent targetable alterations across distinct histologically defined tumor types, coupled with an expanding portfolio of molecularly targeted therapies, demands flexible and comprehensive approaches to profile clinically relevant genes across the full spectrum of cancers. We established a large-scale, prospective clinical sequencing initiative using a comprehensive assay, MSK-IMPACT, through which we have compiled tumor and matched normal sequence data from a unique cohort of more than 10,000 patients with advanced cancer and available pathological and clinical annotations. Using these data, we identified clinically relevant somatic mutations, novel noncoding alterations, and mutational signatures that were shared by common and rare tumor types. Patients were enrolled on genomically matched clinical trials at a rate of 11%. To enable discovery of novel biomarkers and deeper investigation into rare alterations and tumor types, all results are publicly accessible.

Stat3 is tyrosine-phosphorylated through the interleukin-6/glycoprotein 130/Janus kinase pathway in breast cancer

IntroductionSignal transducer and activator of transcription 3 (Stat3) is constitutively tyrosine-phosphorylated in approximately 50% of primary breast carcinomas. A number of different mechanisms responsible for Stat3 activation, including abnormal activation of receptor tyrosine kinases, Src, and Janus kinases (Jaks), have been implicated in breast cancer.MethodsWe examined six breast cancer-derived cell lines expressing high or low levels of tyrosine-phosphorylated Stat3 (pStat3) as well as primary breast cancer specimens.ResultsInhibition of Src or EGFR (epidermal growth factor receptor) tyrosine kinases had no effect on pStat3 levels, whereas pan-Jak inhibitor P6 resulted in complete abrogation of Stat3 phosphorylation and inhibition of growth. Jaks are required for cytokine signaling, and the glycoprotein 130 (gp130) receptor-associated Jaks are known mediators of Stat3 phosphorylation. Blockade of the gp130 receptor or sequestration of the interleukin-6 (IL-6) ligand led to a decrease of pStat3 levels. Conditioned media from those cell lines expressing high levels of pStat3 contained IL-6 and were capable of stimulating Stat3 phosphorylation. We examined IL-6 levels in primary breast tumors and found a positive correlation between pStat3 and IL-6 expression.ConclusionIn summary, a principal mechanism of Stat3 activation in breast cancer is through the IL-6/gp130/Jak pathway.

Prevalence and Co-Occurrence of Actionable Genomic Alterations in High-Grade Bladder Cancer

PURPOSE We sought to define the prevalence and co-occurrence of actionable genomic alterations in patients with high-grade bladder cancer to serve as a platform for therapeutic drug discovery. PATIENTS AND METHODS An integrative analysis of 97 high-grade bladder tumors was conducted to identify actionable drug targets, which are defined as genomic alterations that have been clinically validated in another cancer type (eg, BRAF mutation) or alterations for which a selective inhibitor of the target or pathway is under clinical investigation. DNA copy number alterations (CNAs) were defined by using array comparative genomic hybridization. Mutation profiling was performed by using both mass spectroscopy-based genotyping and Sanger sequencing. RESULTS Sixty-one percent of tumors harbored potentially actionable genomic alterations. A core pathway analysis of the integrated data set revealed a nonoverlapping pattern of mutations in the RTK-RAS-RAF and phosphoinositide 3-kinase/AKT/mammalian target of rapamycin pathways and regulators of G1-S cell cycle progression. Unsupervised clustering of CNAs defined two distinct classes of bladder tumors that differed in the degree of their CNA burden. Integration of mutation and copy number analyses revealed that mutations in TP53 and RB1 were significantly more common in tumors with a high CNA burden (P < .001 and P < .003, respectively). CONCLUSION High-grade bladder cancer possesses substantial genomic heterogeneity. The majority of tumors harbor potentially tractable genomic alterations that may predict for response to target-selective agents. Given the genomic diversity of bladder cancers, optimal development of target-specific agents will require pretreatment genomic characterization.

Prognostic indicators of malignancy in adrenal pheochromocytomas: clinical, histopathologic, and cell cycle/apoptosis gene expression analysis

BACKGROUND Pheochromocytomas are malignant in approximately 10% of patients. The histologic differentiation between benign and malignant tumors is difficult, the latter diagnosed by the presence of metastatic disease or recurrence. AIM To determine if postoperative histologic evaluation using the previously proposed Pheochromocytoma of the Adrenal Gland Scaled Score (PASS) and cell cycle/apoptosis markers can predict patients at risk for recurrence. METHODS Using the Memorial Sloan-Kettering Cancer Center adrenal database, we identified 48 patients with 51 resected pheochromocytomas (1987-2006). A senior endocrine pathologist, blinded to clinical outcome, reviewed the histopathologic characteristics of all cases using the PASS system. This pheochromocytoma scoring system is based on the presence of 12 different histologic parameters, including tumor necrosis, mitotic rate, tumor cell spindling, and the presence of large cell nests. In addition, we constructed a tissue microarray of all 5 malignant tumors and 41 of the benign tumors. By immunostaining of the tissue microarray, we assessed the expression of 7 different cell cycle/apoptosis-related genes (p53, Ki-67, Bcl-2, mdm-2, cyclin D1, p21, and p27). RESULTS Forty-three patients had a benign clinical course while 5 patients harbored a clinically malignant pheochromocytoma. Tumor necrosis (focal or confluent) was a particularly powerful indicator of malignancy present in 4 of 5 patients (80%) with malignant tumors, but only in 3 of 42 cases (7%) with benign neoplasms (P = .0009). The presence of a high mitotic rate (>3/10 high power fields) and tumor cell spindling significantly correlated with malignancy (P = .026 and .041, respectively). High cellularity was more often present in the malignant lesions (P = .050). There was a highly significant difference in PASS scores between benign and malignant cases (P = .0003). All malignant pheochromocytomas had a PASS score >/=6, well above the previously proposed >/=4 cutoff value. Two of the 4 patients testing positive for Ki-67 (>2% nuclear staining) had a clinically malignant course while only 3 (7%) of the 41 cases with lower Ki-67 positivity rate behaved in a malignant fashion (P = .055). Ki-67-positive tumor had a significantly higher chance of harboring tumor necrosis than Ki-67-negative neoplasms (P < .01). There was no difference in staining between benign and malignant pheochromocytomas using p53, Bcl-2, mdm-2, cyclin D1, p21, and p27. CONCLUSIONS (1) A PASS score of <4 predicted benign pheochromocytomas. (2) All malignant pheochromocytomas had a PASS score >/=6, which was significantly higher compared with the benign lesions. Patients with a PASS score >/=4 should be followed closely for recurrence. (3) p53, Bcl-2, mdm-2, cyclin D1, p21, and p27 appear to have no role in predicting the behavior of pheochromocytomas. Ki-67 may help identify those neoplasms at risk for recurrence by prompting the pathologist to look aggressively for adverse histologic features.

Phase II study of everolimus in metastatic urothelial cancer

No recent advances have been made in the treatment of patients with advanced bladder cancer and, to date, targeted therapies have not resulted in an improvement in outcome. The mammalian target of rapamycin pathway has been shown to be up‐regulated in bladder cancer and represents a rational target for therapeutic intervention. In the present phase II study of everolimus, one near‐complete response, one partial response and several minor responses suggest that everolimus possesses biological activity in a subset of patients with bladder cancer. To maximize benefit from targeted agents such as everolimus, the preselection of patients based on molecular phenotype is required.

Arginase-1: a new immunohistochemical marker of hepatocytes and hepatocellular neoplasms.

The distinction of hepatocellular carcinoma (HCC) from metastatic tumor in the liver often presents a diagnostic challenge that carries significant impact on prognostication and therapy. The number of diagnostically useful immunohistochemical markers of hepatocytes is limited to hepatocyte paraffin antigen (HepPar-1), polyclonal carcinoembryonic antigen, and CD10, with α-fetoprotein and glypican-3 labeling HCCs. Arginase-1 (Arg-1) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of arginine to ornithine and urea. We used immunohistochemistry to compare the sensitivity of Arg-1 to that of HepPar-1 in 151 HCCs. We found that the overall sensitivities of Arg-1 and HepPar-1 are 96.0% and 84.1%, respectively. The sensitivities of Arg-1 in well, moderately, and poorly differentiated HCCs are 100%, 96.2%, and 85.7%, respectively, whereas, in comparison, HepPar-1 demonstrated sensitivities of 100%, 83.0%, and 46.4% for well, moderately, and poorly differentiated tumors, respectively. There were no HCCs in our study that were reactive for HepPar-1 but nonreactive for Arg-1. We also examined Arg-1 expression in nonhepatocellular tumors, including many that are potential mimics of HCC (renal cell carcinomas, neuroendocrine tumors, melanomas, gastric adenocarcinomas, and adrenocortical carcinomas) and found that only 2 non-HCC tumors were reactive for Arg-1. Arg-1 represents a sensitive and specific marker of benign and malignant hepatocytes that may ultimately prove to be a useful diagnostic tool in routine surgical pathology practice.