Sameek Roychowdhury

Personalized Oncology Through Integrative High-Throughput Sequencing: A Pilot Study

The mutations present in advanced cancers can be identified by integrative high-throughput sequencing to enable biomarker-driven clinical trials and, ultimately, treatment. First Steps to Personalized Cancer Treatment In an optimistic vision of personalized medicine, each cancer patient is treated with drugs tailored for their particular tumor. This sounds appealing, but is it even possible? Roychowdhury and his colleagues tested this approach by extensively characterizing cancers in several patients and then convening a Sequencing Tumor Board of experts to determine the appropriate treatment. With a combination of whole genome and exome sequencing plus sequencing of transcribed RNA, the authors were able to find informative mutations within 3 to 4 weeks, a short enough time to be useful clinically. To verify that their sequencing strategy would work before testing it on actual patients, they assessed two xenografts established from patients with metastatic prostate cancer. They found that one of these carried the common prostate cancer–specific gene fusion of TMPRSS2 and ERG and another, previously undescribed, gene fusion. Also, the androgen receptor gene was amplified and two tumor suppressors were inactivated. The Board concluded that this pattern of mutations could in theory be treated by combined block of the PI3K and androgen receptor signaling pathways. The authors then turned to an actual patient, a 46 year old with colorectal cancer, who had been unsuccessfully treated. Characterization of his metastatic tumor showed mutations in the oncogene NRAS, the tumor suppressor TP53, aurora kinase A, a myosin heavy chain and the FAS death receptor, plus amplification of CDK8. Of these, the Sequencing Tumor Board concluded that the NRAS and CDK8 aberrations could potentially be matched to clinical trials, although none were available at the time. Similar analysis of another patient with metastatic melanoma revealed a structural rearrangement in CDKN2C and HRas. Although the HRAS mutation has not been described before in melanoma, the Sequencing Tumor Board suggested that combined treatment with PI3K and MEK inhibitors would be suitable for this patient. The good news resulting from these studies was that the patients’ tumors were analyzed with in 24 days for ~

Activating ESR1 mutations in hormone-resistant metastatic breast cancer

3600, well within the cost of routine clinical tests. But aspects need improvement: Additional testing for epigenetic and small RNA variants will allow more informative characterization. Sequencing at higher depth or enrichment methods will be needed for tumors of lower purity. And perhaps most important, we need a broader array of clinical trials, as highlighted by the fact that none was available for these two patients. Individual cancers harbor a set of genetic aberrations that can be informative for identifying rational therapies currently available or in clinical trials. We implemented a pilot study to explore the practical challenges of applying high-throughput sequencing in clinical oncology. We enrolled patients with advanced or refractory cancer who were eligible for clinical trials. For each patient, we performed whole-genome sequencing of the tumor, targeted whole-exome sequencing of tumor and normal DNA, and transcriptome sequencing (RNA-Seq) of the tumor to identify potentially informative mutations in a clinically relevant time frame of 3 to 4 weeks. With this approach, we detected several classes of cancer mutations including structural rearrangements, copy number alterations, point mutations, and gene expression alterations. A multidisciplinary Sequencing Tumor Board (STB) deliberated on the clinical interpretation of the sequencing results obtained. We tested our sequencing strategy on human prostate cancer xenografts. Next, we enrolled two patients into the clinical protocol and were able to review the results at our STB within 24 days of biopsy. The first patient had metastatic colorectal cancer in which we identified somatic point mutations in NRAS, TP53, AURKA, FAS, and MYH11, plus amplification and overexpression of cyclin-dependent kinase 8 (CDK8). The second patient had malignant melanoma, in which we identified a somatic point mutation in HRAS and a structural rearrangement affecting CDKN2C. The STB identified the CDK8 amplification and Ras mutation as providing a rationale for clinical trials with CDK inhibitors or MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase) and PI3K (phosphatidylinositol 3-kinase) inhibitors, respectively. Integrative high-throughput sequencing of patients with advanced cancer generates a comprehensive, individual mutational landscape to facilitate biomarker-driven clinical trials in oncology.

Identification of targetable FGFR gene fusions in diverse cancers.

Breast cancer is the most prevalent cancer in women, and over two-thirds of cases express estrogen receptor-α (ER-α, encoded by ESR1). Through a prospective clinical sequencing program for advanced cancers, we enrolled 11 patients with ER-positive metastatic breast cancer. Whole-exome and transcriptome analysis showed that six cases harbored mutations of ESR1 affecting its ligand-binding domain (LBD), all of whom had been treated with anti-estrogens and estrogen deprivation therapies. A survey of The Cancer Genome Atlas (TCGA) identified four endometrial cancers with similar mutations of ESR1. The five new LBD-localized ESR1 mutations identified here (encoding p.Leu536Gln, p.Tyr537Ser, p.Tyr537Cys, p.Tyr537Asn and p.Asp538Gly) were shown to result in constitutive activity and continued responsiveness to anti-estrogen therapies in vitro. Taken together, these studies suggest that activating mutations in ESR1 are a key mechanism in acquired endocrine resistance in breast cancer therapy.

Identification of recurrent NAB2-STAT6 gene fusions in solitary fibrous tumor by integrative sequencing

Through a prospective clinical sequencing program for advanced cancers, four index cases were identified which harbor gene rearrangements of FGFR2, including patients with cholangiocarcinoma, breast cancer, and prostate cancer. After extending our assessment of FGFR rearrangements across multiple tumor cohorts, we identified additional FGFR fusions with intact kinase domains in lung squamous cell cancer, bladder cancer, thyroid cancer, oral cancer, glioblastoma, and head and neck squamous cell cancer. All FGFR fusion partners tested exhibit oligomerization capability, suggesting a shared mode of kinase activation. Overexpression of FGFR fusion proteins induced cell proliferation. Two bladder cancer cell lines that harbor FGFR3 fusion proteins exhibited enhanced susceptibility to pharmacologic inhibition in vitro and in vivo. Because of the combinatorial possibilities of FGFR family fusion to a variety of oligomerization partners, clinical sequencing efforts, which incorporate transcriptome analysis for gene fusions, are poised to identify rare, targetable FGFR fusions across diverse cancer types.

Implementing personalized cancer genomics in clinical trials.

A 44-year old woman with recurrent solitary fibrous tumor (SFT)/hemangiopericytoma was enrolled in a clinical sequencing program including whole-exome and transcriptome sequencing. A gene fusion of the transcriptional repressor NAB2 with the transcriptional activator STAT6 was detected. Transcriptome sequencing of 27 additional SFTs identified the presence of a NAB2-STAT6 gene fusion in all tumors. Using RT-PCR and sequencing, we detected this fusion in all 51 SFTs, indicating high levels of recurrence. Expression of NAB2-STAT6 fusion proteins was confirmed in SFT, and the predicted fusion products harbor the early growth response (EGR)-binding domain of NAB2 fused to the activation domain of STAT6. Overexpression of the NAB2-STAT6 gene fusion induced proliferation in cultured cells and activated the expression of EGR-responsive genes. These studies establish NAB2-STAT6 as the defining driver mutation of SFT and provide an example of how neoplasia can be initiated by converting a transcriptional repressor of mitogenic pathways into a transcriptional activator.

Integrative Clinical Sequencing in the Management of Refractory or Relapsed Cancer in Youth

The recent surge in high-throughput sequencing of cancer genomes has supported an expanding molecular classification of cancer. These studies have identified putative predictive biomarkers signifying aberrant oncogene pathway activation and may provide a rationale for matching patients with molecularly targeted therapies in clinical trials. Here, we discuss some of the challenges of adapting these data for rare cancers or molecular subsets of certain cancers, which will require aligning the availability of investigational agents, rapid turnaround of clinical grade sequencing, molecular eligibility and reconsidering clinical trial design and end points.

Combination immunotherapy of B-cell non-Hodgkin's lymphoma with rituximab and interleukin-2: a preclinical and phase I study.

IMPORTANCE Cancer is caused by a diverse array of somatic and germline genomic aberrations. Advances in genomic sequencing technologies have improved the ability to detect these molecular aberrations with greater sensitivity. However, integrating them into clinical management in an individualized manner has proven challenging. OBJECTIVE To evaluate the use of integrative clinical sequencing and genetic counseling in the assessment and treatment of children and young adults with cancer. DESIGN, SETTING, AND PARTICIPANTS Single-site, observational, consecutive case series (May 2012-October 2014) involving 102 children and young adults (mean age, 10.6 years; median age, 11.5 years, range, 0-22 years) with relapsed, refractory, or rare cancer. EXPOSURES Participants underwent integrative clinical exome (tumor and germline DNA) and transcriptome (tumor RNA) sequencing and genetic counseling. Results were discussed by a precision medicine tumor board, which made recommendations to families and their physicians. MAIN OUTCOMES AND MEASURES Proportion of patients with potentially actionable findings, results of clinical actions based on integrative clinical sequencing, and estimated proportion of patients or their families at risk of future cancer. RESULTS Of the 104 screened patients, 102 enrolled with 91 (89%) having adequate tumor tissue to complete sequencing. Only the 91 patients were included in all calculations, including 28 (31%) with hematological malignancies and 63 (69%) with solid tumors. Forty-two patients (46%) had actionable findings that changed their cancer management: 15 of 28 (54%) with hematological malignancies and 27 of 63 (43%) with solid tumors. Individualized actions were taken in 23 of the 91 (25%) based on actionable integrative clinical sequencing findings, including change in treatment for 14 patients (15%) and genetic counseling for future risk for 9 patients (10%). Nine of 91 (10%) of the personalized clinical interventions resulted in ongoing partial clinical remission of 8 to 16 months or helped sustain complete clinical remission of 6 to 21 months. All 9 patients and families with actionable incidental genetic findings agreed to genetic counseling and screening. CONCLUSIONS AND RELEVANCE In this single-center case series involving young patients with relapsed or refractory cancer, incorporation of integrative clinical sequencing data into clinical management was feasible, revealed potentially actionable findings in 46% of patients, and was associated with change in treatment and family genetic counseling for a small proportion of patients. The lack of a control group limited assessing whether better clinical outcomes resulted from this approach than outcomes that would have occurred with standard care.

Advancing Precision Medicine for Prostate Cancer Through Genomics

Purpose: Cytokine-induced modulation of innate immunity is being explored to enhance the activity of monoclonal antibodies. Severe combined immunodeficient (SCID) mice engrafted with peripheral blood leukocytes (PBLs) from Epstein Barr virus-seropositive donors develop human B-cell non-Hodgkin’s lymphomas [B-NHLs (hu-PBL-SCID mouse model)]. We used this hu-PBL-SCID mouse model to study the synergism between interleukin (IL)-2 and rituximab. We also conducted a phase I trial of IL-2 and rituximab in relapsed B-NHL to study whether expansion of natural killer (NK) cells and enhanced cellular cytotoxicity could be safely accomplished in vivo. Experimental Design: Hu-PBL-SCID mice were treated with various schedules of rituximab and IL-2, with survival as the end point. Patients with relapsed B-NHL received rituximab (375 mg/m2 weekly × 4) followed by daily low-dose IL-2 (1 MIU/m2/day × 4 weeks) with pulses of intermediate-dose IL-2 (3–15 MIU/m2). Toxicity, NK cell numbers, and cellular cytotoxicity were measured. Results: In the hu-PBL-SCID mouse, the combination of rituximab and IL-2 showed greater activity against B-NHL than either agent alone. Treatment was most effective when IL-2 was given before rituximab. Twelve patients with heavily pretreated B-NHL entered the phase I trial. Toxicity was manageable, and responses were observed. NK cell expansion and enhanced cellular cytotoxicity against a B-cell lymphoma target were observed but did not correlate with response. Conclusions: The combination of IL-2 and rituximab is synergistic against B-NHL in the hu-PBL-SCID model. In the phase I trial, a sequential combination of rituximab and IL-2 was well tolerated and achieved biological end points. Responses were observed.

Failed adoptive immunotherapy with tumor-specific T cells: reversal with low-dose interleukin 15 but not low-dose interleukin 2.

Prostate cancer is the most common type of cancer in men and the second leading cause of cancer death in men in the United States. The recent surge of high-throughput sequencing of cancer genomes has supported an expanding molecular classification of prostate cancer. Translation of these basic science studies into clinically valuable biomarkers for diagnosis and prognosis and biomarkers that are predictive for therapy is critical to the development of precision medicine in prostate cancer. We review potential applications aimed at improving screening specificity in prostate cancer and differentiating aggressive versus indolent prostate cancers. Furthermore, we review predictive biomarker candidates involving ETS gene rearrangements, PTEN inactivation, and androgen receptor signaling. These and other putative biomarkers may signify aberrant oncogene pathway activation and provide a rationale for matching patients with molecularly targeted therapies in clinical trials. Lastly, we advocate innovations for clinical trial design to incorporate tumor biopsy and molecular characterization to develop biomarkers and understand mechanisms of resistance.

Translating cancer genomes and transcriptomes for precision oncology

Adoptive immunotherapy with tumor-specific T cells has emerged as a valid approach for prevention or treatment of diseases, such as melanoma and EBV-associated lymphoma. As interleukin (IL) 15 promotes survival of CD8+ memory CTLs, we hypothesized that it could be used to enhance antitumor immunity in vivo through the maintenance of adoptively transferred memory CTL. To test this, we treated mice bearing P1A+ tumors with adoptively transferred T cells possessing a transgenic Vα8+ T-cell receptor specific for the P1A tumor antigen (called P1CTL). Mice were then randomized to receive daily low-dose IL-15 (0.5 μg/day) or PBS. Mice receiving the transgenic P1CTL and IL-15 experienced a significantly delayed tumor relapse or complete tumor regression (P < 0.002 compared with PBS), with a striking persistence of the CD8+ Vα8+ P1CTL compared with mice receiving the CD8+ Vα8+ P1CTL and PBS vehicle (26.3 versus 5.1% P < 10−5). Animals exhibiting complete tumor regression had a significant population of CD8+ Vα8+ P1CTL (46%) that persisted with IL-15 treatment until 140 days after adoptive transfer and successfully defended them against tumor rechallenge without IL-15. Low-dose IL-2 afforded no protection over vehicle and resulted in lower percentages of T cells with an activated memory phenotype, lower Bcl-2 expression, and lower ex vivo antitumor cytotoxicity compared with mice treated with IL-15. Collectively, the data support the notion that exogenous low-dose IL-15 therapy can enhance and even reverse the limited efficacy of adoptively transferred tumor-specific T-cell therapy and may do so in a fashion that is superior and distinct from exogenous IL-2 therapy.