Jessica Aldrich

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

Genome and Transcriptome Sequencing in Prospective Metastatic Triple-Negative Breast Cancer Uncovers Therapeutic Vulnerabilities

Triple-negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and HER-2. Thirty percent of patients recur after first-line treatment, and metastatic TNBC (mTNBC) has a poor prognosis with median survival of one year. Here, we present initial analyses of whole genome and transcriptome sequencing data from 14 prospective mTNBC. We have cataloged the collection of somatic genomic alterations in these advanced tumors, particularly those that may inform targeted therapies. Genes mutated in multiple tumors included TP53, LRP1B, HERC1, CDH5, RB1, and NF1. Notable genes involved in focal structural events were CTNNA1, PTEN, FBXW7, BRCA2, WT1, FGFR1, KRAS, HRAS, ARAF, BRAF, and PGCP. Homozygous deletion of CTNNA1 was detected in 2 of 6 African Americans. RNA sequencing revealed consistent overexpression of the FOXM1 gene when tumor gene expression was compared with nonmalignant breast samples. Using an outlier analysis of gene expression comparing one cancer with all the others, we detected expression patterns unique to each patients tumor. Integrative DNA/RNA analysis provided evidence for deregulation of mutated genes, including the monoallelic expression of TP53 mutations. Finally, molecular alterations in several cancers supported targeted therapeutic intervention on clinical trials with known inhibitors, particularly for alterations in the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. In conclusion, whole genome and transcriptome profiling of mTNBC have provided insights into somatic events occurring in this difficult to treat cancer. These genomic data have guided patients to investigational treatment trials and provide hypotheses for future trials in this irremediable cancer. Mol Cancer Ther; 12(1); 104–16. ©2012 AACR.

Alzheimer's disease is associated with altered expression of genes involved in immune response and mitochondrial processes in astrocytes

Alzheimers disease (AD) is characterized by deficits in cerebral metabolic rates of glucose in the posterior cingulate (PC) and precuneus in AD subjects, and in APOEε4 carriers, decades before the onset of measureable cognitive deficits. However, the cellular and molecular basis of this phenotype remains to be clarified. Given the roles of astrocytes in energy storage and brain immunity, we sought to characterize the transcriptome of AD PC astrocytes. Cells were laser capture microdissected from AD (n = 10) and healthy elderly control (n = 10) subjects for RNA sequencing. We generated >5.22 billion reads and compared sequencing data between controls and AD patients. We identified differentially expressed mitochondria-related genes including TRMT61B, FASTKD2, and NDUFA4L2, and using pathway and weighted gene coexpression analyses, we identified differentially expressed immune response genes. A number of these genes, including CLU, C3, and CD74, have been implicated in beta amyloid generation or clearance. These data provide key insights into astrocyte-specific contributions to AD, and we present this data set as a publicly available resource.

Extramedullary myeloma whole genome sequencing reveals novel mutations in Cereblon, proteasome subunit G2 and the glucocorticoid receptor in multi drug resistant disease.

Extramedullary disease (EMD) in Multiple Myeloma (MM) is characterized by the detection of monoclonal plasma cells outside the bone marrow niche, and is frequently associated with poor prognosis. Here we describe novel genomic events leading to drug refractory disease in a heavily pretreated 37-year-old IgG-kappa MM patient presenting with progressive, multi-drug refractory EMD. For the first time we report an acquired truncating mutation of Cereblon (CRBN) as well as point mutations in proteasome subunit G2 and the glucocorticoid receptor as an explanation for drug resistance. Initial myeloma treatment for the patient occurred over multiple years and included the immunomodulatory drugs (IMiDs) thalidomide and lenalidomide, the proteasome inhibitor bortezomib, cortiosteroids, radiation, one autologous and two allogeneic transplantations. She experienced extramedullary relapse, presenting as an extensive neck mass and smaller soft tissue nodules in the upper left triceps. The most recent therapy immediately prior to genomic sequencing was hyper-CVAD (hyperfractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone) incorporating alkylating agent cyclophosphamide with transient minor response. The patient was then enrolled in a pilot study utilizing next generation sequencing (NGS) to identify novel markers and potential therapeutic targets. Samples were acquired with patient consent in compliance with Mayo Clinic Institutional Review Board. For this study we completed array comparative genomic hybridization, whole exome, whole genome (insert = 1 kb) and RNA sequencing (RNASeq) of a biopsy taken from the neck mass to thoroughly interrogate the tumour genome of this patient. The presence of mutations of interest was evaluated by capillary sequencing in an expanded cohort of 25 CD138+ MM samples with low CRBN expression. The neck mass pathology confirmed sheets of atypical plasma cells, kappa light chain restriction, CD138+, CD20− and CD45−. Array comparative genomic hybridization revealed multiple copy number abnormalities, most notably del1(p13.2–34.2), monosomy 13 and monosomy X. Therapy subsequent to biopsy for genome sequencing was with pomalidomide and dexamethasone without response. Unfortunately, the patient succumbed to her disease in less than the 12 weeks required at the time for sequencing and data analysis. Sequencing revealed a highly disturbed genome (Figure 1) consisting of 4 somatic insertions/deletions, 38 intra-chromosomal rearrangements, and 35 translocations, including the high risk marker and initiating tumour event t(14;16). Furthermore, 271 nonsynonymous, somatic point mutations were detected in genes including KRAS, PIK3CA, ATM, and NFKB2 (Table I). Importantly, a Q99* truncating mutation as well as a R283K point mutation were observed in CRBN, that we recently demonstrated as essential for the anti-MM action of IMiDs (Zhu, et al 2011). To our knowledge this is the first documented mutation of Cereblon in a primary myeloma sample. Additional sequencing of CRBN in the expanded cohort of 25 patients revealed a synonymous mutation in only one sample. Figure 1 Circos plot depicting genome wide somatic variants, rearrangements and copy number changes derived from next generation sequencing. Numbers with circles around them indicate the following: 1) somatic single nucleotide variation (SNV), 2) location of SNV ... Table I Summary of clinically relevant single nucleotide variations We also observed in the patient biopsy a potentially clinically relevant nonsynonymous point mutation in proteasome assembly chaperone 2, PSMG2 (E171K). PSMG2 is a proteasome assembly protein involved in mammalian 20S proteasome maturation (Hirano, et al 2005). Mutations in proteasome assembly components contribute to proteasome inhibitor resistance (Keats, et al 2007), possibly explaining this patient’s bortezomib-refractory disease. Capillary sequencing of PSMG2 in our expanded cohort revealed no mutations, although exonic deletion of PSMG2 has also been reported in myeloma (Walker, et al 2012). The last nonsynonymous point mutation associated with drug resistance was identified in NR3C1 (G369A), a glucocorticoid receptor. Mutation of NR3C1 has been associated with resistance to steroid therapy (Bray and Cotton 2003), which this patient received and proved refractory. No NR3C1 mutations were identified in our expanded cohort and none have been previously reported in other myeloma genomes (Chapman, et al 2011, Walker, et al 2012). Mutations in NR3C1 have however been described in the glucocorticoid resistant MM.1R cell line (Moalli, et al 1992). Patients with low NR3C1 expression levels who received thalidomide demonstrated better progression-free survival and overall survival than those with low NR3C1 who did not receive thalidomide (Heuck, et al 2012). While these mutations suggest causality of drug-refractory disease, they do not identify pathways that can be exploited with targeted therapies. Additional mutations were observed in pathways for which targeted therapies are available. This patient had mutations in KRAS (G12C) and in ATM (T1985I), both of which affect the signalling of MEK downstream, thus making MEK a therapeutic target of interest in this patient. While there are no approved MEK inhibitors available for MM treatment, more than 100 trials are currently investigating MEK inhibitors, of which three of these trials are being conducted in MM patients (www.clinicaltrials.gov). The patient tumour also contained canonical, activating mutations in PIK3CA (E542K). Interestingly, one study demonstrated that 64% of PIK3CA mutations occur in exon 9, where codon 542 is located. Moreover, 19% of patients with PIK3CA mutations also presented with KRAS mutations, of which 9% are G12C (Janku, et al 2012), found in our patient. The PI3K pathway is vitally important as it regulates downstream targets, such as AKT and MTOR, which are responsible for cell proliferation, growth, survival and metastasis (Bartholomeusz and Gonzalez-Angulo 2012). In addition, a number of clinical trials are currently investigating PIK3 inhibitors (www.clinicaltrials.gov). In summary, this is the first description of CRBN mutations in a primary myeloma sample and furthermore of a “triple negative” MM patient possessing mutations probably contributing to resistance to all three major drug classes utilized in MM therapy. These mutations were not replicated in our validation cohort of 25 patients with low level CRBN expression and functional data have not yet been obtained, thus further investigation is necessary to better understand the mutation frequency and the functional significance of mutation in these genes. In summary, our approach utilizing comprehensive next generation sequencing not only identified mutations suggestive of the patient’s refractory disease, but also revealed unforeseen therapeutic options highlighting the importance of this technology in advancing individualized medicine.

Long insert whole genome sequencing for copy number variant and translocation detection

As next-generation sequencing continues to have an expanding presence in the clinic, the identification of the most cost-effective and robust strategy for identifying copy number changes and translocations in tumor genomes is needed. We hypothesized that performing shallow whole genome sequencing (WGS) of 900–1000-bp inserts (long insert WGS, LI-WGS) improves our ability to detect these events, compared with shallow WGS of 300–400-bp inserts. A priori analyses show that LI-WGS requires less sequencing compared with short insert WGS to achieve a target physical coverage, and that LI-WGS requires less sequence coverage to detect a heterozygous event with a power of 0.99. We thus developed an LI-WGS library preparation protocol based off of Illumina’s WGS library preparation protocol and illustrate the feasibility of performing LI-WGS. We additionally applied LI-WGS to three separate tumor/normal DNA pairs collected from patients diagnosed with different cancers to demonstrate our application of LI-WGS on actual patient samples for identification of somatic copy number alterations and translocations. With the evolution of sequencing technologies and bioinformatics analyses, we show that modifications to current approaches may improve our ability to interrogate cancer genomes.

Clinical Implementation of Integrated Genomic Profiling in Patients with Advanced Cancers

DNA focused panel sequencing has been rapidly adopted to assess therapeutic targets in advanced/refractory cancer. Integrated Genomic Profiling (IGP) utilising DNA/RNA with tumour/normal comparisons in a Clinical Laboratory Improvement Amendments (CLIA) compliant setting enables a single assay to provide: therapeutic target prioritisation, novel target discovery/application and comprehensive germline assessment. A prospective study in 35 advanced/refractory cancer patients was conducted using CLIA-compliant IGP. Feasibility was assessed by estimating time to results (TTR), prioritising/assigning putative therapeutic targets, assessing drug access, ascertaining germline alterations, and assessing patient preferences/perspectives on data use/reporting. Therapeutic targets were identified using biointelligence/pathway analyses and interpreted by a Genomic Tumour Board. Seventy-five percent of cases harboured 1–3 therapeutically targetable mutations/case (median 79 mutations of potential functional significance/case). Median time to CLIA-validated results was 116 days with CLIA-validation of targets achieved in 21/22 patients. IGP directed treatment was instituted in 13 patients utilising on/off label FDA approved drugs (n = 9), clinical trials (n = 3) and single patient IND (n = 1). Preliminary clinical efficacy was noted in five patients (two partial response, three stable disease). Although barriers to broader application exist, including the need for wider availability of therapies, IGP in a CLIA-framework is feasible and valuable in selection/prioritisation of anti-cancer therapeutic targets.

Comprehensive molecular profiling of 718 Multiple Myelomas reveals significant differences in mutation frequencies between African and European descent cases

Multiple Myeloma (MM) is a plasma cell malignancy with significantly greater incidence and mortality rates among African Americans (AA) compared to Caucasians (CA). The overall goal of this study is to elucidate differences in molecular alterations in MM as a function of self-reported race and genetic ancestry. Our study utilized somatic whole exome, RNA-sequencing, and correlated clinical data from 718 MM patients from the Multiple Myeloma Research Foundation CoMMpass study Interim Analysis 9. Somatic mutational analyses based upon self-reported race corrected for ancestry revealed significant differences in mutation frequency between groups. Of interest, BCL7A, BRWD3, and AUTS2 demonstrate significantly higher mutation frequencies among AA cases. These genes are all involved in translocations in B-cell malignancies. Moreover, we detected a significant difference in mutation frequency of TP53 and IRF4 with frequencies higher among CA cases. Our study provides rationale for interrogating diverse tumor cohorts to best understand tumor genomics across populations.

Simultaneous characterization of somatic events and HPV-18 integration in a metastatic cervical carcinoma patient using DNA and RNA sequencing.

Objective Integration of carcinogenic human papillomaviruses (HPVs) into the host genome is a significant tumorigenic factor in specific cancers including cervical carcinoma. Although major strides have been made with respect to HPV diagnosis and prevention, identification and development of efficacious treatments for cervical cancer patients remains a goal and thus requires additional detailed characterization of both somatic events and HPV integration. Given this need, the goal of this study was to use the next generation sequencing to simultaneously evaluate somatic alterations and expression changes in a patient’s cervical squamous carcinoma lesion metastatic to the lung and to detect and analyze HPV infection in the same sample. Materials and Methods We performed tumor and normal exome, tumor and normal shallow whole-genome sequencing, and RNA sequencing of the patient’s lung metastasis. Results We generated over 1.2 billion mapped reads and identified 130 somatic point mutations and indels, 21 genic translocations, 16 coding regions demonstrating copy number changes, and over 36 genes demonstrating altered expression in the tumor (corrected P < 0.05). Sequencing also revealed the HPV type 18 (HPV-18) integration in the metastasis. Using both DNA and RNA reads, we pinpointed 3 major events indicating HPV-18 integration into an intronic region of chromosome 6p25.1 in the patient’s tumor and validated these events with Sanger sequencing. This integration site has not been reported for HPV-18. Conclusions We demonstrate that DNA and RNA sequencing can be used to concurrently characterize somatic alterations and expression changes in a biopsy and delineate HPV integration at base resolution in cervical cancer. Further sequencing will allow us to better understand the molecular basis of cervical cancer pathogenesis.

Abstract CT325: Combination of the PARP inhibitor veliparib (ABT888) with irinotecan in patients with triple negative breast cancer: Preliminary activity and signature of response

Background: The nuclear enzyme PARP is essential in recognition and repair of DNA damage. Preclinical evidence suggests that PARP inhibitors work as sensitizing agents for DNA-damaging agents such as irinotecan. Veliparib is an orally bioavailable PARP 1 and 2 inhibitor. This expansion to a phase I study, which demonstrated veliparib reduces PAR levels in tumor after irinotecan exposure, was conducted to assess the safety, tolerability and preliminary anti-tumor activity of the combination of veliparib and irinotecan in triple negative breast cancer (TNBC) patients (pts), as well as to apply next generation sequencing technologies to define a signature of response. Methods: Pts were enrolled to two breast cancer cohorts: (1) TNBC, germline BRCA-mutant positive and (2) TNBC, non-BRCA mutated (wt). Eligibility included performance status 0-2; ≥ age 18; adequate bone marrow, hepatic and renal function. Cycles were 21 days. Irinotecan was given i.v. 100 mg/m2 over 90 min on Days 1 and 8. Twice daily (BID) oral dosing of 40 mg veliparib occurred Days 2-15 (Cycle 1) and Days 1-15 (subsequent cycles) followed by a 6-day rest. Tumor biopsies were collected at baseline, 4-6 hours after the first dose of irinotecan (day 1) and the combination (day 8) in cycle 1. Whole exome and transcriptome sequencing was performed using both normal and tumor tissue. Circulating tumor cells (CTC) were evaluated using the CellSearch platform. Results: 24 TNBC pts were enrolled, with 20 pts treated and evaluable for response (8 germline BRCA-mutation positive, 10 non-BRCA mutated, 2 suspected deleterious). Median age was 51 (range 31-63). Median number of prior treatments was 4 (range 1-7). Most frequent drug-related toxicities included: leukopenia (60%), neutropenia (60%), nausea (55%), diarrhea (40%), fatigue (40%), anemia (30%), and vomiting (30%). Best responses were as follows: Germline BRCA-mutant positive 7/8 PR (88%; median number of days on study = 330; range 148-594 days), 1/8 PD (12%); suspected deleterious 2/2 PD (100%); non-BRCA mutated 7/10 SD (70%; median number of days on study = 70; range 42-98 days), 3/10 PD (30%). Exploratory molecular profiling has been performed in a subset of these pts and the results will be presented. EpCAM+ CTC numbers were evaluable in 11 of 22 enrolled pts, and nuclear γH2Ax+, a pharmacodynamic biomarker of DNA damage, was identified in a fraction of CTCs from all 11 of these pts. Conclusions: Veliparib in combination with irinotecan was safe and tolerable in TNBC pts. Although the cohort in this trial is small, the preliminary response rate of 88% in pts with germline BRCA mutation is encouraging and higher than that historically reported with PARP inhibitor monotherapy in this population. Deep molecular profiling among BRCA mutant carriers will be validated in a larger, independent cohort to define potential biomarkers of response. Support: NCI U01-CA062487, NCI U01-CA062490, Komen KG120001, NCI R21-CA135572, and HHSN261200800001E. Citation Format: Patricia M. LoRusso, Sara M. Tolaney, Shukmei Wong, Ralph E. Parchment, Robert J. Kinders, Lihua Wang, Jessica Aldrich, Alice Chen, Diane Durecki, Scott A. Boerner, Tina Guthrie, Adam Bowditch, Lance K. Heilbrun, Mary Jo Pilat, David Craig, Dongpo Cai, Tracy Bell, John Carpten, Geoffrey Shapiro. Combination of the PARP inhibitor veliparib (ABT888) with irinotecan in patients with triple negative breast cancer: Preliminary activity and signature of response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT325. doi:10.1158/1538-7445.AM2015-CT325

An integrated framework for reporting clinically relevant biomarkers from paired tumor/normal genomic and transcriptomic sequencing data in support of clinical trials in personalized medicine

The ability to rapidly sequence the tumor and germline DNA of an individual holds the eventual promise of revolutionizing our ability to match targeted therapies to tumors harboring the associated genetic biomarkers. Analyzing high throughput genomic data consisting of millions of base pairs and discovering alterations in clinically actionable genes in a structured and real time manner is at the crux of personalized testing. This requires a computational architecture that can monitor and track a system within a regulated environment as terabytes of data are reduced to a small number of therapeutically relevant variants, delivered as a diagnostic laboratory developed test. These high complexity assays require data structures that enable real-time and retrospective ad-hoc analysis, with a capability of updating to keep up with the rapidly changing genomic and therapeutic options, all under a regulated environment that is relevant under both CMS and FDA depending on application. We describe a flexible computational framework that uses a paired tumor/normal sample allowing for complete analysis and reporting in approximately 24 hours, providing identification of single nucleotide changes, small insertions and deletions, chromosomal rearrangements, gene fusions and gene expression with positive predictive values over 90%. In this paper we present the challenges in integrating clinical, genomic and annotation databases to provide interpreted draft reports which we utilize within ongoing clinical research protocols. We demonstrate the need to retire from existing performance measurements of accuracy and specificity and measure metrics that are meaningful to a genomic diagnostic environment. This paper presents a three-tier infrastructure that is currently being used to analyze an individual genome and provide available therapeutic options via a clinical report. Our framework utilizes a non-relational variant-centric database that is scaleable to a large amount of data and addresses the challenges and limitations of a relational database system. Our system is continuously monitored via multiple trackers each catering differently to the diversity of users involved in this process. These trackers designed in analytics web-app framework provide status updates for an individual sample accurate to a few minutes. In this paper, we also present our outcome delivery process that is designed and delivered adhering to the standards defined by various regulation agencies involved in clinical genomic testing.