Shukmei Wong

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.

Open-access synthetic spike-in mRNA-seq data for cancer gene fusions.

BackgroundOncogenic fusion genes underlie the mechanism of several common cancers. Next-generation sequencing based RNA-seq analyses have revealed an increasing number of recurrent fusions in a variety of cancers. However, absence of a publicly available gene-fusion focused RNA-seq data impedes comparative assessment and collaborative development of novel gene fusions detection algorithms. We have generated nine synthetic poly-adenylated RNA transcripts that correspond to previously reported oncogenic gene fusions. These synthetic RNAs were spiked at known molarity over a wide range into total RNA prior to construction of next-generation sequencing mRNA libraries to generate RNA-seq data.ResultsLeveraging a priori knowledge about replicates and molarity of each synthetic fusion transcript, we demonstrate utility of this dataset to compare multiple gene fusion algorithms’ detection ability. In general, more fusions are detected at higher molarity, indicating that our constructs performed as expected. However, systematic detection differences are observed based on molarity or algorithm-specific characteristics. Fusion-sequence specific detection differences indicate that for applications where specific sequences are being investigated, additional constructs may be added to provide quantitative data that is specific for the sequence of interest.ConclusionsTo our knowledge, this is the first publicly available synthetic RNA-seq data that specifically leverages known cancer gene-fusions. The proposed method of designing multiple gene-fusion constructs over a wide range of molarity allows granular performance analyses of multiple fusion-detection algorithms. The community can leverage and augment this publicly available data to further collaborative development of analytical tools and performance assessment frameworks for gene fusions from next-generation sequencing data.

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.

A somatic reference standard for cancer genome sequencing

Large-scale multiplexed identification of somatic alterations in cancer has become feasible with next generation sequencing (NGS). However, calibration of NGS somatic analysis tools has been hampered by a lack of tumor/normal reference standards. We thus performed paired PCR-free whole genome sequencing of a matched metastatic melanoma cell line (COLO829) and normal across three lineages and across separate institutions, with independent library preparations, sequencing, and analysis. We generated mean mapped coverages of 99X for COLO829 and 103X for the paired normal across three institutions. Results were combined with previously generated data allowing for comparison to a fourth lineage on earlier NGS technology. Aggregate variant detection led to the identification of consensus variants, including key events that represent hallmark mutation types including amplified BRAF V600E, a CDK2NA small deletion, a 12 kb PTEN deletion, and a dinucleotide TERT promoter substitution. Overall, common events include >35,000 point mutations, 446 small insertion/deletions, and >6,000 genes affected by copy number changes. We present this reference to the community as an initial standard for enabling quantitative evaluation of somatic mutation pipelines across institutions.

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

Enrichment of PI3K-AKT–mTOR pathway activation in hepatic metastases from breast cancer

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K–AKT–mTOR pathway is associated with specific sites of breast cancer metastasis. Experimental Design: Next-generation sequencing–based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K–AKT–mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K–AKT–mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions. Results: PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined. Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K–AKT–mTOR signaling network. Clin Cancer Res; 23(16); 4919–28. ©2017 AACR.

Abstract 3371: Aldh expressing stem cells mediate tumor initiation and metastasis in triple negative breast cancers across different ethnicities

TNBC is the only subtype of breast cancer for which there are no approved targeted therapies. In the US, its incidence is highest in women with African ancestry (AA); in western sub-Saharan Africa, single-institution studies show that TNBC constitutes 40- 80% of all breast cancers. Given the Caucasian/AA survival disparity in breast cancer, there is an urgent need to find actionable targets in TNBC of all ethnicities, but especially in TNBC in AA, which are suspected to be more aggressive. Breast cancer stem cells, the small population of cells that have been shown to mediate breast tumor initiation, metastasis, and resistance to conventional therapy have also been reported to mediate the heterogeneity of TNBC and are especially abundant in TNBC in AA women. Here, we sought to better understand the biology of TNBC by finding genes and pathways that are differentially expressed in the stem cell population of patient derived xenografts (PDX) from TNBC from Ghanaian (G), AA and Caucasian (C) women and the effect of these differentially expressed genes on the stem cell phenotype in these primary tumors. We isolated the ALDH+ and the CD44+/CD24- stem cell populations from the bulk cells from 15 PDXs using flow cytometry. We performed RNA sequencing (Illumina HiSeq platform) on the isolated populations and bulk cells (45). Comprehensive bioinformatics analyses led to the identificationof highly significantly differentially expressed genes and pathways between the cell populations. By principal component analysis, the tumors were very heterogeneous. However, the ALDH+ cells separated out from the CD44+/CD24- and the bulk cells. We identified 14 genes that were simultaneously differentially expressed between the ALDH+ vs the CD44+/CD24- as well as ALDH+ VS bulk (p-value Citation Format: Evelyn M. Jiagge, Shukmei Wong, Rabia Gilani, Sean Mcdermott, Lisa Newman, Jessica Bensenhaver, Max Wicha, John Carpten, Sofia Merajver. Aldh expressing stem cells mediate tumor initiation and metastasis in triple negative breast cancers across different ethnicities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3371. doi:10.1158/1538-7445.AM2017-3371

Abstract P2-05-21: The AKT-mTOR pathway as a potential organ-specific drug target signature of hepatic metastases from breast cancer

Background: The identification of organ-specific targetable signatures may help design more effective treatment for patients with metastatic breast cancer (MBC). We took a multi-OMIC approach to assess whether the AKT-mTOR pathway is globally activated during metastatic progression or whether it represents an organ-specific target. Methods: Snap frozen biopsies from 25 MBC patients enrolled in a prospective phase II trial were used. Sites of metastasis were classified as liver (n=8) and others (n=17), the latter including cutaneous, lung, lymph nodes, and intra-abdominal lesions. Signaling analysis of the 25 cases was performed using Reverse Phase Protein Microarray (RPPA) coupled with Laser Capture Microdissection. Activation of the AKT-mTOR pathway was quantified as phosphorylation of AKT (S473) and the mTOR target p70S6 (T389). Matched exome (WES) and RNASeq data were available for 17 of 25 patients, five with liver metastases. Sequencing data was processed using an in-house developed pipeline to identify somatic events including coding mutations, copy number alterations, gene fusions, and differential expression. Activation of the AKT-mTOR pathway and sequencing data were compared between hepatic and non-hepatic lesions using an integrated RPPA and genomic approach. Results: Among liver metastases, AKT was activated in 4 of the 8 (50.0%) patients, while 6 of the 8 cases (75.0%) showed activation of p70S6. Sequencing data revealed mutation of PIK3CA in 4 of the 5 liver metastases (80.0%). Three of the PIK3CA mutated specimens with catalytic domain mutations (codons 1023 and 147) demonstrated co-activation of AKT and p70S6, while the fourth case, containing a helical domain mutation (E542K), had activation of p70S6 only. The PIK3CA wild-type liver metastasis demonstrated low activation of AKT and p70S6. For non-hepatic metastases AKT was activated in 2 of the 17 cases (11.8%) and p70S6 in 5 of the 17 patients (29.4%). Discussion: Although these results need further validation, activation of the AKT-mTOR pathway appears to be a hepatic specific signature in MBC and could be used for the selection of targeted agents for hepatic lesions. Citation Format: Pierobon M, Wong S, Reeder A, Anthony SP, Robert NJ, Northfelt DW, Jahanzeb M, Vocila L, Wulfkuhle J, Dunetz B, Aldrich J, Byron S, Craig D, Liotta L, Petricoin EF, Carpten J. The AKT-mTOR pathway as a potential organ-specific drug target signature of hepatic metastases from breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-21.

Abstract 1518: Multiethnic triple negative breast cancer comparisons of gene expression shows enrichment of distinct pathways in ALDH1+ subpopulations compared to CD44+/CD24-/EpCAM+

Triple negative breast cancers are a heterogeneous group with diverse, usually poorer survival outcomes and are more prevalent in individuals of African descent. Given the 8-12% higher mortality in breast cancers in individuals of African descent, we set out to identify differences in pathway enrichment in patients with TNBC of Caucasian, African American, and African (Ghanaian) ethnicity. Moreover, we analyzed subpopulations of cells previously found to exhibit stem-like characteristics of self-renewal, within each tumor in order delineate the heterogeneity of gene expression patterns also in different subpopulations of cells within tumors of patients of diverse ethnicities. Based on previous work, we hypothesized that there are distinct sub populations of rare cells, which exhibit distinct signaling pathway enrichment that may confer to TNBCs their highly metastatic and drug resistant characteristics. We developed PDX models from TNBC of 5 Ghanaian, 5 African American and 5 Caucasian patients. We collected bulk cells from the PDXs and used fluorescence-activated cell sorting (FACS) to select and collect ALDH+ cells and CD24-CD44+EpCAM+ (CD44+) cells. We extracted RNA from the sorted cell populations and performed RNA-sequencing using the Illumina Next Generation Sequencing platform. Our results showed that the bulk, ALDH1+ and CD44+ populations within each tumor segregate closely together, and we noted no distinct ethnic separation when we performed the analyses either of the bulk or special subpopulations. Upon centering on the mean for each sample in each tumor, thereby abrogating differences between individual tumors, we observe a significant separation between the ALDH1+ and the CD44+/CD24- sub populations. In particular, the following pathways are most differentially enriched in these subpopulations. The numbers between parentheses indicate the numbers of genes significantly altered that contribute to each pathway. ALDH1+ : Wnt (10), MAPK (13), axon guidance (6), GnRH signaling (7), TGFbeta (9), endocytosis(5) CD44+/CD24-/EpCAM+: tRNA synthesis (5), N-glycan biosynthesis (7), RNA degradation (6) We are focusing our experiments on verifying whether the above pathways are indeed active in these subpopulations and we are exploring their biological significance with regards to tumor growth and metastases. Although we did not detect any specific clustering of tumors by ethnicity, our work suggests the presence of an ALDH1+ subpopulation of cells that exhibit characteristic enrichment of pathways distinct from those enriched in CD44+/CD24-/EpCAM+ cells. Citation Format: Evelyn M. Jiagge, Qingxuan Song, Shukmei Wong, Tahra Luther, Michele Dziubinski, Shawn Clouthier, Sean McDermott, Lisa Newman, John Carpten, Jun Li, Max WIcha, Sofia Merajver. Multiethnic triple negative breast cancer comparisons of gene expression shows enrichment of distinct pathways in ALDH1+ subpopulations compared to CD44+/CD24-/EpCAM+. [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 1518. doi:10.1158/1538-7445.AM2015-1518

Abstract 3852: Prevalent loss of BRCA1 and BRCA2 expression in African TNBC suggests their prominent role in sporadic carcinogenesis

BRCAness is a property shared by breast tumors with sporadic BRCA1 mutations or arising in patients with germline BRCA1 mutations, or tumors which down-regulate BRCA1 expression by other mechanisms. Women with African ancestry have been shown to have breast tumors that have characteristics similar to BRCA mutated tumors, but there is scant data on the signaling and regulation of the BRCA1 and BRCA 2 pathways in theses tumors. We have hypothesized that defective expression of key players in the BRCA1 and BRCA2 pathways may contribute to the BRCAness observed in sporadic breast cancers in women with African ancestry. Method: 1. Genomic and transcriptomic studies using Next Generation Sequencing technologies of normal DNA extracted from saliva and tumor DNA and RNA, extracted from frozen tumors were performed on four triple negative breast cancer (TNBC) specimens from Ghanaian women. Whole exome sequencing and RNA-sequencing (RNA-seq) were completed on all patients. 2. Immunohistochemistry staining for BRCA1, BRCA2 and PALB2 protein was completed for 49 Ghanaian TNBC tumor samples. IHC staining was also performed on some of the tumors that were sequenced. IHC staining was performed on PDX tumors derived from Ghanaian TNBC. 3. Generation of patient derived xenografts (PDX) models of TNBC from Ghanaian breast cancer patients for sequencing, q and standard PCR, and Western blot to determine the signaling of BRCA1 and BRCA2 pathway in Ghanaian TNBCs. Results: We discovered novel BRCA2 nonsense variant that will likely result in truncation or no expression. This tumor did not show BRCA2 expression on IHC. All the patients who had previously reported BRCA1 and BCRA2 variants had no expression of the proteins on IHC. Of the 49 Ghanaian TNBC tumors that were stained for BRCA1 and BRCA2, half did not express either BRCA1 or BRCA2, 10% had no expression of BRCA1 alone and none had loss of BRCA2 expression alone. Studies are ongoing on the PDX models to identify the expression of members of the BRCA1 and BRCA2 pathways in TNBC of women with African ancestry. Conclusion: A large proportion of TNBCs from African women have loss of expression of BRCA1 and BRCA2. Identifying the signaling alterations in the BRCA1 and BRCA2 pathways may help to identify novel drug targets for TNBC or may help predict response to PARP inhibitors or related drugs. IHC is an important and relatively inexpensive method for determining patients with BRCA pathway alteration who my benefit from DNA repair targeted therapy. This will be very beneficial for women in Africa who do not have access to BCRA germline testing. Citation Format: Evelyn M. Jiagge, Shukmei Wong, Gabriel Lupu, Mu Qiao, Michele Dziubinski, Lisa A. Newman, John Carpten, Max Wicha, Sofia D. Merajver. Prevalent loss of BRCA1 and BRCA2 expression in African TNBC suggests their prominent role in sporadic carcinogenesis. [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 3852. doi:10.1158/1538-7445.AM2015-3852