Malasi, Smriti

Abstract Adenosquamous cancer of the pancreas (ASCP) is a subtype of pancreatic cancer that has a worse prognosis and greater metastatic potential than the more common pancreatic ductal adenocarcinoma (PDAC) subtype. To distinguish the genomic landscape of ASCP and identify actionable targets for this lethal cancer, we applied DNA content flow cytometry to a series of 15 tumor samples including five patient-derived xenografts (PDX). We interrogated purified sorted tumor fractions from these samples with whole-genome copy-number variant (CNV), whole-exome sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analyses. These identified a variety of somatic genomic lesions targeting chromatin regulators in ASCP genomes that were superimposed on well-characterized genomic lesions including mutations in TP53 (87%) and KRAS (73%), amplification of MYC (47%), and homozygous deletion of CDKN2A (40%) that are common in PDACs. Furthermore, a comparison of ATAC-seq profiles of three ASCP and three PDAC genomes using flow-sorted PDX models identified genes with accessible chromatin unique to the ASCP genomes, including the lysine methyltransferase SMYD2 and the pancreatic cancer stem cell regulator RORC in all three ASCPs, and a FGFR1-ERLIN2 fusion associated with focal CNVs in both genes in a single ASCP. Finally, we demonstrate significant activity of a pan FGFR inhibitor against organoids derived from the FGFR1-ERLIN2 fusion–positive ASCP PDX model. Our results suggest that the genomic and epigenomic landscape of ASCP provide new strategies for targeting this aggressive subtype of pancreatic cancer. Significance: These data provide a unique description of the ASCP genomic and epigenomic landscape and identify candidate therapeutic targets for this dismal cancer.

Abstract Background: Amplification of chromosome 9p24.1 targeting PD-L1, PD-L2, and JAK2 (PDJ amplicon) is present in ~15% of TNBCs and is associated with activation of the JAK/STAT pathway and poor clinical outcomes. In preclinical studies, PD-L1 expression is markedly and rapidly inducible by low-dose IFN-γ in a PDJ amplicon copy-number dependent manner, mimicking an in situ inflammatory response. This suggests PDJ amplification could identify those TNBCs that would respond to ICIs and/or JAK2 targeted-therapies. The goals in this study were to determine the PDJ status of a well annotated cohort of TNBC PDXs with a 3 color FISH assay and to further define the genomes and transcriptomes of PDJ+ and PDJ- TNBCs. Methods: Tissue microarrays (TMAs) of 49 cores from 27 breast cancer PDX samples, including 21 TNBCs were obtained from the Baylor College of Medicine. The TMAs were interrogated with a 3 color FISH assay consisting of three probes that simultaneously target the PDJ amplicon, 9q22, and 9q34.1. The ratios of PDJ to 9q22 and PDJ to 9q34.1 were calculated to distinguish PDJ amplification from polyploidy. In each case, 50 cells were scored. Samples with PDJ signal >4.0, PDJ/9q22 ≥2 and/or PDJ/9q34.1 ≥2 were called positive by FISH. A subset of PDJ+ PDXs was then flow sorted by DNA content and interrogated with 400k whole genome CNV arrays. These results were integrated with existing clinical, exome, and transcriptome data for the TMAs (pdxportal.research.bcm.edu). Results: PDJ amplification was identified in 6/27 PDX samples including 3/21 (14%) TNBCs. Notably the number of PDJ+ cells in each sample ranged from 2-18. Single cells with ≥10 copies were observed in each PDJ+ sample as well as two PDJ- TNBCs. PDJ+ samples had increased expression of PD-L1 and JAK2 in their RNAseq data. Whole genome CNV analysis of flow sorted PDJ+ samples confirmed the presence of the PDJ amplicon and identified co-occurring amplifications and deletions. A similar pattern of PDJ amplicon heterogeneity was seen in a Mayo Clinic Arizona cohort of 70 surgically resected TNBCs. Previous whole genome CNV analysis of flow sorted samples identified 13/70 (18%) PDJ+ cases. FISH analysis of 14 TNBCs from this cohort (PDJ+ and PDJ- cases) validated these results and identified PDJ+ tumors with cells containing >20 copies of the amplicon. Conclusion: Our multicolor FISH assay of PDX and primary resected samples confirms a prevalence of ~15% PDJ+ TNBCs. Previous studies have reported an enrichment of JAK2 amplification in neoadjuvant treated TNBCs. Given the heterogeneous FISH patterns in both PDJ+ and PDJ- samples in our cohorts, we hypothesize that neoadjuvant therapy could enrich the presence of PDJ+ cells and thereby sensitize TNBCs to ICIs and JAK2 targeted therapies. Future work will explore the role of PDJ amplification and the presence of PDJ+ cells in TNBCs in response to neoadjuvant therapy. Citation Format: Alexander S. Roesler, Smriti Malasi, Elizabeth Lenkiewicz, Barbara A. Pockaj, Michael T. Lewis, Lacey E. Dobrolecki, Lori Koslosky, Peter Hartmayer, Karen S. Anderson, Michael T. Barrett. PDJ amplicon heterogeneity in triple negative breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1790.

Diffuse large B‐cell lymphoma (DLBCL) is up to 17‐fold more likely to occur, follows a more aggressive clinical course and frequently presents at advanced stages in HIV infected (+) individuals compared to HIV negative (−) individuals. However, the molecular pathology underpinning the clinical features of DLBCL in HIV(+) patients relative to the general population is poorly understood. We performed a retrospective study examining the transcriptional, genomic and protein expression differences between HIV(+) and HIV(−) germinal center B‐cell (GCB) DLBCL cases using digital gene expression analysis, array comparative genomic hybridization (CGH) and immunohistochemistry (IHC). Genes associated with cell cycle progression ( CCNA2 , CCNB1 , CDC25A , E2F1 ), DNA replication ( MCM2 , MCM4 , MCM7 ) and DNA damage repair, including eight Fanconi anemia genes ( FANCA , FANCD1/BRCA2 , FANCE , FANCG , FANCR/RAD51 , FANCS/BRCA1 , FANCT/UBE2T , FANCV/MAD2L2 ), were significantly increased in HIV(+) GCB‐DLBCL tumors compared to HIV(−) tumors. In contrast, genes associated with cell cycle inhibition ( CDKN1A , CDKN1B ) as well as apoptosis regulating BCL2 family members ( BCL2 , BAX , BIM , BMF , PUMA ) were significantly decreased in the HIV(+) cohort. BCL2 IHC confirmed this expression. Array CGH data revealed that HIV(+) GCB‐DLBCL tumors have fewer copy number variations than their HIV(−) counterparts, indicating enhanced genomic stability. Together, the results show that HIV(+) GCB‐DLBCL is a distinct molecular malignancy from HIV(−) GCB‐DLBCL; with an increased proliferative capacity, confirmed by Ki67 IHC staining, and enhanced genomic stability, the latter of which is likely related to the enhanced expression of DNA repair genes.

Abstract Introduction: Striking clinical responses to immunotherapies in subsets of patients with a variety of solid tumors have prompted the search for predictive biomarkers. Recent studies have proposed that gene specific and genome wide mutation and copy number signatures in tumor cells may be predictive of responses to immune checkpoint blockade. Notably high levels of PD-L1 on tumor cells, a context associated with an adaptive immune response, has been linked to specific oncogenic driver lesions including loss of PTEN, activating KRAS mutation, and MYC amplification, and to the total burden of copy number variants (CNVs) in aneuploid tumors. Triple negative breast cancers (TNBCs) typically have multiple driver mutations and high levels of CNVs in their genomes. Thus there is significant interest in exploiting genomic data for the development of prognostic immunotherapy biomarkers for patients with TNBC. Study Design: We interrogated 62 well annotated surgical resections from patients with TNBC and assessed the associations of genomic lesions with expression of PD-1 and PDL-1 in tumor and non-tumor cells in each sample. We applied a systematic approach to rigorously interrogate the genomes of each TNBC sample. Tumor ploidy was initially measured with DNA content flow cytometry followed by sorting the nuclei of distinct diploid, tetraploid, and aneuploid cell populations from each TNBC. The next level of analysis measured genome wide copy number variants (CNV) with oligonucleotide arrays designed for CNV detection using purified (>95%) tumor populations. This enabled the discrimination and mapping of CNVs including single copy losses and gains, focal amplifications, and homozygous deletions across each TNBC genome. Finally we generated whole exome data in a subset of samples to increase the resolution within loci of interest and to incorporate mutations of individual genes into our genomic signatures. This combined approach provides high resolution measures of TNBC genomes from ploidy, whole chromosome and chromosome arm level CNVs, focal amplicons, breakpoints and homozygous deletions, to the level of gene specific indels and mutations. In parallel whole tissue samples were screened by IHC for PD-1 and PD-L1 using validated antibodies and established scoring methods for staining of tumor and non-tumor cells. Results and Conclusions: High levels of PD-1 and PD-L1 were detected in 5/60 (8.3%) and 16/60 (26.7%) evaluable cases with staining of PD-1 exclusively on non tumor cells, while PD-L1 was primarily on tumor cells; 15/16 (93.8%) cases. These data were then used to test the associations of individual recurring genomic lesions, and the extent and nature of chromosome aberrations on the expression patterns of PD-1 and PD-L1. Homozygous deletion of PTEN or activating mutation in PIK3CA did not correlate with increased expression of either immune checkpoint regulator in TNBC cells. Furthermore tumors with highly aberrant aneuploid genomes and distinct oncogenic drivers frequently express relatively low levels of both proteins suggesting an intrinsic escape from immunosurveillance. Citation Format: Barrett MT, Lenkiewicz E, Malasi S, Yearley JH, Annamalai L, McCullough AE, Anderson KS, Pockaj BA. Genomic lesions and PD-1/PD-L1 expression in resected triple negative breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-05.

Abstract Introduction: Clonal heterogeneity in cancer is associated with resistance to therapies and evolution of metastatic disease. Clinical management of breast cancer relies on the status of estrogen (ER), progesterone (PR), and Her2 receptors in diagnostic biopsies. Results for these biomarkers rely on IHC and FISH assays that incorporate staining intensity and percentage of positive cell numbers for each sample of interest. Heterogeneous results for one or more of these biomarkers suggest the presence of multiple tumor populations within a biopsy. However the impact of clonal heterogeneity on clinical biomarkers has not been rigorously evaluated. Methods: We interrogated the clonal composition of 3 treatment-naïve surgically excised invasive ductal carcinomas. Patient #1 cancer, (pT2, pN0) was grade 3, ER+, PR+, and Her2-- with a germ line BRCA2Q49X mutation. Patient #2 cancer (pT2, pN0) was grade 3, ER+ PR- Her2+. Patient #3 was a locally advanced cancer with both axillary and supraclavicular lymph node (LN) involvement (pT2, pN3c, and was grade 3, ER+PR-Her2+ by core but found to be ER+PR-Her2- on the resection specimen. We applied DNA content flow cytometry to multiple (8-18) mapped biopsies in each case. Each sorted tumor population was interrogated with whole exome sequencing, and whole genome array comparative genomic hybridization (aCGH). Single cell analysis of aneuploid populations present in the primary tumor and lymph nodes in patient 3 was done to further assess clonal heterogeneity. Results: Tumor from patient #1 had a single ploidy with stable copy number profiles including an interstitial 13q deletion that converted a germ line BRCA2Q49X mutation to homozygosity, and a clonal homozygous deletion in Numb in each of 4 tumor biopsies. Tumor from patient #2 had a dominant ploidy (3.2N) in each of 10 (A1-A10) primary tumor biopsies but with a second co-existing ploidy (3.6N) in one (A2) biopsy. A homozygous somatic BRCA2r3129x mutation was identified in all 10 biopsies and in both ploidies; however the genomes had heterogeneous aCGH profiles. In contrast, 12 primary biopsies from Patient #3 contained 6 distinct ploidies with highly aberrant but homogenous genomes, characterized by SARC amplification, homozygous deletion of ROBO1 and ROBO2, and clonal mutations in TP53, NF1, and PIK3CA. One of the ploidies, (5.8N) was present in an adjacent node and another (5.0N) in both the adjacent and distant nodes. Single cell analyses of the 5.0N and 5.8N populations revealed that allele frequencies of driver mutations were stable in the tumor and become homozygous in the LNs. There were no Her2 amplicons or mutations in biopsies analyzed in Patients #2 and #3 further contradicting the heterogeneous staining for ER, PR and Her2. Conclusion: Tumor heterogeneity includes variations in ploidies, copy number aberrations, and allele frequencies. However even highly aberrant aneuploid genomes can be stable within multiple primary and lymph node sites. Rigorous interrogation of flow sorted tumor populations, in contrast to inferring heterogeneity in bulk samples, identifies driver aberrations in single biopsies, challenges heterogeneous results with clinical biomarkers, and distinguishes allelic heterozygosity from tumor heterogeneity in highly aberrant aneuploid tumor genomes. Citation Format: Barrett MT, Lenkiewicz E, Malasi S, Webster T, Wison Sayres MA, McCullough AE, Anderson KS, Pockaj BA. Clonal heterogeneity in breast cancer and its impact on clinical biomarkers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-12.

Abstract Introduction: Triple negative breast carcinoma (TNBC) is a subtype of breast cancer with a paucity of therapeutic targets and a poor prognostic phenotype. In a retrospective cohort, we sought to determine the prevalence of the amplicon targeting the 9p24.1 locus, resulting in over-expression of PD-L1, PD-L2, and JAK2 (PDJ), and potentially actionable therapeutic targets. We then probed the genomic landscape of these PDJ positive tumors and identified co-occurring copy number aberrations, including focal amplifications and homozygous deletions. The presence of the PDJ amplicon and selected co-occurring aberrations provide a unique description of a clinically relevant subtype of TNBC. Methods: We evaluated fresh frozen and formalin-fixed paraffin embedded tumor samples from 64 patients with triple negative breast cancer whom underwent definitive surgical resection. Clinical annotation was available in 60 of the samples. Tumor populations (diploid, tetraploid, and aneuploidy) were sorted from each biopsy using DNA content flow cytometry. Each sorted sample was interrogated with oligonucleotide array comparative genomic hybridization (aCGH). All microarray slides were scanned using an Agilent 2565C DNA scanner and the images were analyzed with Agilent Feature Extraction version 10.7. The aCGH data was assessed with a series of QC metrics then analyzed using an aberration detection algorithm. Results: We detected a high level (log2ratio greater than or equal to 2) amplicon targeting 9p24.1 in 18 of 64 patients (28%) genomic profiles with triple negative breast carcinoma. In the PDJ positive population, we detected 8 of 18 patients (44%) with co-amplification targeting myc at 8q24, 3 of 18 patients (17%) with co-amplifications targeting EGFR at 7p11, 3 of 18 patients (17%) with co-amplifications targeting PIK3CA. These co-occurring genomic events in PDJ positive tumors may provide clinically actionable targets. Other selected amplifications detected included NOTCH3, KRAS, RUNX1, TUBAL3, FGFR2, AKT1, AKT2, YPEL2, PBXL7, KIT. We detected PTEN homozygous deletion in 2 of 18 patients (11%) PDJ positive tumors. Other homozygous deletions identified in the genomic landscape included FAT1, SOX3, Park2, TNFAIP3, GPC3, RB1, and CREBBP. Conclusions: In our retrospective analysis, the amplification of chromosome 9p24.1 involving PD-L1, PD-L2, and JAK2 is present in approximately 28% of triple negative breast cancer patients. The genomic landscape of these PDJ positive TNBCs include recurring high-level focal amplifications and targeted homozygous deletions of clinically relevant genes. The clinical implications of these data are under current investigation using model systems and are in early phase clinical trials. The efficacy of immune checkpoint inhibitors including nivolumab, pembrolizumab, and JAK2 inhibitors including ruxolitinib, in PDJ positive triple negative breast carcinoma is intriguing and remains to be elucidated. Citation Format: Gawryletz CD, Anderson KS, Cunliffe HE, Northfelt DW, McCullough AE, Lenkiewicz E, Malasi S, Pockaj BA, Barrett MT. The genomic landscape of PD-L1, PD-L2, Jak2 (PDJ) amplified triple negative breast carcinoma. [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 P6-03-13.