Anupama Praveen-Kumar

BACKGROUND: Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors. METHODS: We retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers. RESULTS: We observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations in TP53, PLXNB3, Zinc finger genes, and fewer mutations in SDC2, CDH1, PIK3CA, SVIL, and PTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA-protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, including KIAA1522, DCK, FOXO3, MYO9B, ARID1A, EPRS, ZC3HAV1, and RBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors. CONCLUSIONS: This study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

Breast cancer in women <40, accounting for ~5% of all breast cancer cases diagnosed in the U.S., is more aggressive and associated with worse outcomes compared to breast cancer in older women. We performed a first-ever integrated proteogenomic study from a matched cohort of laser-microdissected tumors of 34 young (<40 years) and 34 older (≥60 years) women to identify molecular features that may underlie the worse outcomes in young women. Progression-free interval was shorter in young women, and their tumors were enriched for more aggressive molecular subtypes. Our multi-omic analysis identified distinct clusters between age groups in luminal but not basal-like cancers. Notably, GATA3 mutations were enriched in luminal tumors from young women while TP53 and PIK3CA mutations were more common in luminal tumors from older women. Young women's tumors exhibited lower estrogen receptor (ER) expression yet paradoxically enhanced ER response pathways and increased expression of tamoxifen-resistance-associated genes (IRS1, FERMT1). Immune pathway activity and immune scores were lower in tumors from young women, whereas proliferative and MYC pathways were notably elevated, identifying potential therapeutic targets. Transcriptomic data from TCGA and METABRIC confirmed our findings, with 10 of 11 observed pathways corroborated. Finally, differential expression of four immune-related surface proteins also suggested the potential of age-specific responses to immune-based therapies. Together, these findings may contribute to the understanding of the molecular mechanisms underlying worse outcomes in young women and offer new insight to therapeutic strategies.

Abstract Introduction: Laser microdissection (LMD) is a valuable method to isolate target populations of cells for molecular analysis. LMD of breast tumor samples can isolate breast tumor cells whereas bulk processing of tumor tissue will incorporate surrounding non-cancerous cells and bias tumor expression profiling. Here, we evaluated the advantage of using LMD breast tumors for RNA-Seq over bulk processing. Methods: Tissue samples for the in-house dataset were from breast cancer patients consented by a HIPAA-compliant, IRB-approved protocol of the Clinical Breast Care Project. A total of 118 primary breast tumors embedded in OCT (Optimum Cutting Temperature) were selected and processed by LMD. Total RNA and protein were extracted using the illustra triplePrep kit. Paired-end RNA sequencing of 118 cases was performed using the Illumina HiSeq platform and the reads were preprocessed using a PERL-based pipeline involving PRINSEQ, GSNAP and HTSeq. The Cancer Genome Atlas (TCGA) primary breast cancer RNA-Seq data for 1097 tumors, bulk processed was downloaded. Differential expression of genes (DEG) was assessed using DESeq2. Significance was described for DEG with fold change &amp;gt;2 and p-adjusted value of 0.05. Results: A total of 24,518 genes with a mean expression of ≥ 10 (~9%) raw counts across 118 tumor samples were identified in the in-house LMD dataset. In TCGA breast cancer RNA-Seq, 14,281 genes with a mean expression of ≥ 100 (~9%) raw counts across 1097 tumor samples were identified. The conventional PAM50 classifier was used for intrinsic subtyping of in-house data, yielding 36 Basal-like, 14 HER2-enriched, 43 Luminal A, 22 Luminal B and 3 Normal-like calls. The provided PAM50 calls for TCGA were 192 Basal-like, 82 HER2-enriched, 566 Luminal A, 217 Luminal B and 40 Normal-like calls. Within commonly expressed 13,165 genes, LMD (in-house) and bulk (TCGA) processing exhibited approximately 40-78% non-overlap in significantly differentially expressed genes (SDEG) among the conventional intrinsic subtypes. 21 unique stromal genes were present in SDEG unique to TCGA whereas there were only 5 SDEG unique to in-house dataset. We validated the results with 34 patients that had both LMD and bulk processing RNA-Seq data and found the non-overlap genes percentage to be even greater from 46-85%. The observed percentages of non-overlapping genes in the whole datasets were also validated in the 34 overlapping cases when using IHC subtypes. Overall high positive correlation is observed among the stromal genes present in SDEG unique to TCGA suggesting strong stromal contribution in bulk processing. Pathway analysis of SDEG unique to LMD data suggested alterations in known cancer pathways (B-cell immune response, RNA metabolism and splicing, phagocytosis, and signaling components). Conclusion: Analysis of The Cancer Genome Atlas breast cancer RNA-Seq data set (based on bulk tissue processing) suggested contribution of stromal signature genes and important differences from LMD specimens. Thus, tumor selection via LMD may allow us to unveil signals that are more specific to cancer cells. Disclaimer: The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of Uniformed Services University of the Health Sciences (USUHS), The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., the Department of Defense (DoD), the Departments of the Army, Navy, or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Citation Format: Praveen-Kumar Raj-Kumar, Lori A. Sturtz, Albert J. Kovatich, Brenda Deyarmin, Jeffrey A. Hooke, Leigh Fantacone-Campbell, Anupama Praveen-Kumar, Jianfang Liu, James Craig, Leonid Kvecher, Jennifer Kane, Jennifer Melley, Stella Somiari, Stephen C. Benz, Justin Golovato, Shahrooz Rabizadeh, Patrick Soon-Shiong, Richard Mural, Craig D. Shriver, Hai Hu. Evaluation of laser microdissected primary breast tumors for RNA Seq over bulk processing and validated with cohort control [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-09.

Abstract Introduction: RNA-Seq based gene expression profiling of breast tumor samples is widely used to subgroup patients and to identify gene signatures of prognostic value. However, tumor samples are highly heterogeneous, and so bulk processing of tumor tissue will consist of several different cell types. Here, we evaluated the advantage of using laser microdissected (LMD) breast tumors for RNA-Seq over bulk processing. Methods: Patients for the in-house dataset were duly consented under an IRB-approved protocol of the Clinical Breast Care Project. A total of 118 primary breast tumors embedded in OCT (Optimum Cutting Temperature) were selected and processed by LMD. Total RNA and protein were extracted using the Illustra triplePrep kit. Paired-end RNA sequencing of 118 cases was performed using the Illumina HiSeq platform and the reads were preprocessed using a PERL-based pipeline involving PRINSEQ, GSNAP and HTSeq. The Cancer Genome Atlas (TCGA) primary breast cancer RNA-Seq data for 1097 samples was downloaded. Differential expression of genes (DEG) was assessed using DESeq2. Significance was described for DEG with fold change &amp;gt;2 and p-adjusted value of 0.05. Results: A total of 24,518 genes with a mean expression of ≥ 10 raw counts across 118 tumor samples were identified in the in-house LMD dataset. In TCGA breast cancer RNA-Seq, 14,281 genes with a mean expression of ≥ 100 raw counts across 1097 tumor samples were identified. The conventional PAM50 classifier was used for intrinsic subtyping of in-house data, yielding 36 Basal-like, 14 HER2-enriched, 43 Luminal A, 22 Luminal B and 3 Normal-like calls. The provided PAM50 calls were used for TCGA which are 192 Basal-like, 82 HER2-enriched, 566 Luminal A, 217 Luminal B and 40 Normal-like calls. Within commonly expressed 13,165 genes, LMD and bulk processing exhibited approximately 40-78% non-overlap in significantly differentially expressed genes (SDEG) among the intrinsic subtypes. 21 unique stromal genes were present in SDEG unique to TCGA whereas there were only 5 SDEG unique to in-house dataset. Overall high positive correlation is observed among the stromal genes present in SDEG unique to TCGA suggesting strong stromal contribution in bulk processing. Pathway analysis of SDEG unique to LMD data suggested alterations in known cancer pathways (B-cell immune response, RNA metabolism and splicing, phagocytosis, and signaling components). Conclusion: Analysis of The Cancer Genome Atlas breast cancer RNA-Seq data set (based on bulk processing tissue) suggested contribution of stromal signature genes and important differences from LMD specimens. Thus, tumor selection via LMD can result in better expression profiling by RNA-Seq which has the potential to uncover many cancer genes and pathways. The views expressed in this abstract are those of the author and do not reflect the official policy of the Department of Army/Navy/Air Force, Department of Defense, or U.S. Government. Citation Format: Praveen-Kumar Raj-Kumar, Lori A. Sturtz, Albert J. Kovatich, Brenda Deyarmin, Jeffrey A. Hooke, Leigh Fantacone-Campbell, Anupama Praveen-Kumar, Jianfang Liu, James Craig, Leonid Kvecher, Jennifer Kane, Jennifer Melley, Stella Somiari, Stephen C. Benz, Justin Golovato, Shahrooz Rabizadeh, Patrick Soon-Shiong, Richard J. Mural, Craig D. Shriver, Hai Hu. Evaluation of laser microdissected primary breast tumors for RNA-Seq over bulk processing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3402.