Distinct Tumor Populations Research Articles

Pathologic complete response to KEYNOTE522 and HER2-directed therapy for synchronous TNBC and HER2+ breast cancer

Simultaneous presentation of two separate primary breast cancers of differing histology at initial diagnosis is an uncommon phenomenon; it is even rarer to find these pathologically distinct populations within the same biopsy. Here we report the case of a patient diagnosed with clearly demarcated, pathologically heterogenous triple negative breast cancer (TNBC) and HER2+ breast cancer that was treated with a hybrid chemoimmunotherapy regimen combining elements of Keynote-522 and a standard HER2-directed neoadjuvant regimen, yielding apathologic complete response by the time of surgery with no notable adverse events. Molecular analysis of the histologically distinct tumor populations confirmed molecular evidence of differential HER2 expression but also suggested clonal relatedness of the two tumor populations based upon mutational profile, with phenotypic divergence potentially resulting from copy number alterations in NF1. Overall, this case highlights a rare histologic phenomenon that was successfully treated by combining both TNBC and HER2 directed neoadjuvant therapies.

Tumor heterogeneity and tumor-microglia interactions in primary and recurrent IDH1-mutant gliomas

The isocitrate dehydrogenase (IDH) gene is recurrently mutated in adult diffuse gliomas. IDH-mutant gliomas are categorized into oligodendrogliomas and astrocytomas, each with unique pathological features. Here, we use single-nucleus RNA and ATAC sequencing to compare the molecular heterogeneity of these glioma subtypes. In addition to astrocyte-like, oligodendrocyte progenitor-like, and cycling tumor subpopulations, a tumor population enriched for ribosomal genes and translation elongation factors is primarily present in oligodendrogliomas. Longitudinal analysis of astrocytomas indicates that the proportion of tumor subpopulations remains stable in recurrent tumors. Analysis of tumor-associated microglia/macrophages (TAMs) reveals significant differences between oligodendrogliomas, with astrocytomas harboring inflammatory TAMs expressing phosphorylated STAT1, as confirmed by immunohistochemistry. Furthermore, inferred receptor-ligand interactions between tumor subpopulations and TAMs may contribute to TAM state diversity. Overall, our study sheds light on distinct tumor populations, TAM heterogeneity, TAM-tumor interactions in IDH-mutant glioma subtypes, and the relative stability of tumor subpopulations in recurrent astrocytomas.

Fine needle aspiration of salivary gland carcinomas with high-grade transformation: A multi-institutional study of 22 cases and review of the literature.

High-grade transformation (HGT) is a rare process whereby conventional low- to intermediate-grade salivary gland carcinomas (SGC) transform into high-grade, poorly or undifferentiated malignancies with focal or complete loss of their conventional histomorphologic features. Because tumors with HGT are associated with a worse prognosis than their conventional counterparts, preoperative recognition of HGT may be of benefit for optimal patient management. Using a multi-institutional approach, we describe the largest fine needle aspiration (FNA) cohort of salivary gland carcinomas with HGT. The archives of 9 large academic medical centers were searched, and 22 cases of SGC with HGT were identified by surgical excision accompanied by preoperative FNA. Clinical and cytomorphologic features were retrospectively reviewed. The male-to-female ratio was 14:8, and the mean patient age was 60.2 years. The average tumor size was 3.6 cm, and 19 cases were from the parotid gland. Acinic cell carcinoma with HGT was the most common tumor subtype, comprising 12 cases with HGT, followed by adenoid cystic carcinoma, secretory carcinoma, and other subtypes. Eighteen cases were classified as malignant; however, a specific diagnosis of HGT was not made. Sixteen cases contained a high-grade cytologic component, and 7 cases had a mixture of both conventional and high-grade components retrospectively. SGC with HGT should be considered in the differential diagnosis of a salivary gland aspirate exhibiting high-grade cytomorphologic features. The presence of distinct tumor populations, conventional and high-grade, should prompt consideration of HGT, especially when the conventional component is acinic cell carcinoma or adenoid cystic carcinoma.

Abstract PO-053: MYC Influences metastatic heterogeneity in pancreatic cancer

Abstract Tumor heterogeneity - resulting from genetic and epigenetic alterations acquired during tumor progression - is a critical driver of phenotypic diversity in most cancers. A lethal consequence of tumor heterogeneity is the acquisition of metastatic traits by tumor cells, leading to poor clinical outcomes. This remains a major problem in pancreatic ductal adenocarcinoma (PDAC), which continues to have the worst prognosis of any major cancer type. While most cases of PDAC present with metastatic disease at the time of diagnosis, the patterns and burden of metastasis can vary widely, with some patients exhibiting a limited metastatic burden while others have more extensive spread, which impacts clinical outcomes. However, the biological and functional differences that drive metastatic heterogeneity are poorly understood. One barrier to understanding metastatic heterogeneity has been a paucity of model systems that capture this natural variation and allow for direct assessment of paired primary tumors and metastases. We previously developed an autochthonous model of PDAC – the KPCX model – that employs multiplexed fluorescence-based labeling to track the contribution of multiple distinct tumor populations to metastasis. Importantly, this technique allows for ascertainment of primary-metastatic lineage relationships in vivo, so that primary tumor clones with substantial metastatic potential can be distinguished with those having poor metastatic potential. To understand the factors underlying differences in metastatic potential, we analyzed paired primary tumors and metastases in the KPCX model and from a cohort of 398 PDAC patients. Genomic and transcriptomic analysis of murine and human metastatic PDAC revealed an association between the highly metastatic state and gene amplification or transcriptional upregulation of MYC and its transcriptional targets. Functional assessments showed that MYC promotes metastasis by recruiting tumor associated macrophages (TAMs), leading to greater bloodstream intravasation. Consistent with these findings, metastatic progression in human PDAC was also associated of MYC signaling pathways and enrichment for MYC amplification in metastasis. Collectively, these results implicate MYC activity as a major determinant of metastatic burden and heterogeneity in advanced PDA. Citation Format: Ravikanth Maddipati, Robert J. Norgard, Timour Baslan, Komal S. Rathi, Amy Zhang, Pichai Raman, Max D. Wengyn, Taiji Yamazoe, Jinyang Li, David Balli, Michael J. LaRiviere, Ian W. Folkert, Ian D. Millstein, Jonathan Bermeo, Erica L. Carpenter, Scott Lowe, Christine Iacobuzio-Donahue, Faiyaz Notta, Ben Z. Stanger. MYC Influences metastatic heterogeneity in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-053.

Abstract 4135: A polyclonal origin for pancreatic cancer metastases

Abstract Cancer genome studies demonstrate that tumors are comprised of multiple sub-clones with varied genetic and phenotypic properties. Metastases are often depicted as arising from these subpopulations through seeding by single cells, leading to clonally-derived lesions at distant sites. Alternatively, it has been suggested that metastases can also arise from the collective migration of tumor cells, resulting in formation of polyclonal metastases. To better understand the dynamics of metastasis formation in native tumors, we employed the multi-color “Confetti” lineage tracing system, in an autochthonous mouse model of pancreatic ductal adenocarcinoma (PDAC). Through the stochastic expression of distinct fluorescent proteins in any given cell, we tracked the contribution of different tumor subpopulations to malignant progression. We find that early pre-malignant lesions exhibit significant clonal heterogeneity but this clonal diversity decreases during progression to frank carcinoma. Furthermore our data provide the first direct evidence for polyclonal metastasis in the context of a naturally-evolving tumor and suggest that seeding and colonization of metastatic sites involves interactions between at least two distinct tumor populations. We also observe that clonal evolution during metastatic growth varies significantly between the sites of metastatic invasion, resulting in either monoclonal or polyclonal outgrowth. These results provide and unprecedented window into the dynamic changes in clonality that accompany tumor progression and suggest that heterotypic interactions between tumor subpopulations and microenvironmental factors unique to each tissue site contribute to the clonal evolution of metastases. Citation Format: Ravikanth Maddipati, Ben Z. Stanger. A polyclonal origin for pancreatic cancer metastases. [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 4135. doi:10.1158/1538-7445.AM2015-4135

Endoscopic ultrasound-guided fine-needle aspiration diagnosis of mixed endocrine somatostatinoma of the minor papilla in von Recklinghausen disease

Von Recklinghausen disease (VRD) is an autosomal-dominant disorder with variable penetrance. VRD is associated with a variety of benign and malignant neoplastic lesions [1]. The association with duodenal neuroendocrine tumors, particularly somatostatinomas, is widely recognized [1–3]. Mixed endocrine tumors are a particular category of neuroendocrine tumors and include at least two distinct tumor populations, one of which is endocrine [3,4]. We describe a case of mixed neuroendocrine tumor with somatostatin expression arising from the minor papilla in a patient with VRD, diagnosed by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). A 61-year-oldwomanwith VRDwas referred to our institute for endoscopic resection of an adenomatous polyp of themajor papilla. Duodenoscopy revealed a 2.5-cm polyp in the area of the minor papilla (● Fig.1a,b). EUS showed that the lesion arose from thesubmucosaof theminor papilla and obstructed the dorsal pancreatic duct (● Fig.2a). Endoscopic biopsy and EUS-FNAwith a standard 19-gauge needle were done (● Fig.2b). While superficial biopsy specimens showed tubular adenoma with mild dysplasia (● Fig.3a), small core specimens obtained with EUS-FNA showed small monomorphic cubic cells with immunohistochemical expression of chromogranin, synaptophysin, and somatostatin (● Fig.3b). Computed tomography showed two hypervascular lesions, 25 and 4mm in size, in the second part of the duodenum (● Fig.4), with the largest one obstructing the dorsal pancreatic duct in the context of pancreas divisum. The results of an octreotide scanwere negative. Surgery was recommended, but the patient refused. The diagnosis was difficult because of the presenceof amixedendocrine tumorcomposed of epithelial adenoma and somatostatinoma. A precise diagnosis is important because adenomas with mild dysplasia are an indication for endoscopic resection, but somatostatinomas associated with VRD are an indication for surgery, especiallywhen the diameter of a tumor is larger than 2cm, because of the high degree of malignant potential with regional lymph node involvement [4–6]. In this case, bothmorphologic EUS and FNAwere essential for a correct diagnosis and management, and they should be considered for all patients with VRD and duodenal lesions.

Abstract 3423: The genomic evolution of prostate cancer under the selective pressure of anti-androgen therapy

Abstract Background The implementation of novel technologies such as array comparative genomic hybridization (aCGH) and next-generation sequencing has led to a deeper understanding of the genomic nature of cancer. However, these analyses have classically been done without respecting intra-tumor heterogeneity. Here, we applied a methodology that allows us analyzing the genomic profile of distinct tumor populations from individual tumors and their clonal evolution during the progression to castration-resistant disease, thus enabling the discovery of novel genetic alterations in the course of the development of therapy resistance. Methods Matched pre- and post- hormone treated fresh frozen and/or formalin fixed prostate cancer samples were selected from our biobank. Clonal tumor populations were flow-sorted according to their nuclear DNA content. Sorted tumor populations were subjected to whole genome CGH and to full exome sequencing analyses by the use of Agilent SurePrint 2x400k microarrays and the SureSelect All Exon Kit, respectively. Target aberrations were evaluated using a prostate cancer tissue micro array (TMA) and fluorescence in situ hybridization as well as immunohistochemistry. Results The de-convolution of the genomic evolution in the investigated tumors shows that genetic aberrations and mutations are newly occurring under the selective pressure of anti-androgen therapy. More importantly, judging on the temporal dynamics of changes in allelic frequencies, we cannot only observe events that become more abundant, but also cancer associated mutations that are negatively selected for. We are currently following up several of these targets, of which at least one shows significant association with overall survival and differences in its expression pattern between hormone naïve and castration resistant cancer. Conclusions Genomic profiling of distinct clonal tumor populations during prostate cancer progression allows for analysis of intra-tumoral heterogeneity and the underlying clonal evolution. Importantly, this approach identifies genomic aberrations that were selected for under the pressure of hormone ablation therapy. Citation Format: Joël Roman Gsponer, Tanja Dietsche, Alexander Rufle, Thomas Lorber, Darius Juskevicius, Valeria Perrina, Elisabeth Lenkiewicz, Tobias Zellweger, Alexander Bachmann, Michael T. Barrett, Cyrill A. Rentsch, Christian Ruiz, Lukas Bubendorf. The genomic evolution of prostate cancer under the selective pressure of anti-androgen therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3423. doi:10.1158/1538-7445.AM2014-3423

Abstract 3181: The genomic evolution of prostate cancer under the selective pressure of anti-androgen therapy.

Abstract Background: The implementation of novel technologies such as array comparative genomic hybridization (aCGH) and next-generation sequencing has led to a deeper understanding of the genomic nature of cancer. However, these analyses have classically been done without respecting intra-tumor heterogeneity. Here, we applied a methodology that allows us analyzing the genomic profile of distinct tumor populations from individual tumors and their clonal evolution during the progression to castration-resistant disease. Methods: Matched pre- and post- hormone treated fresh frozen and/or formalin fixed prostate cancer samples were selected from our biobank. Clonal tumor populations were flow-sorted according to their nuclear DNA content. Sorted tumor populations were subjected to whole genome CGH and to full exome sequencing analyses by the use of Agilent SurePrint 2x400k microarrays and the SureSelect All Exon Kit, respectively. Results: The genomic analyses of the tumor samples underscore the presence of significant intra-tumoral heterogeneity. The analysis of matched tumor specimens allowed us to identify two particular patterns of tumor evolution during the progression after treatment: First, a more parallel pattern of tumor evolution, in which the ancestor population is breeding multiple aneuploid tumor clones. In this case, only the 2N ancestor population is able to withstand therapy by the acquisition of few specific genomic aberrations whereas the aneuploid populations are eradicated. Second, a more sequential pattern of tumor evolution with a tumor population that evolves out of a continuous line of clones. This population shows increasing signs of genomic instability over time, with a punctual event of chromothripsis (a massive destruction and rearrangement of chromosomal structures) resulting in castration-resistant clonal tumor populations. Conclusions: Genomic profiling of distinct clonal tumor populations during prostate cancer progression allows for analysis of intra-tumoral heterogeneity and the underlying clonal evolution. Importantly, this approach identifies genomic aberrations that were selected for under the pressure of hormone ablation therapy. Citation Format: Joël R. Gsponer, Tanja Dietsche, Alexander Rufle, Elisabeth Lenkiewicz, Tobias Zellweger, Alexander Bachmann, Daniel D. Von Hoff, M T. Barrett, Cyrill Rentsch, Christian Ruiz, Lukas Bubendorf. The genomic evolution of prostate cancer under the selective pressure of anti-androgen therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3181. doi:10.1158/1538-7445.AM2013-3181

Genomic analysis and selected molecular pathways in rare cancers

It is widely accepted that many cancers arise as a result of an acquired genomic instability and the subsequent evolution of tumor cells with variable patterns of selected and background aberrations. The presence and behaviors of distinct neoplastic cell populations within a patient's tumor may underlie multiple clinical phenotypes in cancers. A goal of many current cancer genome studies is the identification of recurring selected driver events that can be advanced for the development of personalized therapies. Unfortunately, in the majority of rare tumors, this type of analysis can be particularly challenging. Large series of specimens for analysis are simply not available, allowing recurring patterns to remain hidden. In this paper, we highlight the use of DNA content-based flow sorting to identify and isolate DNA-diploid and DNA-aneuploid populations from tumor biopsies as a strategy to comprehensively study the genomic composition and behaviors of individual cancers in a series of rare solid tumors: intrahepatic cholangiocarcinoma, anal carcinoma, adrenal leiomyosarcoma, and pancreatic neuroendocrine tumors. We propose that the identification of highly selected genomic events in distinct tumor populations within each tumor can identify candidate driver events that can facilitate the development of novel, personalized treatment strategies for patients with cancer.

Geographic distribution of co-dominant DNA stemlines in breast carcinoma.

Breast carcinomas often contain multiple DNA stemlines in flow cytometric DNA histograms. However, due to mixing during tissue disaggregation the microanatomical relationship between the cells which comprise distinct stemlines is unclear. We performed image cytophotometric DNA analysis (IA) on two separate areas of intact tissue sections of 19 breast carcinomas which were selected on the basis of flow cytometric (FCM) DNA content heterogeneity (i.e., multiple stemlines). For comparison, similar analyses were performed on seven tumors with unimodal FCM DNA histograms. Six of the 7 tumors (86%) with unimodal FCM histograms were also unimodal in both IA DNA histograms. Among tumors with heterogeneous FCM DNA histograms, the presence of multiple stemlines was confirmed in IA DNA histograms in 16/19. In nine of these 16 cases, multiple DNA stemlines having similar DNA indices were present in both areas of neoplasm examined with IA. The remaining seven cases displayed unimodal IA histograms in both areas, however DNA indices differed between the two histograms. These findings imply that cell populations corresponding to flow cytometrically detected DNA stemlines are often intimately admixed, even within geographically separated portions of breast tumors. This pattern suggests that productive interactions between genetically distinct tumor populations may lead to stable co-dominance of ancestral clones during progression of some breast carcinomas.