Presence Of Multiple Subclones Research Articles

Cell Mass and Stiffness As Integrative Biomarkers of Cell State in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) manifests extensive clinical phenotypic heterogeneity, ranging from indolent to aggressive, rapidly progressive disease. Molecular, genomic, transcriptomic, and immunophenotypic variation, as well as the presence of multiple subclones at diagnosis reflect the clinical diversity seen among MCL patients and make it challenging to define therapies that optimally align with individual patient risk. Biophysical phenotypes of cells, such as mass, density and stiffness, are valuable markers of changes in their intrinsic properties and closely associated with cellular states and functions, which have proven useful in elucidating fundamental processes in cell growth and development. We integrate single-cell biophysical features, which provide more granularity than canonical immunophenotypes, to further refine the biological characterization of MCL with the goal of defining rapid ex vivo biomarkers of clinical response or resistance. Methods: We utilize a device called suspended microchannel resonator (SMR) to perform biophysical measurements (mass, stiffness and density) of individual cells. We characterized the biophysical properties of MCL cells from representative sites, including peripheral blood, bone marrow and lymph nodes, of human clinical specimens and, additionally, spleen from six patient-derived xenografts (PDX) models of MCL. Furthermore, to identify genes that covary with lymphoma cell biophysical heterogeneity, we linked the biophysical measurements of individual MCL cells from the matched spleen, peripheral blood, bone marrow, and liver tissues of three carefully curated PDX models of MCL to downstream single-cell RNA-sequencing (SMR-scRNA-Seq). Results: We observed inter- and intra-tumor heterogeneity in mass and stiffness within each lymphoma specimen. Interestingly, these biophysical features reflect underlying histopathologic phenotypes, with higher mass and higher stiffness measured in blastoid and pleomorphic diseases as opposed to the smaller classical variant. By coupling biophysical measurement with transcriptomic profiling at single-cell resolution, we found that mass is strongly associated with the expression of genes annotated for cell division, cell cycle, and B-cell activation such as CCNB1, CDK1, CENPF, BLK, CD79A. Similarly, cell stiffness correlated with B-cell activation signatures and B-cell receptor signaling pathway activity, especially in the highly proliferative models. Functional genomics assays are ongoing to validate the contribution of these target pathways to MCL biophysical changes. To further define how B-cell activation impacts cellular biophysical properties, we profiled the biophysical properties of normal mature B-cell developmental stages from naïve B-cells to plasma cells. We found that cells at each differentiation stage possess a unique combination of mass, stiffness, and density. Highlighting the dynamic nature of these properties, B-cells undergo significant biophysical change after activation, with buoyant mass quadrupling upon activation and then rapidly decreasing by half. Preliminary data further reveal that mature B-cells lose approximately 25% of their stiffness after activation before reverting to their original stiffness after immunoglobulin class switching and even increasing in stiffness at the plasma cell stage. Most MCL cells are conventionally thought to derive from naïve pre-germinal center B cells, but our analysis revealed biophysical profiles overlapping strongly with more mature B cell states, suggesting that lymphomagenesis may entail reprogramming the machinery regulating cell mass and stiffness. We are now generating biophysical, transcriptomic, and genomic measurements within primary specimens from patients with MCL undergoing combined therapy targeting BTK, BCL2, and CD20 to assess their potential for inclusion within integrated predictive biomarkers. Conclusions: We established the biophysical landscape of MCL and non-malignant B-lymphoid cells to sharpen biological characterization and resolve tumor heterogeneity. We did so using a unique single-cell platform integrating transcriptomic and biophysical features as rapid, quantitative, and label-free measurements of live-cell state and phenotypic composition in pursuit of functionally integrated predictive biomarkers.

The Evolutionary Landscape of Localized Prostate Cancers Drives Clinical Aggression

The majority of newly diagnosed prostate cancers are slow growing, with a long natural life history. Yet a subset can metastasize with lethal consequences. We reconstructed the phylogenies of 293 localized prostate tumors linked to clinical outcome data. Multiple subclones were detected in 59% of patients, and specific subclonal architectures associate with adverse clinicopathological features. Early tumor development is characterized by point mutations and deletions followed by later subclonal amplifications and changes in trinucleotide mutational signatures. Specific genes are selectively mutated prior to or following subclonal diversification, including MTOR, NKX3-1, and RB1. Patients with low-risk monoclonal tumors rarely relapse after primary therapy (7%), while those with high-risk polyclonal tumors frequently do (61%). The presence of multiple subclones in an index biopsy may be necessary, but not sufficient, for relapse of localized prostate cancer, suggesting that evolution-aware biomarkers should be studied in prospectivestudies of low-risk tumors suitable for active surveillance.

Genetic Evidence for Early Peritoneal Spreading in Pelvic High-Grade Serous Cancer.

Most pelvic high-grade serous (HGS) carcinomas have been proposed to arise from tubal primaries that progress rapidly to advanced disease. However, the temporal sequence of ovarian and peritoneal metastases is not well characterized. To establish the sequence of metastases, phylogenetic relationships among the ovarian and peritoneal carcinomas were determined from single-nucleotide variations (SNVs) in nine tumor regions from each patient with pelvic HGS carcinomas. Somatic SNVs from each tumor sample were used to reconstruct phylogenies of samples from each patient. Variant allele frequencies were used to reconstruct subclone phylogenies in each tumor sample. We show that pelvic HGS carcinomas are highly heterogeneous, only sharing less than 4% of somatic SNVs among all nine carcinoma implants in one patient. TP53 mutations are found in all nine carcinoma implants in each patient. The phylogenetic analyses reveal that peritoneal metastases arose from early branching events that preceded branching events for ovarian carcinomas in some patients. Finally, subclone phylogenies indicate the presence of multiple subclones at each tumor implant and early tumor clones in peritoneal implants. The genetic evidence that peritoneal implants arose before or concurrently with ovarian implants is consistent with the emerging concept of the extra-ovarian origin of pelvic HGS cancer. Our results challenge the concept of stepwise spatial progression from the fallopian primary to ovarian carcinomas to peritoneal dissemination and suggest an alternative progression model where peritoneal spreading of early clones occurs before or in parallel with ovarian metastases.

Abstract PR08: Evidence for extra-ovarian origin and peritoneal metastases that precede ovarian carcinomas in high-grade serous ovarian cancer.

Abstract High-grade serous (HGS) ovarian cancer is the most lethal form of epithelial ovarian cancer because it represents the most common histologic subtype and usually present at an advanced stage. Due to its advanced stage presentation, HGS ovarian cancer has been described as arising de novo from undefined precursor lesions that progress rapidly to advanced disease. To gain insights into clonal heterogeneity and progression to distant metastases, we analyzed single nucleotide variations (SNVs) from nine regions of carcinomas from four patients with HGS ovarian cancer. Three primary sites, three metastases from omentum, and three metastases from bowel were collected from each patient and subjected to RNA sequencing and SNV analysis. Although a modest number (23.62%) of SNVs are shared within the same anatomic site, only limited number (1.26%) of SNVs are shared among all 9 sites, suggesting a high degree of intratumor heterogeneity in these patients. Truncal mutations, such as TP53 mutations, are shared among all sites and observed at high allele frequencies. In addition, mutations in genes associated with epigenetic modifiers, such as SETD2, CHD8, KDM5A, SMARCA1 and ARID5B are observed as truncal mutations in these patients. Phylogenetic tree reconstruction of tumors from various sites suggests a concurrent ovarian and peritoneal metastases in one patient and peritoneal metastases preceding the ovarian carcinomas in three other patients. Finally, subclone inference from multi-region sequencing indicates the presence of multiple subclones at each site in these patients. Dominant subclones, present at metastases, are often ancestral clones in the clonal hierarchy, further providing evidence that peritoneal spreading occurs early in the clonal expansion. Collectively, these results provide evidence for early peritoneal spreading that often precedes ovarian carcinomas in HGS ovarian cancer. This abstract is also presented as Poster B44. Citation Format: Jeremy Chien, Viji Shridhar, Andrea Mariani. Evidence for extra-ovarian origin and peritoneal metastases that precede ovarian carcinomas in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr PR08.

Detection of Dual IDH1 and IDH2 Mutations by Targeted Next-Generation Sequencing in Acute Myeloid Leukemia and Myelodysplastic Syndromes

Studies in myeloid neoplasms have described recurrent IDH1 and IDH2 mutations as primarily mutually exclusive. Over a 6-month period of clinical testing with a targeted next-generation sequencing assay, we evaluated 92 patients with acute myeloid leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia and identified a subset of 21 patients (23%) who harbored mutations in either IDH1 or IDH2. Of the 21 patients with IDH mutations, 4 (19%) were found to have single nucleotide variants in both IDH1 and IDH2. An additional patient included in the study was found to have two different IDH2 mutations. The mutations were typically present at different variant allelic frequencies, with one predominating over the other, consistent with the presence of multiple subclones in a single patient. In one case, the variant allelic frequencies in both IDH1 and IDH2 were equally low in the setting of a high percentage of blasts, suggesting that the IDH mutations were unlikely to be present in the founding clone. Given these data, we conclude that dual IDH1/2 mutations likely were previously underestimated, a finding that may carry important treatment implications.

Intratumor heterogeneity of DNA copy number aberrations in gastric and oesophageal adenocarcinomas.

78 Background: DNA copy number aberrations (CNAs) are common in oesophageal and gastric adenocarcinomas (OGCs) and display extensive inter-tumour heterogeneity. CNA patterns define gastric cancer molecular subtypes and ERBB2 amplifications, present in a small fraction of patients with OGC, are predictive for ERBB2-targeted drug sensitivity. Together, this suggests a critical role of CNAs determining OGC tumour biology and clinical outcomes. Despite this, predictive and prognostic CNA biomarkers have not been identified for the majority of OGCs, precluding the development of effective personalized therapy approaches for these aggressive tumours. Intra-tumour heterogeneity, characterized by the presence of multiple subclones with distinct genetic profiles within an individual cancer, can hinder the accurate molecular analysis and classification of tumours. The aim of this pilot study was to assess whether chemotherapy-naïve localized OGCs display intra-tumour macroheterogeneity of CNA profiles. Methods: Tissue specimens from four tumour regions representing the macroscopic spatial extent of each of five OGCs were systematically collected after surgical resection. DNA extracted from these FFPE specimens was analysed by molecular inversion probe SNP arrays for high resolution CNA detection. Results: Comparison of genome wide copy number and B-allele frequency profiles suggested highly concordant CNA profiles across the regions from individual primary tumours. Eight driver CNAs leading to amplification of the MET, KRAS, ERBB2, PIK3CA or FGFR2 oncogenes were identified in 4/5 tumours. Only one out of these eight driver CNA’s, harbouring the KRAS oncogene, was heterogeneous within a tumour. Conclusions: Although chromosomal instability is thought to be common in this tumour type, this pilot study suggests that macroheterogeneity is limited and that CNA profiles assessed from a single tumour biopsy are likely to be representative of the dominant CNA profile of localized OGCs. Thus, clinical correlative CNA analyses may be possible from single biopsies of localized OGCs. Mutational heterogeneity and microheterogeneity in microdissected and single cells are currently being investigated.

Genomic Analysis of Serial Samples from CLL Patients Identifies Clonal Events Associated with Disease Progression

CLL is a highly heterogeneous disorder with some patients developing progressive disease shortly after diagnosis while other patients have stable disease for decades. In addition to established clinical prognosticators, recent genomic studies have identified that intra-tumoral genetic heterogeneity (the presence of multiple subclones within an individual patient), is also correlated with adverse outcome. However, few studies have performed longitudinal sequencing of uniform cohorts of CLL patients to delineate the contribution of subclonal events to CLL progression. Here we performed extensive DNA and RNA-sequencing of 208 CLL samples from 69 CLL patients over time (average of 3.2 samples/patient), all of whom initially presented with untreated CLL without indication for therapy. The goals of this effort were to identify genomic alterations (1) predictive of disease progression at time of initial presentation and (2) enriched in serial samples with disease progression.Of the 69 patients, 49 patients developed progressive disease requiring initiation of therapy (“progressors”) while 20 did not progress over a similar period of observation (“non-progressors”). We sequenced the entire coding region of 405 genes known to be mutated in cancer as well as the RNA of 265 genes involved in fusions. Adaptor ligated sequencing libraries were captured by solution hybridization and sequenced with Illumina HiSeq in a CLIA-certified laboratory. Median exon coverage for DNA sequencing was 466X (range 315-714X) and >20,000,000 unique pairs for RNA sequencing. The average period between initial and serial sample was 589 days; the median clinical follow-up following last sample was 1,045 days. All clinical variables including IGHV mutational status, cytogenetics/FISH were known for each patient and progressor and non-progressor samples had identical frequencies of IGHV unmutated patients.Analysis of the overall cohort across all samples identified mutations in 78 genes with the 10 most mutated being NOTCH1 (30.4% of patients), KRAS (24.6%), SF3B1 (20.3%), TP53 (18.8%), ATM (15.9%), FBXW7 (10.1%), BCOR (8.7%), XPO1 (7.2%), MLL2 (7.2%), BRAF (5.8%), and AXIN1 (5.8%). Paired bone marrow (BM) and peripheral blood (PB) CLL cells were sequenced in 15 patients. Interestingly, the constellation of genomic alterations differed between BM and PB in one-third of cases, and the variant allele frequency (VAF) of mutations common to both compartments differed by >10% in 60% of patients.Frequent mutations were identified in genes not previously described to be mutated at a rate >5% in CLL. For example, mutations activating MAP kinase signaling (KRAS, BRAF, NRAS, and MAP2K1) were identified in 24% of the cohort and largely occurred in a mutually exclusive manner. The majority of MAP kinase pathway mutations were subclonal with a median VAF of 5% (range 1-47%) (Figure A), possibly explaining why these mutations have not been appreciated in prior studies at lower sequencing depth. Additional recurrent mutations not previously described as recurrent in CLL but identified here, included loss-of-function mutations in AXIN1, a negative regulator of WNT/b catenin signaling, and SPEN, a transcriptional regulator that opposes Notch signaling.Comparison of mutational frequencies in non-progressors (55 samples from 20 patients), initial samples in progressors (54 samples from 49 patients), and progression samples (104 samples from 49 patients) revealed clear differences in the somatic mutations present at each state (Figure B). Mutations in TP53 and CREBBP were significantly enriched in progressors while mutations in AXIN1 and ZRSR2 were enriched in non-progressors.We next attempted to identify changes in clonal architecture with progression. Using a comprehensive sequencing approach in a CLIA-certified CAP-accredited laboratory, we were able to identify mutations occurring with disease progression in 24/49 patients who developed disease progression, some of which were potentially clinically actionable. In contrast, only 2 of 31 non-progressor patients developed new genomic alterations in serial samples (p<0.0001).The high-depth comprehensive combined DNA and RNA sequencing approach utilized here in an initially untreated CLL cohort with serial samples delineated genomic predictors associated with disease progression and underscore the utility of this strategy to detect clonal evolution in CLL. [Display omitted] DisclosuresNahas:Foundation Medicine Inc.: Employment. He:Foundation Medicine: Employment. Donahue:Foundation Medicine: Employment. Otto:Foundation Medicine, Inc.: Employment. Dogan:Foundation Medicine, Inc: Consultancy. Lipson:Foundation Medicine, Inc.: Employment. van den Brink:Foundation Medicine, Inc: Consultancy. Miller:Foundation Medicine, Inc: Employment. Abdel-Wahab:Foundation Medicine Inc.: Consultancy.

Deciphering intratumor heterogeneity and temporal acquisition of driver events to refine precision medicine.

The presence of multiple subclones within tumors mandates understanding of longitudinal and spatial subclonal dynamics. Resolving the spatial and temporal heterogeneity of subclones with cancer driver events may offer insight into therapy response, tumor evolutionary histories and clinical trial design.

Identification of Multiple Subclones in Peripheral T-Cell Lymphoma, Not Otherwise Specified with Genomic Aberrations

Abstract 298Peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) is a heterogeneous group of nodal and extra-nodal mature T-cell lymphomas that do not belong to any of the hitherto characterized T cell lymphoma subtypes. Adult T-cell leukemia/lymphoma (ATLL) is a mature T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). PTCL, NOS with genomic aberrations has been shown to resemble lymphoma-type ATLL in terms of genomic aberration patterns, histopathology and prognosis (Clin Cancer Res., 15:30–38). We have recently shown that a majority of patients with acute-type ATLL have multiple subclones that were likely produced in lymph nodes (Blood, 117:5473–5478). In this study, high-resolution oligo-array comparative genomic hybridization (CGH) was employed to determine whether PTCL, NOS with genomic aberrations also possesses multiple subclones as found in ATLL. Thirteen cases of PTCL, NOS were available for 44K high-resolution array CGH analysis. These samples analyzed in this study were shown to be monoclonal by Southern blot analysis. We obtained average log2 ratios of regions where aberrations continued over 400 probes and showed a linear average ratio of >0.1 or <-0.1. The log2 ratio imbalance was defined when a patient had a different average log2 ratio for gain or loss regions. The results showed that 11 of the 13 cases (84.6%) had a log2 ratio imbalance, suggesting that multiple subclones exist in PTCL, NOS with genomic aberrations. As previously demonstrated, the average log2 ratio of aberrant regions reflected the ratio of tumors (Blood, 117:5473–5478). In one case, as represented in Figure 1, all tumor cells had regions of loss on chromosomes 3 and 13. Fifty-four percent of tumor cells had a region of loss on chromosome 16, and 83% of tumor cells had loss on chromosome 17. It is therefore speculated that clonal evolution took place as shown in Figure 2. In an effort to examine the association between multiple subclones and prognosis, we used our previous BAC array data for 24 cases and found that the presence of multiple subclones was associated with a poor prognosis (p=0.0279) (Figure 3). Our finding concerning the presence of multiple subclones in PTCL, NOS and ATLL should assist greatly in revealing the mechanism of lymphoma development, progression and drug-resistance associated with these diseases. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Clonal evolution of adult T-cell leukemia/lymphoma takes place in the lymph nodes

AbstractAdult T-cell leukemia/lymphoma (ATLL) is the neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). We performed oligo-array comparative genomic hybridization (CGH) against paired samples comprising peripheral blood (PB) and lymph node (LN) samples from 13 patients with acute ATLL. We found that the genome profiles of the PB frequently differed from those of the LN samples. The results showed that 9 of 13 cases investigated had a log2 ratio imbalance among chromosomes, and that chromosome imbalances were more frequent in LN samples. Detailed analysis revealed that the imbalances were likely caused by the presence of multiple subclones in the LN samples. Five of 13 cases showed homozygous loss regions in PB samples, which were not found in the LN samples, indicating that tumors in the PB were derived from LN subclones in most cases. Southern blot analysis of TCRγ showed that these multiple subclones originated from a common clone. We concluded that in many ATLL cases, multiple subclones in the LNs originate from a common clone, and that a selected subclone among the LN subclones appears in the PB.