Clonal Evolution Of Tumor Cells Research Articles

Novel Insights into the Initiation, Evolution, and Progression of Multiple Myeloma by Multi-Omics Investigation

The evolutionary history of multiple myeloma (MM) includes malignant transformation, followed by progression to pre-malignant stages and overt malignancy, ultimately leading to more aggressive and resistant forms. Over the past decade, large effort has been made to identify the potential therapeutic targets in MM. However, MM remains largely incurable. Most patients experience multiple relapses and inevitably become refractory to treatment. Tumor-initiating cell populations are the postulated population, leading to the recurrent relapses in many hematological malignancies. Clonal evolution of tumor cells in MM has been identified along with the disease progression. As a consequence of different responses to the treatment of heterogeneous MM cell clones, the more aggressive populations survive and evolve. In addition, the tumor microenvironment is a complex ecosystem which plays multifaceted roles in supporting tumor cell evolution. Emerging multi-omics research at single-cell resolution permits an integrative and comprehensive profiling of the tumor cells and microenvironment, deepening the understanding of biological features of MM. In this review, we intend to discuss the novel insights into tumor cell initiation, clonal evolution, drug resistance, and tumor microenvironment in MM, as revealed by emerging multi-omics investigations. These data suggest a promising strategy to unravel the pivotal mechanisms of MM progression and enable the improvement in treatment, both holistically and precisely.

Tumor heterogeneity and immune-evasive T follicular cell lymphoma phenotypes at single-cell resolution.

T follicular helper (TFH) cell lymphomas (TFHLs) are characterized by TFH-like properties and accompanied by substantial immune-cell infiltration into tumor tissues. Nevertheless, the comprehensive understanding of tumor-cell heterogeneity and immune profiles of TFHL remains elusive. To address this, we conducted single-cell transcriptomic analysis on 9 lymph node (LN) and 16 peripheral blood (PB) samples from TFHL patients. Tumor cells were divided into 5 distinct subclusters, with significant heterogeneity observed in the expression levels of TFH markers. Copy number variation (CNV) and trajectory analyses indicated that the accumulation of CNVs, together with gene mutations, may drive the clonal evolution of tumor cells towards TFH-like and cell proliferation phenotypes. Additionally, we identified a novel tumor-cell-specific marker, PLS3. Notably, we found a significant increase in exhausted CD8+ T cells with oligoclonal expansion in TFHL LNs and PB, along with distinctive immune evasion characteristics exhibited by infiltrating regulatory T, myeloid, B, and natural killer cells. Finally, in-silicoand spatial cell-cell interaction analyses revealed complex networking between tumor and immune cells, driving the formation of an immunosuppressive microenvironment. These findings highlight the remarkable tumor-cell heterogeneity and immunoevasion in TFHL beyond previous expectations, suggesting potential roles in treatment resistance.

Osimertinib Resistance: Molecular Mechanisms and Emerging Treatment Options.

The development of tyrosine kinase inhibitors (TKIs) targeting the mutant epidermal growth factor receptor (EGFR) protein initiated the success story of targeted therapies in non-small-cell lung cancer (NSCLC). Osimertinib, a third-generation EGFR-TKI, is currently indicated as first-line therapy in patients with NSCLC with sensitizing EGFR mutations, as second-line therapy in patients who present the resistance-associated mutation T790M after treatment with previous EGFR-TKIs, and as adjuvant therapy for patients with early stage resected NSCLC, harboring EGFR mutations. Despite durable responses in patients with advanced NSCLC, resistance to osimertinib, similar to other targeted therapies, inevitably develops. Understanding the mechanisms of resistance, including both EGFR-dependent and -independent molecular pathways, as well as their therapeutic potential, represents an unmet need in thoracic oncology. Interestingly, differential resistance mechanisms develop when osimertinib is administered in a first-line versus second-line setting, indicating the importance of selection pressure and clonal evolution of tumor cells. Standard therapeutic approaches after progression to osimertinib include other targeted therapies, when a targetable genetic alteration is detected, and cytotoxic chemotherapy with or without antiangiogenic and immunotherapeutic agents. Deciphering the when and how to use immunotherapeutic agents in EGFR-positive NSCLC is a current challenge in clinical lung cancer research. Emerging treatment options after progression to osimertinib involve combinations of different therapeutic approaches and novel EGFR-TKI inhibitors. Research should also be focused on the standardization of liquid biopsies in order to facilitate the monitoring of molecular alterations after progression to osimertinib.

Advances in single-cell sequencing technology in the field of hepatocellular carcinoma.

Tumors are a class of diseases characterized by altered genetic information and uncontrolled growth. Sequencing technology provide researchers with a better way to explore specific tumor pathogenesis. In recent years, single-cell sequencing technology has shone in tumor research, especially in the study of liver cancer, revealing phenomena that were unexplored by previous studies. Single-cell sequencing (SCS) is a technique for sequencing the cellular genome, transcriptome, epigenome, proteomics, or metabolomics after dissociation of tissues into single cells. Compared with traditional bulk sequencing, single-cell sequencing can dissect human tumors at single-cell resolution, finely delineate different cell types, and reveal the heterogeneity of tumor cells. In view of the diverse pathological types and complex pathogenesis of hepatocellular carcinoma (HCC), the study of the heterogeneity among tumor cells can help improve its clinical diagnosis, treatment and prognostic judgment. On this basis, SCS has revolutionized our understanding of tumor heterogeneity, tumor immune microenvironment, and clonal evolution of tumor cells. This review summarizes the basic process and development of single-cell sequencing technology and its increasing role in the field of hepatocellular carcinoma.

Investigation of the Heterogeneity Evolution and Drug Resistance Mechanism of Diffuse Large B-Cell Lymphoma Under the Treatment of a Novel HDAC Inhibitor LAQ824

Aims:Diffuse large B-cell lymphoma (DLBCL), the most frequent malignant lymphoma subtype, is a group of highly invasive diseases with great heterogeneity in genomic alterations, clinical characteristics, morphological manifestations, treatment response and prognosis. Although most DLBCL patients can be cured by immunochemotherapy, nearly 40% of DLBCL patients still develop drug resistance and relapse. For relapsed/ refractory (R/R) DLBCL patients, there is still no optimal treatment. The heterogeneity and clonal evolution of tumor cells are the core driving forces for the occurrence and development of DLBCL, and the root causes for their refractory, recurrence and drug resistance. In this study, we screened out a novel small molecule compound effectively killing DLBCL cells, and analyzed its potential mechanism of anti-tumor. Meanwhile, by using single cell sequencing technology, we try to further investigate the heterogeneity and clona evolution and drug resistance mechanism of DLBCL under different drug pressure, explore core driver factors of drug resistance, evaluate and develop new treatment strategies.Methods:In this study, GEXSCOPE microfluidic platform was used for single-cell transcriptome sequencing. Seruat software was used for cell type recognition and clustering analysis. In order to further investigate the molecular mechanism of LAQ824 inducing the apoptosis of DLBCL cells and explore the target of LAQ824, antibody chip was performed to detect the phosphorylation of related signaling pathway. Gene expression was detected by real-time qPCR and Western blot. ChIP, RNA interfering (RNAi) and dual-luciferase activity assay were performed to validate the potential drug resistance targets for LAQ824. Moreover, WES of 21 DLBCL cell lines were performed to map mutations and analyze the correlation between related mutations and LAQ824 resistance. In this study, we established DLBCL animal models using NOD SCID mice transplanted with DLBCL cell lines, by which we could evaluate the tumor inhibition efficiency of LAQ824 alone and/or combination with other small molecular inhibitors.Results:Using GDSC database, we screened out Dacinostat (LAQ824), a novel HDAC inhibitor, was highly sensitive that could effectively induce the apoptosis of most DLBCL cells at low concentrations. Functional assay showed that LAQ824 could inhibit cell proliferation and promote apoptosis of tumor cells. LAQ824 treatment could significantly upregulate the acetylation level of histone H3 within a certain concentration range, and the DNA damage repair function of DLBCL cells was supressed by inhibiting Chk2 expression, thus significantly inducing cell apoptosis and effectively killing DLBCL cells. Meanwhile, through single-cell sequencing analysis, it was found that c-Fos could be activated under certain drug pressure of LAQ824. As a potential drug-resistant core driver gene, the expression level of c-Fos is highly correlated with IC50 of LAQ824 and the prognosis of patients with DLBCL, which can be used as a sensitivity indicator of LAQ824. Treatment with c-Fos inhibitor combined with LAQ824 can significantly improve the tumor inhibition rate, validated both in vitro and in vivo, which is expected to alleviate the recurrence and drug resistance of DLBCL patients.Conclusions:In general, we explores potential therapeutic drugs for DLBCL parients, adjusts and explores new clinical treatment strategies on this basis, and provides theoretical basis and data support for the realization of individualized precise treatment and the solution of DLBCL recurrence and drug resistance. DisclosuresNo relevant conflicts of interest to declare.

Features of tumor heterogeneity in regional metastasis of breast cancer

The review looked at the issues of tumor heterogeneity in breast cancer. Tumor heterogeneity is classified according to the main feature demonstrating regional differences within a tumor (for example, heterogeneity of clinical manifestations, histological heterogeneity, heterogeneity of protein expression, etc.) and by tumor regions (differences between primary tumors and metastases, differences between cell clones within a single tumor node, etc.). Temporal heterogeneity is also distinguished, which manifests itself in the clonal evolution of tumor cells. The review covers the heterogeneity in the expression of four biomarkers from the gold standard for immunohistochemical staining of breast cancer: estrogen receptors, progesterone receptors, Her2/neu and Ki67 in primary tumor tissue and regional metastases. According to various studies, discordance in estrogen receptor status of primary tumor cells and metastases was observed with a frequency of 4 to 62%, progesterone receptors from 12 to 54%, Her2/neu from 0 to 24%, Ki67 from 4 to 39%. The results of studies of changes in the expression levels of individual markers in breast cancer metastases, as well as the heterogeneity of surrogate subtypes of tumor tissue in metastasis, are briefly described. Possible reasons for heterogeneity in the expression of key prognostic and predictive markers by primary tumor and metastatic cells, such as artificial factors at the preanalytic and analytic stages of the study, polyclonality of the primary tumor before metastasis, clonal evolution of tumor cells during metastasis, selection of tumor clones under the therapy are highlighted.

Features of 18F-fluorodeoxyglucose uptake in multiple myeloma

Background. Multiple myeloma (MM) is a kind of malignancy from malignant plasma cells with high intra- and interpatient variability, because of complex clonal evolution of tumor cells. Positron emission tomography combined with computed tomography with 18F-fluorodeoxyglucose (18F-FDG-PET/CT) plays a major role in MM visualization, but there are evidences of non-FDG-avid cases of MM. Taking into account the increasing role of 18F-FDG-PET/CT in MM, special criteria for risk-group stratification was elaborated. These criteria are based on comparison of radiotracer uptake in tumor tissue, mediastinal blood pool and liver.Objective: the study of 18F-FDG uptake in MM and solitary plasmacytoma before antitumor treatment to assess the applicability of criteria based on the ratio of activity in tumor tissue and liver.Materials and methods. We reviewed 65 18F-FDG-PET/CT scans of patients with MM and solitary plasmacytoma before treatment.Results. In our cohort we identified 2 tumor cases of a plasma cell nature, which amounted to 2 % among all B-NHLs. In one case, the process was located in the nasal cavity and clinically manifested itself with nosebleeds. The second case is a lesion of the mouth floor, primarily with the ulcer formation. In the first cases, at diagnosis, the immunohistochemistry (IHC) test was performed after patient’s chemotherapy and radiation treatment, which distorted the tumor immunophenotype. In the second cases with extensive process in maxillary sinuses, a complete and very detailed IHC test was carried out; however the data did not allow for a definitive diagnosis. Difficulties apparently arose in the interpretation of CD38 expression – main marker of plasmacytic line cells, as well as due to the unusual morphology.Conclusion. Therefore, 5-point scale is eligible for MM tumor assessment in 52 % of patients and feasible in 32 % of patients, but in 16 % patients alternative criteria are required.

A method for reliable detection of genomic point mutations based on single-cell target-sequencing

The analysis of genomic point mutations is one of the research strategies to explore the clonal evolution of tumor cells. At present, clonal evolution of tumor cells is mainly determined by bulk sampling and sequencing of different sections of the tumor. Since this approach analyzes a mixture of different cell types, it may not accurately represent the clonal evolution of specific tumor cell populations and likely miss low frequency mutations, especially when the sequencing depths are not sufficient. To address this issue, we have developed a strategy to analyze genomic point mutations from prostate basal cell carcinoma (BCC) tissues at single-cell resolution. Firstly, we optimized the single-cell whole genome amplification procedure with HepG2 cells. Then the single cells from BCC tissue were captured by a microfluidic chip of Fluidigm and processed for whole-genome amplification. Both SCUBE3 and MST1L genomic mutations were obtained by whole exome sequencing. Finally, we examined the genomic mutations through single-cell targeted amplification and Sanger sequencing. The established method successfully reconfirmed the mutations of SCUBE3 and MST1L in BCC at single cell level. The strategy established in this study could provide a useful tool for determining the clonal evolution of tumor cells based on genomic mutations at single-cell resolution.

Inconsistent Intersample ALK FISH Results in Patients with Lung Cancer: Analysis of Potential Causes.

ALK FISH analyses of multiple specimens occasionally yield inconsistent intersample results in lung cancer patients, posing clinical challenges requiring intensive analysis of all potential causative pre- and post- analytic factors. In this study, 19 patients (8M/11F) with inconsistent intersample ALK FISH results were analyzed, representing 4.9% of patients assessed ≥ twice in our institution. Fifteen patients received ALK tyrosine kinase inhibitor(s) (TKIs). Nine patients died, and ten were alive for 8 to 74-month follow-ups (median, 40 months). Through strict and stringent laboratory and case-review policies, all postanalytic factors were excluded. Correlating clinical information, ALK results obtained by RNA sequencing (RNA-seq) and other concurrent tests, several pre-analytic factors were determined. A suboptimal specimen was likely the cause in three patients, supported by the failure of one or more concurrent tests or discrepant results between FISH and RNA-seq. ALK inhibition by TKIs might have been responsible for the change of ALK status from positive to negative in eight patients. Other potential explanations include the existence of multiple primary lung cancer lesions, tumor heterogeneity, and the clonal evolution of tumor cells, related or not to ALK TKI therapy. This study is helpful for both pathologists and clinicians encountering inconsistent and/or discrepant intersample results.

Abstract A31: Tumor stroma in the development of acquired cancer therapy resistance

Abstract Despite the onset of new treatment strategies, mortality rates for breast cancer are estimated at approximately 600,000 women per year, mostly due to acquired resistance to therapy. Chemotherapy, often combination therapy, includes DNA-damaging agents such as epirubicin, an epimer of doxorubicin. Although chemotherapy has been reported to reduce recurrence rates by 50% over two years, eight-year survival rates are approximately 30% and overall mortality is 20-25%. Studies aiming to overcome resistance to therapy have focused mainly on in vitro malignant cell assays. These have failed to deliver appropriate targets to overcome resistance because they have failed to account for the influence of the tumor microenvironment on chemoresistance. Published evidence indicates that resistance to therapy is not malignant cell-intrinsic, but instead depends on DNA-damaging chemotherapy-induced changes in paracrine signals from endothelial cells. Our current research has established that the doxorubicin treatment acutely affects endothelial cells with long-term effects on tumor cell resistance to therapy in vivo. Endothelial cells are heterogenous, and the effect of DNA damage on endothelial cell evolution and how this regulates malignant cell resistance to therapy is unknown. We have observed that orthotopic murine mammary tumors arising from the same source of primary mammary tumor cells show differential chemotherapy responses. RNA seq analysis of tumors after acute doxorubicin treatment reveal highest levels of differential transcript expression in endothelial cells compared with cancer-associated fibroblasts or malignant cells. In vitro assays show that conditioned media from tumor endothelial cells treated with chemotherapy can protect tumor cells from DNA damage and cell death. Current analysis now focuses on analyzing the complexity of endothelial cell regulation of tumor cell response to chemotherapy and how this could model clonal evolution of tumor cells. We aim to create a chemoresistance stromal evolution signature, to determine the individual components of this and commonalities between cancers and possible drug effects. Thus, our data could change the paradigm of drug resistance and define how stromal compartments regulate acquired resistance. Citation Format: Jesus Gomez-Escudero, Eleni Maniati, Freddie Whiting, Jun Wang, Trevor Graham, Kairbaan Hodivala-Dilke. Tumor stroma in the development of acquired cancer therapy resistance [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A31.

The KRAS mutation status and resistance to cetuximab in patients with squamous cell carcinoma of oral cavity.

e15514 Background: The standard treatment of squamous cell carcinoma of the tongue and the mucosa of the bottom of the oral cavity (SCCTOM) is chemotherapy (CT) in combination with cetuximab (mC, anti-EGFR antibody). However, not all patients manage to achieve positive results. A common cause of resistance to anti-EGFR antibodies is the constitutive activation of mediators of the underlying signaling pathways. Therefore, the aim of the study was to analyze mutations in the KRAS using the Digital Droplet PCR method in patients with SCCTOM on the background of CT in combination with targeted therapy or standard CT. Methods: The study used the QX200 system (Bio-Rad) and the ddPCRtm KRAS Screening Multiplex Kit (7 mutations). The study included 60 patients with T3-4N0-1M0 SCCTOM. The main group consisted of 30 patients (cisplatin, 5-fluorouracil+cetuximab). The control group consisted of 30 patients (CT). For research, cfDNA from blood plasma was used. Analysis of the data was performed using QuantaSoft v1.7.4. Results: The frequency of the KRAS mutant type (mtKRAS) is 43% before treatment and 32% after treatment (n = 60). In the group of patients CT+mC the incidence of mtKRAS before treatment was 40%, after treatment - 20%. In the group of patients CT without mC the incidence of mtKRAS before treatment was 47%, after treatment - 43%. Decrease of 20% (p = 0.00089) in the frequency of mtKRAS after CT+mC was found, while it was 23% (p = 0.00009) lower than the frequency of mtKRAS after CT without mC. After treatment, in the CT+mC group of patients, the number of mtKRAS DNA copies increased 1.5 times (p = 0.048). Analysis of clinical response to therapy allowed us to divide the main and control groups of patients into 2: sensitive and resistant to therapy. After treatment, a change in the mtKRAS ratio was not found in the subgroup sensitive to mC compared with the period before treatment, while the frequency of mtKRAS was reduced by 3 times (p = 0.009). In the mC-resistant subgroup, the mtKRAS ratio increased 1.9 times compared to the period before treatment (p = 0.009), while the frequency of mtKRAS increased 3.5 times compared with the mC-sensitive group (p = 0.0045). Conclusions: Prior to treatment, patients resistant to CT+mC were characterized by an increased occurrence of mutations in the KRAS and an apparently large number of tumor cells carrying mutations. The use of mCs caused a change in the direction of the clonal evolution of tumor cells - leading to an increase in the number of cells carrying mtKRAS (conditions were created for the elimination of cells with mutation in EGFR and KRAS wild type).

Abstract 570: A glioblastoma translational pipeline: discovery of novel tumor antigens that drive GBM recurrence

Abstract Background: Glioblastoma (GBM) is the most common malignant primary adult brain tumor, characterized by extensive cellular and genetic heterogeneity. Even with surgery, temozolomide chemotherapy and radiation, tumor re-growth and patient relapse are inevitable, with a median survivorship of just 15 months. Genomic profiling studies have shown that clonal evolution within GBM may be driven by cancer treatment, such that the recurrence may no longer resemble the genetic landscape of the original primary tumor. Furthermore, intratumoral heterogeneity associated with clonal evolution complicates biomarker discovery and treatment personalization and underlies treatment failure. Thus, modeling clonal heterogeneity and evolution to understand cancer progression is critical for the development of effective therapeutic approaches. We aim to identify new therapeutic targets that drive clonal evolution in treatment-refractory GBM and develop novel and empirical therapeutic paradigms targeting recurrent GBM. Experimental Procedure: We employed a transcriptomic, proteomic and functional genomics approach to discover and validate genes that drive GBM recurrence. Using a therapy-adapted patient-derived xenograft (PDX) model of treatment-refractory GBM, we profiled the transcriptomic and proteomic landscape of treatment-naïve primary GBM through conventional chemotherapy and radiation therapy, and into recurrence. To complement the transcriptomic data, we used an unbiased genome-wide CRISPR-Cas9 screening platform to identify genes essential for self-renewal in recurrent GBM, as well as to identify novel sensitizers and suppressors of conventional therapy. Furthermore, we coupled cellular DNA barcoding technology with our PDX model to profile the clonal evolution of tumor cells through therapy. Results: Integrative analysis of deep sequencing and surface proteomics of tumor cells harvested at tumor formation, minimal residual disease after chemoradiotherapy, and tumor recurrence from the PDX model resulted in the identification of novel therapeutic targets in treatment-refractory GBM. Using CRISPR, potential targets were knocked out in patient-derived GBMs in order to characterize the effect on self-renewal and tumor formation. We report the successful barcoding of patient-derived primary, treatment-naïve GSCs at a single cell resolution that were expanded into clonal populations, intracranially engrafted in immunodeficient mice and treated with SoC therapy. Conclusion: We have generated a translational pipeline from initial target discovery, through target validation, to building new biotherapeutics against novel targets, and preclinical testing in our PDX model of treatment-resistant GBM. A promising lead panel of biotherapeutic modalities is being translated into early clinical development, generating targeted therapies and hope for future GBM patients. Note: This abstract was not presented at the meeting. Citation Format: Parvez Vora, Chitra Venugopal, Chirayu Chokshi, Maleeha Qazi, Nazanin Tatari, Kevin Brown, Nicholas Yelle, Jarrett Adams, David Tieu, Mathieu Seyfrid, Mohini Singh, Neil Savage, Minomi Subapanditha, David Bakhshinyan, Laura Kuhlmann, Thomas Kislinger, Sachdev Sidhu, Jason Moffat, Sheila Kumari Singh. A glioblastoma translational pipeline: discovery of novel tumor antigens that drive GBM recurrence [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 570.

Abstract 2900: Dissection of clonal heterogeneity unmasks pre-existing chemoresistance and new metabolic vulnerabilities in pancreatic cancer

Abstract Adaptive drug-resistance mechanisms allow human tumors to evade treatment through selection and expansion of treatment-resistant clones. Modeling the functional heterogeneity of tumors can unmask critical contributions of distinct tumor cell sub-populations toward identifying rational drug combinations. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that in vitro adherent cultures and in vivo tumors are maintained by a common set of long term self-renewing tumorigenic cells that can be used to establish clonal replica tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of clonal dynamics and adaptive responses to therapeutic challenge over time, we uncovered that the tumorigenic compartment of pancreatic tumors maintains a multitude of functionally heterogeneous subpopulations of cells with differential degrees of sensitivity to therapeutics. High-throughput isolation and deep characterization of unique clonal lineages showed genetic and transcriptomic diversity underlying the functionally diverse subpopulations, positioning the origins of tumor heterogeneity within the long-term self-renewing compartment. Molecular annotation of gemcitabine-naïve clonal lineages with distinct responses to treatment in the context of CRTs generated signatures that can predict the response to chemotherapy and exposed pre-existing functional mechanisms of clonal resistance, primarily associated to DNA damage tolerance and mitochondrial respiration (OXPHOS). Further transcriptomic and metabolic characterization of residual tumor cells in patient derived xenograft models as well as in patients after chemoradiation showed that resistant cells that contribute to tumor relapse are metabolically rewired to upregulate OXPHOS. Combining a novel inhibitor of oxidative phosphorylation (IACS-10759) developed at the MD Anderson Institute for Applied Cancer Science, and currently in phase I clinical trial in acute myeloid leukemia and solid tumors, with standard of care drugs drastically reduces tumor clonal complexity, underscoring the promise of inhibiting mitochondrial respiration as a new therapeutic strategy to prolong patient survival by eradicating resistant clones that survive chemoradiation. Our study, correlating genomic and transcriptomic traits with specific functional phenotypes, uncovered new mechanisms that underlie intra-tumor sub-clonal heterogeneity, influence treatment response to drugs and sustain tumor relapse. Citation Format: Sahil Seth, Chieh-Yuan Li, Sara Loponte, I-Lin Ho, Denise Corti, Luigi Sapio, Edoardo Del Poggetto, Michael Peoples, Tatiana Karpinets, Frederick S. Robinson, Shan Jiang, Prasanta Dutta, Joseph Marszalek, Maria E. Di Francesco, Timothy P. Heffernan, Virginia Giuliani, Pratip K. Bhattacharya, Giannicola Genovese, Andrew Futreal, Giulio Draetta, Andrea Viale, Alessandro Carugo. Dissection of clonal heterogeneity unmasks pre-existing chemoresistance and new metabolic vulnerabilities in pancreatic cancer [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 2900.

MODERN IDEAS ABOUT CLONAL EVOLUTION OF SARCOMAS

The progress in drug treatment of sarcomas is incredibly slow: primary, it is hindered by often developing resistance of tumors to standard chemotherapy with drugs in the maximum tolerated dosages. The clonal evolution of the tumor mass generates a high genetic heterogeneity of cancer cells, which underlies resistance to chemo- and radiotherapy. The consequence of such chemoresistance is local distribution and dissemination of the process, which, in turn, significantly reduces the quality of life of the patient and increases the probability of an adverse outcome of the disease. The purpose of this article is to highlight the modern understanding of the clonal evolution of tumor cells and the effect of this process on the clinical prospects of cancer patients.

Abstract P3-01-09: Tracing clonal evolution of circulating tumor cells and cell-free DNA in a metastatic breast cancer patient

Abstract Background: Copy number variation analysis has been shown as a valuable tool to infer clonal evolution of tumor cells. While bulk genomic analysis of FFPE tissue can provide information about the characteristics of a primary tumor or a metastatic nodule, these biopsies are rarely repeated due to their invasive nature. Liquid biopsies on the other hand, provide minimally invasive access to tumor derived cells and DNA and can therefore be used to monitor genomic changes of the tumor longitudinally and in real time. Here we evaluate the kinetics of liquid biopsy biomarkers and monitor genomic changes in single circulating tumor cells (CTCs) and cell-free DNA (cfDNA) from 17 longitudinal blood draws collected from a metastatic breast cancer patient over seven treatment regimes. FFPE tissue from the primary tumor and two metastatic sites in liver and bone are available for comparative analysis. Methods: We used the high definition – single cell assay (HD-SCA) platform to detect and enumerate CTCs of the blood of one metastatic breast cancer patient followed for 4 years. CTCs were characterized morphometrically and single CTCs of each draw were sequenced for copy number variation (CNV). cfDNA was extracted from plasma and tissue samples of the primary breast, bone and liver metastasis were micro-dissected. Extracted DNA from tissue samples and plasma underwent CNV analysis and were compared with CNV data of CTCs. Results: CTCs were identified in 14/17 draws and varied from 0 to >3000 cells/ml, spiking at two different times concordant with clinical progression. Changes in cell free tumor DNA fraction (ctDNA) correlated strongly with CTC count. Comparative CNV analysis of CTCs, cfDNA and FFPE tissue confirmed the tumorous origin of both cells and cfDNA. Single cell CNV analysis found four genomically related sub-clones in the patient's blood, three of which were present at time of enrollment, while the fourth clone emerged 3.5 years later concordant with the decline in the patient's health. Genomic copy number losses included tumor suppressor genes such as ARID1A, FOXP1, ATM, BRCA2, RB1, CDH11 and CDH1. Genomic gains were limited but included the proto-oncogenes BCL9, ABL2 and MDM4. It is striking that the sub-clonal structure persisted nearly unchanged for 3.5 years throughout various hormone and cytotoxic treatments, indicating high genomic stability. Despite their genomic differences, cells from the different clones are indistinguishable by immunofluorescence or morphometric analysis. CNV profiles of cfDNA reflect the most abundant CTC clone at each time-point. Conclusion: Using the HD-SCA assay cellular (CTC), and soluble (cfDNA) fractions of liquid biopsies can be compared at the molecular level over a long time course, and verified by comparison with tissue biopsy. This work confirms that liquid biopsies can be used to access the molecular state of a patient's cancer in near real-time and provide insight into the response to treatment years after the original characterization of the primary tissue. Here we show in a single patient example the power of single CTC analysis and highlight how CNV analysis of liquid biopsies could serve as minimally invasive tool to monitor tumor evolution longitudinally. Citation Format: Welter L, Xu L, McKinley D, Dago AE, Restrepo-Vassalli S, Rodriguez Lee M, Kolatkar A, Nieva J, Hicks J, Kuhn P. Tracing clonal evolution of circulating tumor cells and cell-free DNA in a metastatic breast cancer patient [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-09.

Pre-existing Functional Heterogeneity of Tumorigenic Compartment as the Origin of Chemoresistance in Pancreatic Tumors

Adaptive drug-resistance mechanisms allow human tumors to evade treatment through selection and expansion of treatment-resistant clones. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that invitro cultures and invivo tumors are maintained by a common set of tumorigenic cells that can be used to establish clonal replica tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of adaptive responses to therapeutics, we uncovered a multitude of functionally heterogeneous subpopulations of cells with differential degrees of drug sensitivity. High-throughput isolation and deep characterization of unique clonal lineages showed genetic and transcriptomic diversity underlying functionally diverse subpopulations. Molecular annotation of gemcitabine-naive clonal lineages with distinct responses to treatment in the context of CRTs generated signatures that can predict the response to chemotherapy, representing a potential biomarker to stratify patients with pancreatic cancer.

Abstract 1177: Clonal dissection of pancreatic tumors unmasks functional and genomic heterogeneous long-term self-renewing compartments at the origin of treatment resistance

Abstract Intrinsic and adaptive drug-resistance mechanisms allow human tumors to evade treatment through the demonstrated expansion of treatment-resistant clones. Thus, tumors are complex, dynamic ecosystems wherein populations of cells harboring both founder clones and unique, subclonal mutations coexist and progressively evolve. Modeling this functional heterogeneity of tumors can uncover critical contributions of distinct tumor cell sub-populations toward identifying rational drug combinations. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that in vitro adherent cultures and in vivo tumors are maintained by a common set of long-term self-renewing cells that can be used to establish Clonal Replica Tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of clonal dynamics and adaptive responses to therapeutic challenge across different animals over time, we uncovered that the long term self-renewing compartment of pancreatic cancer is represented by a multitude of functionally heterogeneous subpopulations of cells with differential degrees of sensitivity to therapeutics. Consistent with the stem cell hypothesis, although tumors respond to treatments and undergo a transient regression, their clonal complexity at the time of relapse is only partially compromised, implying that many tumorigenic cells survive the treatment and sustain tumor relapse. Moreover, our ability to track the same cell populations in different animals enabled us to demonstrate that the clonal composition of relapsed pancreatic tumors varied across the different drug treatment groups (gemcitabine, MEK1 inhibitor and dual PI3K/mTOR inhibitor), suggesting that the compartment of long-term self-renewing tumorigenic cells is highly functionally diverse in mediating drug resistance to different therapies. Notably, high-throughput isolation and deep characterization of unique clonal lineages isolated through CRTs demonstrated that individual self-renewing populations display a remarkable genetic and molecular heterogeneity that can account for the differential functional responses and adaptation to perturbations. So, our findings portend a model in which the genomic and functional heterogeneity within human tumors is maintained, propagated and recapitulated entirely by distinct pools of long-term self-renewing cells. This concept has important implications for the efficacy of pharmacological combinations, which has historically been ascribed to their synergistic effects to abrogate the emergence of resistance, may instead be linked to the ability of mechanistically unrelated drugs to delay relapse by targeting multiple populations of tumorigenic cells simultaneously. Citation Format: Sahil Seth, Chieh-Yuan Li, I-Lin Ho, Denise Corti, Sara Loponte, Luigi Sapio, Edoardo Del Poggetto, Michael Peoples, Tatiana Karpinets, Giannicola Genovese, Andrew Futreal, Giulio Draetta, Alessandro Carugo, Andrea Viale. Clonal dissection of pancreatic tumors unmasks functional and genomic heterogeneous long-term self-renewing compartments at the origin of treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1177.

Monitoring of treatment responses and clonal evolution of tumor cells by circulating tumor DNA of heterogeneous mutant EGFR genes in lung cancer

ObjectivesEpidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have dramatic effects on EGFR-mutant non-small-cell lung cancer (NSCLC). However, most patients experience disease recurrences, approximately half of which are T790M-mediated. Monitoring EGFR status with re-biopsy has spatiotemporal limitations. Patients and methodsEGFR circulating tumor DNA (ctDNA) in serial plasma samples was amplified and 105 of them were sequenced with a next-generation sequencer. Plasma mutation (PM) score was defined as the number of reads containing deletions/substitutions in 105EGFR cell free DNA (cfDNA). ResultsPM scores of various EGFR mutations showed dynamic, case-specific changes during EGFR-TKI treatments in 52 patients. The effects of the treatment on EGFR ctDNA were evaluated in 38 patients with elevated pre-treatment PM scores. The ctDNA responses correlated well with radiologic responses in radiologic good responders, whereas correlation was poor in non-responders. In addition to the peaks for the most prevalent ctDNA, small peaks of ctDNA with different types of activating EGFR mutations or the T790M mutation (early T790M ctDNA) appeared transiently in 10.5% and 26.3%, respectively. Early T790M ctDNA disappeared in all patients, including 7 who eventually developed acquired resistance accompanied by elevated levels of T790M ctDNA. ConclusionsMonitoring ctDNA is useful in evaluating treatment responses and monitoring driver oncogene status in NSCLC. ctDNA revealed clonal heterogeneity and genetic processes of cancer evolution in individual patients. The simple presence of the T790M mutation may be insufficient to confer EGFR-TKI resistance to tumor cells.

Genetic Heterogeneity and Clonal Evolution of Tumor Cells and their Impact on Precision Cancer Medicine

The efficacy of targeted therapies in leukemias and solid tumors depends upon the accurate detection and sustained targeting of initial and evolving driver mutations and/or aberrations in cancer cells. Tumor clonal evolution of the diverse populations of cancer cells during cancer progression contributes to the longitudinal variations of clonal, morphological, anatomical, and molecular heterogeneity of tumors. Moreover, drug-resistant subclones present at initiation of therapy or emerging as a result of targeted therapies represent major challenges for achieving success of personalized therapies in providing meaningful improvement in cancer survival rates. Here, I briefly portray tumor cell clonal evolution at the cellular and molecular levels, and present the multiple types of genetic heterogeneity in tumors, with a focus on their impact on the implementation of personalized or precision cancer medicine.

Genome Wide SNP Array (SNPa) Analysis Reveals Clonal Evolution During Clinical Course in Waldenstrom's Macroglobulinemia (WM)

AbstractAbstract 297 Background.Current models of cancer progression are based on evolution and clonal selection i.e. evolution of tumour cells inducing expansion of cells that acquire genetic lesions over time, related to ongoing mechanisms of genomic instability. An initiating event may be followed by gene mutation, copy number alteration (CNA) or copy neutral loss of heterozygosity (CN-LOH) that drive tumor progression and emergence of mechanisms of resistance to drugs. WM is a lymphoproliferative disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells that secrete monoclonal IgM antibody. The clinical course is characterized either by an indolent or smoldering status or a symptomatic profile that needs chemotherapy to control the progression of tumour cells. The majority of patients (pts)evolved from indolent to symptomatic, and the mechanisms of progression of WM are not fully understood to date. We hypothesized that we could gain insights into clonal evolution underlying disease progression of WM on a paired serial analysis of WM samples using genome wide SNPa, that allow both the detection of LOH and CNA. Method.BM samples of 19 untreated pts with WM (12 males, mean age: 67 years, 11 symptomatic pts) were analysed. All patients had Genome-Wide Human SNP Array 6.0 (Affymetrix chips) on at least two sequential tumor samples > 6 months apart (42 samples from 19 patients with two to three points). Tumoral DNA was extracted following CD19 B cells selection. Paired samples (tumor/normal T lymphocytes) were used as an intra-individual reference to identify germline polymorphisms. Size, position and location of genes were identified with UCSC Genome Browser HG18 assembly, LOH and CNA using genotyping console 3.02 software (Affymetrix). FISH analysis was performed to detect deletion 6q; 13q14, 11q22, TP53, trisomy 4 and 12 chromosomal aberrations using Vysis probes. P53 and MYD88 mutation were analyzed by sanger sequencing. Results.At initial sampling, SNPa detected a total of 76 CNA genetic aberrations (range 0 – 24 per genome) including 22 gains and 54 losses; 85% of patients had MYD88 L265P mutation. During the follow-up of all indolent WM that remained indolent, we haven’t observed any new genetic aberrations, gain or loss (either CNA or CN-LOH) (n=8, mean follow-up: 63 months, range: 16–107 months). Among the 11 remaining patients (mean follow-up: 44.2 months, range: 7–92 months), a clonal evolution was observed in 6 cases. Three of them were symptomatic and acquired CNA or CN-LOH during clinical evolution: one case with acquisition of several CN-LOH, including one at 3p22 involving MYD88 locus associated to a mutation, one case with an additional deletion 7q31 which was observed in a patient with a chromothripsis at diagnosis, one case with emergence of subclonal del17p. The two remaining patients evolved from smoldering to symptomatic with a major variation in CNA number. Finally, loss of CNAs displayed at diagnosis was observed in one patient after chemotherapy and during indolent follow-up. In 5 cases, no detectable CNA changes were identified between the initial and subsequent sample at relapse. Conclusion.Our results using high resolution SNP array support the hypothesis that a symptomatic WM disease will favour genetic clonal evolution of tumor cells. In our study, 3 distinct genotypic patterns were observed: (i) absence of genomic variation in stable smoldering WM disease or in symptomatic relapsed patient. (ii) unstable genotype in symptomatic or asymptomatic patients switching to symptomatic suggesting high risk tumors that are less stable and more prone to change with time. (iii) loss of abnormalities suggesting that chemotherapy eradicated the dominant clone or that a genetically distinct relapsed clone has emerged. This study refines our understanding of the dynamic genetic changes in progressive WM. Further confirmation of the role of some candidate genes is underway. Disclosures:No relevant conflicts of interest to declare.