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Background: Neoadjuvant anti-PD-(L)1 therapy confers an improvement in pathological complete response (pCR) rate in unselected TNBC. However, given the potential for long-term ...morbidity from immune related adverse events (irAE), it is important to optimize the risk-benefit ratio for the use of these novel agents in the curative neoadjuvant setting. Suboptimal clinical response to neoadjuvant therapy (NAT) by sonography is associated with low rates of pCR rate (2-5%, GeparTrio and Aberdeen trials). Here, we report the results of a single arm phase II study of atezo and nab-p as the second phase of NAT in pts with TNBC with suboptimal clinical response to AC (NCT02530489). Methods: Pts with stage I-III TNBC showing suboptimal response to 4 cycles of doxorubicin and cyclophosphamide (AC), defined as disease progression or a <80% reduction in tumor volume by sonography, were eligible. Pts received atezo (1200mg IV, Q3 weeks x 4), and nab-p (100mg/m2 IV, Q1 week, x 12) as the second phase of NAT before undergoing surgery followed by adjuvant atezo (1200mg IV, Q3 weeks, x 4 cycles). This single arm, two-stage Gehan-type study was designed to detect an improvement in pCR from 5% to 20% in order to deem the regimen worthy of further study in a large, randomized, phase II/III trial; success was defined as pCR in 8 out of 37 pts enrolled. In a subset of pts, sufficient baseline tumor tissue was available for stromal TIL assessment (n=29). Results: 34 pts were enrolled from 2/2016-12/2020. Among the 33 pts who have completed NAT, the pCR rate was 30% (10/33, 95% CI: 16-49%) and the pCR/RCB-I rate was 42% (14/33, 95% CI: 25-61%). Clinicopathological characteristics are described in the table below. Treatment-related adverse events (all grades) occurring in ≥ 20% of pts include fatigue (73%), anemia (55%), peripheral sensory neuropathy (55%), neutropenia (48%), rash (42%), ALT elevation (39%), AST elevation (33%), nausea (30%), anorexia (24%), diarrhea (21%), myalgia (21%). Discontinuation of atezo due to irAEs occurred in 4 pts (12%, nephritis n=2; adrenal insufficiency n=1; hepatitis n=1); 2 of these pts had pCR. Conclusions: This study met its primary endpoint, demonstrating a promising signal of activity in this high risk pt population (pCR=30% vs 5% in historical controls). The 12% discontinuation rate due to irAEs confirms that further evaluation of a strategy administering immunotherapy only to pts with high risk disease not responding to AC warrants further investigation. Exploratory genomic and immunological correlative studies are ongoing. Clinical trial information: NCT02530489. Table: see text
Abstract
Background: The LAR subtype of triple negative breast cancer (TNBC) was defined using the Vanderbilt genomic signature in cell lines. The characteristics of this population remain unclear.
...Methods: We collected the clinicopathological, molecular and imaging characteristics of the LAR population who were enrolled in the ARTEMIS trial, a prospective trial to treat women with TNBC to receive neoadjuvant anthracycline therapy followed by experimental arms based on biomarker versus taxane. The ultrasonography-based response assessment was performed at baseline, after 2 and 4 cycles of anthracycline chemotherapy. The Vanderbilt genomic signature was used to classify the TNBC into the basal-like 1, basal-like 2, immunomodulatory, mesenchymal, mesenchymal stem-like and luminal androgen receptor (LAR) subtypes. The clinical data for this analysis included age, race, menopausal status, TNM stage and BRCA mutation status. Pathological and molecular characteristics included histologic subtype, nuclear grade, Ki67, vimentin expression, androgen receptor (AR) immunohistochemical (IHC) nuclear staining, stromal tumor infiltrating lymphocytes (TIL) percentage and residual cancer burden (RCB) at surgery.
Results: Total 28 patients with the LAR signature were analyzed. The characteristics are showed in the table:
AgeRaceno. (%)Clinical Stageno. (%)AR IHC StainingBRCA Mutationno. (%)Median (yr)55White19 (68)IA1 (4)Median (%)52No mutation16 (57)Distribution(%)no. (%)Asian3 (11)IIA6 (21)Distribution (%)no(%)Mutation present018-402 (7)Black2 (7)IIB10 (36)<259 (32)VUS1 (4)41-6012 (43)Hispanic3 (11)IIIA6 (21)25-505 (18)Test not indicated6 (21)>6014 (50)American Indian1 (3)IIIB051-751 (3,5)Unknown5 (18)IIIC5 (18)76-10012 (43)Not available1 (3,5)Menopauseno. (%)Histologic subtypeno. (%)Ki67Stromal TILno. (%)Vimentin expressionno. (%)Postmenopausal23 (82)IDC24 (87)Median (%)40<5%10 (36)< 1%24 (85)Perimenopausal2 (7)ILC2 (7)Distribution (%)no. (%)5% - <50 %16 (57)≥1%3 (11)Premenopausal3 (11)Apocrine Carcinoma1 (3)<205 (18)≥50%1 (3,5)N/A1 (4)Metaplastic carcinoma1 (3)≥2013 (46)N/A1 (3,5)N/A10 (36)T stageno. (%)Lymph node involvementno. (%)Nuclear gradeno. (%)Tumor reduction (%)no. (%)RCBno(%)T1c3 (11)Yes22 (79)11 (4)≥ 7012 (43)pCR-111 (39)T213 (46)No6 (21)26 (21)< 7014 (50)2-315 (54)T311 (39)321 (75)N/A2 (7)N/A2 (7)T41 (4)Experimental armno. (%)Enzalutamide8 (29)Atezolizumab4 (14)None16 (57)
Relevant characteristics are: median age of 55 years, 82% postmenopausal, 89% breast tumor size > 2cm, 79% with lymph node involvement, no BRCA mutation detected, 75% nuclear grade III, 46% with Ki67 ≥ 20%, median AR IHC staining of 52%, 85% vimentin expression of <1%, 93% had stromal TIL <50%, 39% RCB 0-1 and 54% RCB 2-3. We found that 43% had ≥ 70% reduction in size after 4 cycles of AC. Univariate and multivariate analysis using characteristics did not reveal contributing factor to the RCB.
Conclusion: The LAR subgroup harbors unique characteristics that require further confirmation in larger cohorts.
Citation Format: James Crespo, Seth Sahil, Elizabeth Ravenberg, Lei Huo, Kenneth Hess, Lumarie Santiago, Beatriz Adrada, Gaiane Rauch, Damodaran Senthil, Rashmi Murthy, Jennifer Litton, Debu Tripathy, Naoto Ueno, Stacy Moulder, Bora Lim. Characterization of the LAR subtype triple negative breast cancer population 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 1409.
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Background: The heterogeneity of TNBC results in varied responses to NAST: 30-40% of patients (pts) have pathologic complete response (pCR) with excellent prognosis. Those with ...residual disease, have a much higher risk of recurrence. Longitudinal profiling assesses biologic response to NAST and mechanisms of resistance. Methods: Pts with stage I-III TNBC began a planned 4 cycles of Adriamycin-based chemo (AC). Biopsies were performed pre (mandatory) and post (optional) AC. Volumetric change by ultrasound (VUS) at completion of AC (or progression) was calculated. Pts with sensitive disease received subsequent taxane-based (T) therapy. Pts with insensitive disease were offered phase II trials. Pathologic response was assessed at surgical resection in 47 pts. Matched samples, pre and post AC (N = 48 pts) underwent transcriptomic and genomic profiling. Samples were classified into six previously identified ARTEMIS subtypes of TNBC (ART-Type). Immune deconvolution and estimation was performed using RNA-Seq profiles. Differential pathway-level analysis was performed comparing pre and post AC samples. Results: There was heterogeneity in response to AC with 4 predominate patterns of biologic response (Table). In 48% of cases the ART-Type of the tumor switched after AC, with androgen receptor like (LAR) and immune modulatory (IM) showing greatest stability. Tumors with enrichment in EMT or those with no significant dysregulation after AC (Groups C + D) were associated with less immune modulation and lower rates of pCR compared to those with depleted EMT (A and B) (8.7% vs 45.8%, p = 0.0078). Conclusions: Molecular profiling of longitudinal TNBC samples reveals distinct response patterns in tumors and their micro-environments upon treatment with AC. These patterns were indicative of pathologic response in this cohort; however, they require validation in a separate cohort. Clinical trial information: NCT02276443. Table: see text
Abstract
Chemotherapy/ targeted therapy are both known to trigger evolution of treatment resistant clones that can lead to relapse. Allogeneic stem cell transplant (alloSCT) for refractory Chronic ...Lymphocytic Leukemia (CLL) patients is associated with better outcomes. We hypothesized that allogeneic T-cell immunotherapies, including alloSCT and donor lymphocyte infusion (DLI) would impact tumor evolution through the application of selective immunologic pressure with reciprocal changes in the T-cell compartment. Here, we tested a cohort of 24 heavily pre-treated CLL patients treated. Treatments consisted of alloSCT alone, or with follow-up DLI, which are two established mediators of effective Graft versus Leukemia (GVL). Our cohort included 11 patients who relapsed (denoted as non-responder, NR) after alloSCT and 13 patients who had complete response (CR) after alloSCT, with 11/13 patients showing durable CR with a median post-transplant overall survival (OS) of 9.8 years. We mapped the evolutionary trajectories of tumor cells by whole exome sequencing (WES) of sort purified CLL in post-transplant relapsed patients. To investigate changes in immune repertoire and gene expression post-transplant, CD3 positive T-cells from peripheral blood and bone marrows of CLL patients at complete donor chimerism were analyzed both at bulk and at the single cell level. We found evidence of subclonal leukemic evolution in the majority of our CLL patient cohort after nonmyeloablative HLA-matched alloSCT. Different patterns of CLL evolution were observed, and these changes included putative CLL drivers in every case. In all of the 11 patients with longitudinal post-alloSCT samples available, we observed branched CLL evolution in 4 patients, linear evolution in 4 patients, and no evolution in 3 patients. These data suggest that differential sensitivity of leukemic subclones to allogeneic T cell killing may underlie the branched and linear evolution that we observed, and therefore can shape leukemic subclonal architecture after transplant. Of note, we found that clonal CLL was more responsive to alloSCT in comparison to CLL with subclonal disease architecture.To identify T-cells with GVL potential, we first cataloged potential neoantigens by screening mutated regions in CLL with in silico HLA binding prediction models. Neoantigen specific T-cells were then sorted from longitudinal peripheral blood samples using tetramers, followed by identification of GVL specific TCR in both bulk and single cell setting. We were able to identify T-cells that coevolved with specific tumorigenic lesions in a subset of CLL patients. Taken together, our results suggest that donor-derived antigen-specific T-cells mediate clonal selection of CLL with concurrent changes in allogeneic T-cells, and that these changes can be monitored in longitudinal patient samples.
Citation Format: Celine Kerros, John P. Miller, Xizeng Mao, Haven R. Garber, Hannah C. Beird, Jianhua Zhang, Jason Roszik, Paul Leonard, Li Zhao, Sahil Seth, Pei Lin, Huandong Sun, William G. Wierda, Issa F. Khouri, Karen Clise-Dwyer, Andrew Futreal, Shoudan Liang, Koppikar Priya, Jeffrey Molldrem. Deep profiling of T-cell repertoire and tumor heterogeneity in chronic lymphocytic leukemia patients following allogeneic T-cell therapy abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1516.
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Background: The luminal androgen receptor (LAR) subtype of TNBC has a low pathologic complete response (pCR) rate after NAT. We determined the pCR rate of the enzalutamide and ...paclitaxel (ZT) regimen for pts with anthracycline-insensitive AR
+
TNBC (NCT02689427), and related biomarkers. Methods: ARTEMIS (NCT02276443) is a non-randomized trial to determine if NAT can be used to personalized therapy. Pts received 4 cycles of doxorubicin-based NAT (AC). Pts with insensitive disease by imaging were offered clinical trials as the second phase of NAT based upon molecular profiling of pre-treatment biopsies. Immunohistochemistry (IHC) of AR
+
≥10% was the threshold for selecting ZT (enzalutamide 160 or 120 mg PO qD + paclitaxel 80 mg/m
2
qW for 12 cycles). pCR was determined by surgery after NAT. Trial had two-stage Phase II design, and we report the completed first stage. We evaluated the concordance between Vanderbilt LAR subtype by molecular profiling (microarray and RNAseq) and IHC %AR
+
cells. Frequency of PI3K pathway alterations within the LAR subtype was assessed. Results: 267 pts had tumors profiled by IHC, 220 by microarray, 187 by RNAseq and 197 by whole exome sequencing. 96 pts had post-AC RNAseq. LAR scores from both RNAseq and microarray profiling (n = 139) were highly concordant (R = 0.89, P < 0.001) and identified ~10% of TNBCs tested as LAR. The %AR
+
cells from IHC correlated with LAR subtype scores according to RNAseq (R = 0.6, P < 0.001), with a cut-point of ≥30% AR
+
having the best concordance with LAR subtype. Unlike other subtypes, by serial profiling, LAR TNBCs did not change subtype signatures after exposure to AC. LAR TNBCs had low rates of pCR (23%) and high rates of PI3K pathway activating aberrations (85%); however PI3K aberrations did not correlate with pCR. Seventeen patients with AC-insensitive TNBC received ZT. Five of 15 patients (33.3%) had responses (pCR or RCB-I). Toxicities are Grade (Gr) 4 syncope (n = 1), Gr3 abnormal liver function (n = 2), Gr3 neutropenia (n = 4). IHC & LAR subtype scores did not statistically associate with response to ZT (P = 0.8, P = 0.9). However, all responders to ZT had an upregulated androgen response pathway (ssGSEA Z > 1) as measured by transcriptomic analysis in pre-treatment biopsies analysis (P = 0.05, ppv = 0.56, npv = 1). Conclusions: The LAR TNBC subtype has a low pCR rate to NAT. Among pts with AC-insensitive TNBC, baseline upregulated androgen response pathway and LAR subtype may benefit from the ZT regimen, potentially by PI3K targeting. Clinical trial information: NCT02689427 .
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Background: In TNBC patients (pts) receiving NAST, increasing tumor infiltrating lymphocytes (TILs) is associated with higher pathologic complete response (pCR) rates. However, ...since the presence of TIL do not consistently predict pCR, the current study was undertaken to more fully characterize the immune cell response and its association with pCR. Methods: T cell receptor (TCR) sequencing, PD-L1 immunohistochemistry and multiplex immunofluorescence were performed on prospectively collected pre-NAST tumor samples from 98 pts with stage I-III TNBC enrolled in ARTEMIS (NCT: 02276443). TCR clonality was calculated using Shannon’s entropy. PD-L1+ was defined as ≥1% immune cell staining. Response to NAST was defined using the residual cancer burden (RCB) index. Associations between TCR clonality, immune phenotype, and response were examined with the Wilcoxon rank sum test, Spearman’s rank correlation and multivariable logistic regression using stepwise elimination (threshold p > 0.2), as appropriate. Results: The pCR rate was 39% (38/98). pCR was associated with higher TCR clonality (median = 0.2 in pts with pCR vs 0.1 in pts with residual disease, p = 0.05). Notably, the association between pCR and higher TCR clonality was observed in pts with ≥5% TIL (n = 61; p = 0.05) but not in pts with < 5% TIL (n = 37; p = 0.87). Among pts with ≥5% TIL, TCR clonality emerged as the only independent predictor of response in a multivariable model of tumor immune characteristics (odds ratio/0.1 increase in TCR clonality: 3.0, p = 0.021). PD-L1+ status was associated with higher TCR clonality (median = 0.2 in PD-L1+ vs 0.1 in PD-L1-, p = 0.004). Higher TCR clonality was associated with higher CD3+ (rho = 0.32, p = 0.0018) and CD3+CD8+ (rho = 0.33, p = 0.0013) infiltration but lower expression of PD-1 on CD3+ (rho = -0.24, p = 0.021) and CD3+CD8+ cells (rho = -0.21, p = 0.037). Conclusions: In TNBC, a more clonal T cell population is associated with an immunologically active microenvironment (higher CD3+ and CD3/8+ T cell; lower PD-1+CD3+ and PD-1+CD3/8+ T cell; PD-L1+) and favorable response to NAST, especially in pts with ≥5% TIL, suggesting a role for deep immune phenotyping in further refining the predictive value of TILs.
Abstract
Despite the impressive clinical efficacy of immunotherapy in some patients, many still do not respond or progress following an initial response. The molecular mechanisms underlying the tumor ...resistance in those non-responders remain largely undefined. To address this issue, we set out to perform high-throughput unbiased pooled shRNA screens to identify critical genes that confer immune resistance. Patient-derived melanoma cells were transduced with barcoded pooled lentiviral shRNA libraries that targeted the human kinome, followed by exposure to cytotoxicity mediated by autologous tumor infiltrating lymphocytes. Tumor cells were then subject to genomic deep sequencing and integrated analysis to identify depleted barcodes and corresponding genes. One identified candidate of particular interest is Aurora Kinase A (AURKA), a cell cycle regulator that has previously been shown to contribute to tumorigenesis and correlate with poor prognosis for cancer patients. Importantly, our independent screening with a bioactive compound library also implicated Aurora Kinase as an immune resistance candidate against T cell immunotherapy. Further studies showed that suppression of Aurora kinase activity with a pan inhibitor - AMG900, exhibited a synergistic cytotoxic effect with tumor infiltrating lymphocyte-mediated killing on autologous tumor cells from patients. Furthermore, our Nanostring analysis of tumor biopsies from patients that received adoptive T cell therapy revealed significantly increased expression of AURKA in tumors from non-responding patient when compared with responder counterparts. These results have substantiated the validity of our pooled shRNA screening platform. Further investigations are ongoing to elucidate the underpinnings of Aurora kinase-mediated tumor resistance to immunotherapy and to functionally and physiologically validate other putative targets we have identified.
Citation Format: Zhe Wang, Shruti Malu, Weiyi Peng, Jodi McKenzie, Rina Mbofung, Leila Williams, Sahil Seth, Tim Heffernan, Patrick Hwu. Systems-level interrogation of resistance mechanisms to immunotherapy through pooled shRNA screens. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1441.
Abstract
Triple-negative breast cancer (TNBC) is an aggressively metastatic subtype that can only be treated by chemotherapy. Nearly 50% of patients have residual disease after neoadjuvant ...chemotherapy (NACT) and have extremely poor prognoses. Recent genome sequencing studies have revealed extensive intratumoral heterogeneity (ITH) in treatment-naïve TNBC. However, the functional contribution of ITH to chemoresistance in TNBC is unknown. To understand how this occurs, we are generating patient derived xenograft (PDX) models from treatment-naïve TNBC in order to identify and characterize tumor cell populations that are responsible for chemoresistance with the ultimate goal of selectively targeting resistant tumor clones.
Through an IRB-approved clinical trial using standard techniques, samples (primary tumor, skin metastasis, and blood for germline reference) were obtained from a patient with newly diagnosed, untreated metastatic TNBC. Subsequently, this patient was found to have disease resistant to chemotherapy. Tumor cells were implanted into the humanized mammary fat pad of NOD/SCID mice to establish PDX models of the primary (PIM1-P) and metastatic (PIM1-M) tumors. RNA sequencing and whole-exome sequencing (∼300X) were performed on the patient's primary and metastatic tumors and the first and third passage PDX tumors. Mouse sequences were computationally subtracted from the PDX data and which was then processed according to Genome Analysis Toolkit best practices workflow. Variants were called using MuTect and copy number alterations were estimated by ExomeCN. This revealed high concordance between the genomic profiles of the patient and PDX models. While there were 81 somatic non-silent mutations shared in the patient and PDX, only a few fell in known cancer genes, including TP53 (V143fs), ELF4 (L593H), and ARID3A (R351P). Modeling of clonal clusters with ABSOLUTE revealed ITH in the patient tumor that is preserved in the PDX.
Both the patient and PDX model (PIM1-P) exhibited progressive disease when treated with paclitaxel. Only partial responses were observed in mice treated with doxorubicin plus cyclophosphamide (AC), consistent with residual disease after standard NACT for patients with TNBC. AC treatment resulted in an ∼60% reduction in tumor volume which was not enhanced by repeated cycles. Tumors rapidly re-grew when treatment was halted or if treated tumor cells were engrafted into the mammary fat pads of new recipient mice. This suggests that a subpopulation of AC-resistant tumor cells is present in PIM1-P tumors. To dissect this subpopulation, we established conditions for simultaneous tracking of thousands of PIM1-P tumor clones in vivo using a high-complexity library of up to 30 million unique DNA barcodes. Mice engrafted with barcoded PIM-1P tumors are being treated with AC to identify and characterize AC-resistant tumor cells present in PIM1.
Citation Format: Gloria V. Echeverria, Sahil Seth, Shirong Cai, Stacy Moulder, William F. Symmans, Timothy P. Heffernan, Jeffrey Chang, Helen Piwnica-Worms. Identifying and targeting chemoresistant subclones in triple negative breast cancer. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2406.
Abstract
Colorectal cancer (CRC) is a leading cause of cancer-related morbidity and mortality with significantly heterogeneous features and drug responses. Recently, the international Colorectal ...Cancer Subtyping Consortium identified four robust consensus molecular subtypes of CRC (CMS1-4) using large-scale gene expression data. These findings may enable us to identify molecularly homogenous subsets of CRC patients and accelerate effective drug development strategies. To identify potential therapeutic targets and novel selective vulnerabilities in CRC molecular subtypes, we developed an in vivo loss-of-function genomic screen using CRC patient-derived xenografts (PDXs) for each molecular subtype. Our PDX-derived CRC models underwent comprehensive integrated molecular characterization of mRNA profiles, DNA mutations, and histochemical profiles upon confirmed serial retransplantation to determine whether characteristics of the subtypes are recapitulated in vivo. Because the original CMS classification algorithm was trained and validated using Affymetrix data, profiling the PDX-derived cell lines using this technology provided the most robust analysis of the CMS subtypes. In vivo pooled short hairpin RNA (shRNA) screens rely on specific elimination of individual shRNAs in a cell population and require that the infected tumor cell population is adequately endowed with engraftment capacity when implanted into recipient mice. Therefore, we determined the transduction efficiency of the PDX models, the frequency of tumor-initiating cells, and the maximum library complexity allowed by each model. To identify targets that represent selective vulnerabilities in specific CRC molecular subtypes, we screened each model in vivo with an shRNA library targeting about 200 genes specifically belonging to U.S. Food and Drug Administration-approved targeted therapies (FDAome; 10 shRNAs/gene ). We leveraged redundant shRNA activity analysis to evaluate “hits” (or top-scoring genes) emerging from our screening. We further applied ranking-based analytics in combination with integromic approaches (use of computational packages to unravel relationships between -omics) to inform on selective CMS specific top-scoring genes. One of the benefits of using an FDAome library is the direct correspondence of target genes with clinically available drugs. We therefore tested these drugs for validation in fully annotated PDXs. These efforts, in association with systematic profiling of the CMS subtypes at the patient level through adaptation of NanoString technology, may enable us to stratify CRC patients who will benefit from selective U.S. Food and Drug Administration-approved drugs and to rapidly design successful preclinical and clinical trials in CRC patients.
Citation Format: Akira Inoue, Bahar Salimian Rizi, Alessandro Carugo, Sahil Seth, Christopher Bristow, Giannicola Genovese, Andrea Viale, David G. Menter, Scott Kopetz, Giulio F. Draetta. Identifying selective vulnerabilities in colorectal cancer molecular subtypes using in vivo functional genomic screens abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 414. doi:10.1158/1538-7445.AM2017-414
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.
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