Consequences Of Intratumor Heterogeneity Research Articles

Abstract P1-06-07: Clinical and molecular relevance of intra-tumor genetic heterogeneity in breast cancer: Integrative analysis of data from The Cancer Genome Atlas

Abstract Background: Intra-tumor heterogeneity (ITH) plays a pivotal role in driving breast cancer progression and therapeutic resistance. Emerging evidence has indicated that the extent of genetic heterogeneity may serve as a clinically useful biomarker. While several studies have suggested the prognostic value of ITH in several cancer types, the clinical significance of genetic ITH and molecular portraits that correlated with different ITH levels were poorly understood in breast cancer. The establishment of algorithms estimating genetic ITH based on sequencing of bulk tumor DNA offered us an opportunity to explore the clinical implication of ITH in large breast cancer cohorts and, for the first time, to use integrative genomic analyses to reveal molecular portraits related to intra-tumor genetic heterogeneity. Methods: We assessed 916 female breast cancer patients from The Cancer Genome Atlas. Mutant-allele tumor heterogeneity (MATH) values were calculated from whole-exome sequencing data. We used integers nearest to the tertiles of the MATH values as cutoff points to divide the patients into three groups nearly equal in size. The association between MATH value and clinical characteristics was evaluated, followed by survival analyses in these different MATH groups. We then compared the rates of total non-silent somatic mutations among the different MATH groups, and further determined the mutations independently associated with high MATH by logistic regression adjusting for T classification and clinical subtypes. Similar methods, superadding somatic copy number alteration (SCNA) burden in logistic model, were used to evaluate SCNA events that were significantly associated with high MATH level. Gene enrichment between the high and rest MATH groups was analyzed using Gene Set Enrichment Analysis. Results: The patients were divided into low (MATH value lower than 33), intermediate (MATH between 33 and 46) and high (MATH higher than 46) MATH groups. High T stage, African American race, and triple-negative or basal-like subtype were associated with a higher MATH level (all P<0.001). In hormone receptor-positive and human epidermal growth factor receptor-negative patients, the high MATH group showed a tendency toward a worse overall survival (P=0.052); however, while in triple-negative breast cancer, both high and low MATH indicated a worse outcome (P=0.032). Furthermore, the TP53 mutation rate increased as MATH was elevated (P<0.001), whereas CDH1 mutations were correlated with a lower level of MATH (P=0.002). Several focal and arm-level SCNA events were more common in the high MATH group, including Chr8q24 with only the MYC gene in the “peak” region. Similarly, high MATH was associated with gene set enrichment related to the MYC pathway and proliferation. Conclusion: Our study extended the knowledge concerning the clinical role of ITH in breast cancer, especially the distinct pattern of prognostic values in different clinical subtypes, which may help promote the clinical utilization of genetic ITH. Our attempt at exploring the molecular features related to ITH might provide clues for the source and consequences of ITH, inspiring subsequent experiments investigating the laws underling tumor heterogeneity. Citation Format: Ma D, Jiang Y-Z, Liu X-Y, Liu Y-R, Yu K-D, Shao Z-M. Clinical and molecular relevance of intra-tumor genetic heterogeneity in breast cancer: Integrative analysis of data from The Cancer Genome Atlas [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-06-07.

Abstract 2386: Microfluidic single cell exome-seq and RNA-seq analysis of tumor composition

Abstract Human breast tumors have been shown to exhibit extensive inter- and intra-tumor heterogeneity. While recent advances in genomic technologies have allowed us to deconvolute this heterogeneity, few studies have addressed the functional consequences of diversity within tumor populations. Here, we identified an index case for which we have derived a patient-derived xenograft (PDX) as a renewable tissue source to identify subpopulations and perform functional assays. On pathology, the tumor was an invasive ductal carcinoma which was hormone receptor-negative, HER2-positive (IHC 2+, FISH average HER2/CEP17 2.4), though the FISH signal was noted to be heterogeneous. On gene expression profiling of bulk samples, the primary tumor and PDX were classified as basal-like. We performed single cell RNA and exome sequencing of the PDX to identify population structure. Using a single sample predictor of breast cancer subtype, we have identified single basal-like, HER2-enriched and normal-like cells co-existing within the PDX tumor, a finding replicated in several independent experiments. Genes differentially expressed between these subpopulations are involved in proliferation and differentiation. One population was characterized by high MYC and the other high EGFR/KRT14. These findings were validated using immunohistochemistry on PDX and primary tumor material. Further functional experiments showed that that EGFR subpopulation has stem cell character istics and forms metastasis in the animal model. Microfluidic whole genome amplification followed by whole exome capture of 81 single cells, along with exome sequencing of the germline, primary and post treatment tumor and whole PDX showed that BRCA1 and TP53 are mutated in all single cells, as well as a number of sub-clonal mutations that are being investigated further. Loss of heterozygocity was observed in 16 TCGA cancer driver genes and novel mutations in 7 known cancer driver genes. Careful comparison of the exome sequencing data allowed the association of driver gene mutation prevalence with tumor progression. These findings are important in our understanding the functional consequences of intra-tumor heterogeneity with respect to clinically important phenotypes such as invasion, metastasis and drug-resistance. Citation Format: Ioannis Ragoussis, Paul Savage, Yu-Chang Wang, Timothee Revil, Dunarel Badescu, Sadiq Saleh, Ernesto Iacucci, Nicolas Bertos, Anie Monast, Attila Omeroglou, Dongmei Zuo, Morag Park. Microfluidic single cell exome-seq and RNA-seq analysis of tumor composition. [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 2386.

Pan-cancer analysis of the extent and consequences of intratumor heterogeneity.

Intratumor heterogeneity (ITH) drives neoplastic progression and therapeutic resistance. We used the bioinformatics tools 'expanding ploidy and allele frequency on nested subpopulations' (EXPANDS) and PyClone to detect clones that are present at a ≥10% frequency in 1,165 exome sequences from tumors in The Cancer Genome Atlas. 86% of tumors across 12 cancer types had at least two clones. ITH in the morphology of nuclei was associated with genetic ITH (Spearman's correlation coefficient, ρ = 0.24-0.41; P < 0.001). Mutation of a driver gene that typically appears in smaller clones was a survival risk factor (hazard ratio (HR) = 2.15, 95% confidence interval (CI): 1.71-2.69). The risk of mortality also increased when >2 clones coexisted in the same tumor sample (HR = 1.49, 95% CI: 1.20-1.87). In two independent data sets, copy-number alterations affecting either <25% or >75% of a tumor's genome predicted reduced risk (HR = 0.15, 95% CI: 0.08-0.29). Mortality risk also declined when >4 clones coexisted in the sample, suggesting a trade-off between the costs and benefits of genomic instability. ITH and genomic instability thus have the potential to be useful measures that can universally be applied to all cancers.

Abstract A1-54: Pan-cancer analysis of the etiology and consequences of intratumor heterogeneity

Abstract This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR03) of the Conference Proceedings. Citation Format: Noemi Andor, Trevor A. Graham, Claudia Petritsch, Hanlee P. Ji*, Carlo C. Maley*. Pan-cancer analysis of the etiology and consequences of intratumor heterogeneity. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-54.

Abstract PR03: Pan-cancer analysis of the etiology and consequences of intra-tumor heterogeneity

Abstract Tumors are typically mosaics of mutant clones that have evolved from a common ancestral cell1–3. This intra-tumor heterogeneity (ITH) is thought to drive both neoplastic progression and acquired therapeutic resistance4–6. Availability of just one sample per tumor and moderate sequencing depth have limited systematic analysis of ITH during previous TCGA pan-cancer analyses7–9, confining the study of ITH to a small numbers of tumor samples and cancer types10–13. The molecular and histopathological causes of ITH and its prognostic significance have thus far remained uncertain14–17. To overcome these limitations, we used an analysis method called EXPANDS18 that estimates the proportion of cells harboring specific mutations from exome sequencing data, as well as other methods that quantify ITH15,19. We extrapolate the number of genetically diverse clonal subpopulations in 1,165 primary tumors among 12 different cancer types from TCGA and investigate mechanisms underpinning ITH as well as the correlation of ITH and genomic instability with prognosis. Lastly we validate the prognostic significance of genomic instability in an independent, high-density SNP-array dataset consisting of 2010 tumor samples, across seven additional cancer types. We found evidence of ITH in the vast majority of tumors. Driver gene mutations, prevalence of copy number gains and tumor microenvironment composition were significantly associated with increased ITH. Mutations in driver genes tended to have a characteristic clone size, suggesting differential fitness effects of those mutations. The detection of a mutation in a driver gene that typically appears in a small clone was a predictor of poor survival. In general, ITH was a universal biomarker of prognosis: across cancer types poor prognosis was associated with intermediate, rather than very low or high, levels of ITH. This was also true for the fraction of tumor genome affected by copy number alterations16,20: tumors with intermediate copy number burden (50 to 75% of the genome affected by copy number alterations) progressed faster than tumors with higher copy number burden, independent of cancer type. Chemo-radiation therapy administration was more efficient in decelerating tumor progression among patients with intermediate copy number burden than among patients with low or high copy number burden. These results were validated and confirmed in the independent SNP-array dataset. This study suggests a tradeoff exists between the costs and benefits of genomic instability21,22 that impacts both the evolvability and fitness of the tumor cell population. In the future, this tradeoff might be exploited to improve survival. In summary, we have shown that ITH is a universal feature of human cancers that predicts survival.

Abstract LB-163: Pan-cancer analysis of the causes and consequences of Intra-tumor heterogeneity

Abstract Tumors are typically mosaics of mutant clones that have evolved from a common ancestral cell. This intra-tumor heterogeneity (ITH) is thought to drive both neoplastic progression and acquired therapeutic resistance. Availability of just one sample per tumor and moderate sequencing depth have limited systematic analysis of ITH during previous TCGA pan-cancer analyses, confining the study of ITH to a small numbers of tumor samples and cancer types. The molecular and histopathological causes of ITH and its prognostic significance have thus far remained uncertain. To overcome these limitations, we used an analysis method called EXPANDS that estimates the proportion of cells harboring specific mutations from exome sequencing data, as well as other methods that quantify ITH. We extrapolate the number of genetically diverse clonal subpopulations in 1,165 primary tumors among 12 different cancer types from TCGA and investigate mechanisms underpinning ITH as well as the correlation of ITH and genomic instability with prognosis. Lastly we validate the prognostic significance of genomic instability in an independent, high-density SNP-array dataset consisting of 2010 tumor samples, across seven additional cancer types. We found evidence of ITH in the vast majority of tumors. Mutations in driver genes tended to have a characteristic clone size, suggesting differential fitness effects of those mutations. The detection of a mutation in a driver gene that typically appears in a small clone was a predictor of poor survival. Driver gene mutations, tumor microenvironment composition and prevalence of copy number gains were significantly associated with increased ITH. In general, the prevalence of copy number alterations was a universal biomarker of prognosis: tumors with intermediate copy number burden (50 to 75% of the genome affected by copy number alterations) progressed faster than tumors with higher copy number burden, independent of cancer type. Chemo-radiation therapy administration was more efficient in decelerating tumor progression among patients with intermediate copy number burden than among patients with low or high copy number burden. These results were validated and confirmed in the independent SNP-array dataset. This study suggests a tradeoff exists between the costs and benefits of genomic instability that impacts both the evolvability and fitness of the tumor cell population. In the future, this tradeoff might be exploited to improve survival. Citation Format: Noemi Andor, Trevor A. Graham, Athena C. Aktipis, Claudia Petritsch, Hanlee P. Ji, Carlo C. Maley. Pan-cancer analysis of the causes and consequences of Intra-tumor heterogeneity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-163. doi:10.1158/1538-7445.AM2015-LB-163

Maintaining Tumor Heterogeneity in Patient-Derived Tumor Xenografts.

Preclinical models often fail to capture the diverse heterogeneity of human malignancies and as such lack clinical predictive power. Patient-derived tumor xenografts (PDX) have emerged as a powerful technology: capable of retaining the molecular heterogeneity of their originating sample. However, heterogeneity within a tumor is governed by both cell-autonomous (e.g., genetic and epigenetic heterogeneity) and non-cell-autonomous (e.g., stromal heterogeneity) drivers. Although PDXs can largely recapitulate the polygenomic architecture of human tumors, they do not fully account for heterogeneity in the tumor microenvironment. Hence, these models have substantial utility in basic and translational research in cancer biology; however, study of stromal or immune drivers of malignant progression may be limited. Similarly, PDX models offer the ability to conduct patient-specific in vivo and ex vivo drug screens, but stromal contributions to treatment responses may be under-represented. This review discusses the sources and consequences of intratumor heterogeneity and how these are recapitulated in the PDX model. Limitations of the current generation of PDXs are discussed and strategies to improve several aspects of the model with respect to preserving heterogeneity are proposed.

Abstract LB-129: Identifying tumor subpopulations and the functional consequences of intratumor heterogeneity using single-cell profiling of breast cancer patient-derived xenografts

Abstract Human breast tumors have been shown to exhibit extensive inter- and intra-tumor heterogeneity. While recent advances in genomic technologies have allowed us to deconvolute this heterogeneity, few studies have addressed the functional consequences of diversity within tumor populations. Here, we identified an index case for which we have derived a patient-derived xenograft (PDX) as a renewable tissue source to identify subpopulations and perform functional assays. On pathology, the tumor was an invasive ductal carcinoma which was hormone receptor-negative, HER2-positive (IHC 2+, FISH average HER2/CEP17 2.4), though the FISH signal was noted to be heterogeneous. On gene expression profiling of bulk samples, the primary tumor and PDX were classified as basal-like. We performed single cell RNA and exome sequencing of the PDX to identify population structure. Using a single sample predictor of breast cancer subtype, we have identified single basal-like, HER2-enriched and normal-like cells co-existing within the PDX tumor. Genes differentially expressed between these subpopulations are involved in proliferation and differentiation. Functional studies distinguishing these subpopulations are ongoing. Microfluidic whole genome amplification followed by whole exome capture of 81 single cells showed high and homogeneous target enrichment with &amp;gt;75% of reads mapping uniquely on target. Variant calling using GATK and Samtools revealed founder mutations in key genes as BRCA1 and TP53, as well as subclonal mutations that are being investigated further. Loss of heterozygocity was observed in 16 TCGA cancer driver genes and novel mutations in 7 cancer driver genes. These findings may be important in understanding the functional consequences of intra-tumor heterogeneity with respect to clinically important phenotypes such as invasion, metastasis and drug-resistance. Citation Format: Paul Savage, Sadiq M. Saleh, Ernesto Iacucci, Timothe Revil, Yu-Chang Wang, Nicholas Bertos, Anie Monast, Hong Zhao, Margarita Souleimanova, Keith Szulwach, Chandana Batchu, Atilla Omeroglu, Morag Park, Ioannis Ragoussis. Identifying tumor subpopulations and the functional consequences of intratumor heterogeneity using single-cell profiling of breast cancer patient-derived xenografts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-129. doi:10.1158/1538-7445.AM2015-LB-129

Epigenetic regulation in RCC: opportunities for therapeutic intervention?

Renal cell carcinoma (RCC) is a constellation of malignancies of different histological subtypes arising from the renal parenchyma. The clear cell histological subtype (ccRCC) accounts for around 75% of RCCs and is characterized by distinct genetic abnormalities, of which the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is the most common. Inactivation of other tumor suppressor genes such as SETD2, KDM6A, KDM5C and PBRM1 has been reported in ccRCC--notably, the proteins encoded by these genes are involved in histone and chromatin regulation. Furthermore, the PBRM1 and SETD2 genes are located on the short arm of chromosome 3 near the VHL locus. Chromatin and histones modify gene expression and, as a consequence, their function is tightly regulated. Data from RNA interference (RNAi) assays suggest that loss of function of PBRM1 drives proliferation and growth of ccRCC, but the clinical relevance of this is unclear and restoring the function of these genes for therapeutic purposes is likely to be challenging. An improved understanding of histone and chromatin regulation in RCC biology and the consequences of intratumor heterogeneity might identify novel targets in RCC and present alternative therapeutic opportunities.