Level Of Intratumor Heterogeneity Research Articles

Analysis of genomic and immune intratumor heterogeneity in linitis plastica via multiregional exome and T-cell receptor sequencing.

The molecular landscape and the intratumor heterogeneity (ITH) architecture of gastric linitis plastica (LP) are poorly understood. We performed whole-exome sequencing (WES) and T-cell receptor (TCR) sequencing on 40 tumor regions from four LP patients. The landscape and ITH at the genomic and immunological levels in LP tumors were compared with multiple cancers that have previously been reported. The lymphocyte infiltration was further assessed by immunohistochemistry (IHC) in LP tumors. In total, we identified 6339 non-silent mutations from multi-samples, with a median tumor mutation burden (TMB) of 3.30 mutations per Mb, comparable to gastric adenocarcinoma from the Cancer Genome Atlas (TCGA) cohort (P = 0.53). An extremely high level of genomic ITH was observed, with only 12.42%, 5.37%, 5.35%, and 30.67% of mutations detectable across 10 regions within the same tumors of each patient, respectively. TCR sequencing revealed that TCR clonality was substantially lower in LP than in multi-cancers. IHC using antibodies against CD4, CD8, and PD-L1 demonstrated scant T-cell infiltration in the four LP tumors. Furthermore, profound TCR ITH was observed in all LP tumors, with no T-cell clones shared across tumor regions in any of the patients, while over 94% of T-cell clones were restricted to individual tumor regions. The Morisita overlap index (MOI) ranged from 0.21 to 0.66 among multi-regions within the same tumors, significantly lower than that of lung cancer (P = 0.002). Our results show that LP harbored extremely high genomic and TCR ITH and suppressed T-cell infiltration, suggesting a potential contribution to the frequent recurrence and poor therapeutic response of this adenocarcinoma.

Chromosomal Instability, Selection and Competition: Factors That Shape the Level of Karyotype Intra-Tumor Heterogeneity.

Simple SummaryEach cancer consists of billions of cells. These cells are far from identical; hence, the population of cells that constitute a tumor is heterogeneous. A salient property that varies between cells in a tumor is their karyotype, the number and configuration of the chromosomes. The level of karyotype heterogeneity can be used to predict the survival of a patient. In this review, we describe the processes that shape the level of karyotype heterogeneity in a cancer.Intra-tumor heterogeneity (ITH) is a pan-cancer predictor of survival, with high ITH being correlated to a dismal prognosis. The level of ITH is, hence, a clinically relevant characteristic of a malignancy. ITH of karyotypes is driven by chromosomal instability (CIN). However, not all new karyotypes generated by CIN are viable or competitive, which limits the amount of ITH. Here, we review the cellular processes and ecological properties that determine karyotype ITH. We propose a framework to understand karyotype ITH, in which cells with new karyotypes emerge through CIN, are selected by cell intrinsic and cell extrinsic selective pressures, and propagate through a cancer in competition with other malignant cells. We further discuss how CIN modulates the cell phenotype and immune microenvironment, and the implications this has for the subsequent selection of karyotypes. Together, we aim to provide a comprehensive overview of the biological processes that shape the level of karyotype heterogeneity.

Prognostic Value of Molecular Intratumor Heterogeneity in Primary Oral Cancer and Its Lymph Node Metastases Assessed by Mass Spectrometry Imaging

Different aspects of intra-tumor heterogeneity (ITH), which are associated with the development of cancer and its response to treatment, have postulated prognostic value. Here we searched for potential association between phenotypic ITH analyzed by mass spectrometry imaging (MSI) and prognosis of head and neck cancer. The study involved tissue specimens resected from 77 patients with locally advanced oral squamous cell carcinoma, including 37 patients where matched samples of primary tumor and synchronous lymph node metastases were analyzed. A 3-year follow-up was available for all patients which enabled their separation into two groups: with no evidence of disease (NED, n = 41) and with progressive disease (PD, n = 36). After on-tissue trypsin digestion, peptide maps of all cancer regions were segmented using an unsupervised approach to reveal their intrinsic heterogeneity. We found that intra-tumor similarity of spectra was higher in the PD group and diversity of clusters identified during image segmentation was higher in the NED group, which indicated a higher level of ITH in patients with more favorable outcomes. Signature of molecular components that correlated with long-term outcomes could be associated with proteins involved in the immune functions. Furthermore, a positive correlation between ITH and histopathological lymphocytic host response was observed. Hence, we proposed that a higher level of ITH revealed by MSI in cancers with a better prognosis could reflect the presence of heterotypic components of tumor microenvironment such as infiltrating immune cells enhancing the response to the treatment.

Clear Cell Renal Cell Carcinomas with Aggressive Behavior Display Low Intratumor Heterogeneity at the Histological Level.

Intratumor heterogeneity (ITH) is an inherent characteristic of most tumors and its detection remains a key task for pathologists. However, the clinical significance of the degree of development of this feature is still poorly understood. A series of 28 clear cell renal cell carcinomas (CCRCC) have been exhaustively analyzed with two different sampling protocols [multisite tumor sampling (MSTS) and total sampling] to evaluate to what point the level (low vs. high) of histological ITH detected in routine practice influences tumor behavior and patients' survival. All CCRCC (n = 14) pursuing an aggressive clinical course presented low levels of ITH. A significant worse survival was detected in CCRCC with low ITH (p < 0.001). The simple quantification of the level of ITH using extensive sampling protocol may be of help in predicting tumor evolution, since all CCRCC with aggressive behavior demonstrated low levels of histological ITH.

Abstract P4-07-29: Reactive Oxygen Species (ROS) pathway is associated with aggressive cancer biology, elevated immune response, and with worse survival in ER-positive/HER2-negative breast cancer

Abstract Background: Reactive oxygen species (ROS) in tumor microenvironment are known to promote many aspects of cancer progression. On the other hand, excessive accumulation of ROS can induce cancer cell apoptosis. To our knowledge, clinical relevance of ROS response signaling in breast cancer has never been studied in multiple large patient cohorts. Here, we hypothesized that the high ROS pathway score reflect tumor aggressiveness, thus, it predicts worse survival in breast cancer patients. Methods: A total of 6245 breast cancer patients from three independent cohorts (GSE96058, METABRIC, and TCGA) were analyzed. We defined the ROS pathway score by the degree of enrichment by Gene Set Variant Analysis (GSVA) and median was used to divide high vs low score groups in each cohort. Results: The ROS score was significantly associated with aggressive clinical factors, such as triple-negative breast cancer (TNBC) subtype, Nottingham histological grade, American Joint Committee on Cancer (AJCC) Stage and lymph node metastasis consistently in both GSE96058 and METABRIC (all p &amp;lt; 0.001). High ROS score was significantly associated with worse overall survival (OS) in the GSE96058 (hazard ratio (HR) = 3.59, 95% confidence interval (CI); 2.47-5.20, p &amp;lt; 0.001), as well as OS (HR = 1.55, 95%CI; 1.16-2.07, p = 0.002), disease-free survival (HR = 1.63, 95%CI; 1.10-2.43, p = 0.016), and disease-specific survival (HR = 2.57, 95%CI; 1.75-3.77, p &amp;lt; 0.001) in the METABRIC cohort. Subgroup analysis revealed that a high ROS score was significantly associated with worse OS in ER-positive/HER2-negative breast cancer in both cohorts. High ROS ER-positive/HER2-negative breast cancer was significantly associated with high level of intratumor heterogeneity (p = 0.013), homologous recombination deficient (HRD) (p &amp;lt; 0.001), and mutation-related score (silent and non-silent mutation rate (p = 0.003 and 0.011, respectively), fraction altered (p = 0.038), and single-nucleotide variant (SNV) neoantigens (p = 0.006)) in the TCGA cohort. In agreement, high ROS ER-positive/HER2-negative breast cancer significantly enriched cell proliferation-related gene sets; E2F targets, G2M checkpoint, and MYC targets v1 and v2 consistently in both GSE96058 and METABRIC cohorts (all false discovery rate (FDR) &amp;lt; 0.25). On the other hand, high ROS ER-positive/HER2-negative tumors were associated with elevated infiltration of multiple immune cells, including anti-cancer immune cells (CD8+ T cell, CD4+ memory T cell, M1 macrophage, dendritic cell, gamma-delta T cell), as well as pro-cancer immune cells (regulatory T cell, T helper type2 cell, and M2 macrophage) and with high level of cytolytic activity (CYT) consistently in both cohorts (all p &amp;lt; 0.001). Those tumors also enriched immune-related pathway (Interferon (IFN)-α response, IFN-γ response, allograft rejection, complement, IL6/JAK/STAT3 signaling, and inflammatory response) consistently in both cohorts (all FDR &amp;lt; 0.25). Finally, high ROS ER-positive/HER2-negative breast cancer was significantly associated with high expression of immune checkpoint molecules including PD-1, PD-L1 and PD-L2, CTLA4, IDO1, LAG3, BTLA, HLA-A, and TIGIT, in (all p &amp;lt; 0.005). Conclusion ROS pathway is associated with cancer aggressiveness, elevated immune response, and with worse survival in ER-positive/HER2-negative breast cancer. Citation Format: Masanori Oshi, Rongrong Wu, Akimitsu Yamada, Li Yan, Takashi Ishikawa, Itaru Endo, Kazuaki Takabe. Reactive Oxygen Species (ROS) pathway is associated with aggressive cancer biology, elevated immune response, and with worse survival in ER-positive/HER2-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-29.

Abstract P5-03-01: Conflicting roles of EGFR expression by subtypes in breast cancer

Abstract Epidermal growth factor receptor (EGFR) is one of the receptors that belongs to the epidermal growth factor family of receptor tyrosine kinases (ErbBs). Several malignancies including breast cancer that express EGFR have poor prognosis. Our study examined the EGFR expression among 5176 breast cancer patients from multiple independent breast cancer cohorts and its contribution to tumor immune microenvironment in different subtypes. We found that, EGFR expression in triple negative breast cancer (TNBC) was the highest among breast cancer subtypes in GSE96058 and METABRIC cohorts when compared to other subtypes (both p &amp;lt; 0.001), which is in agreement with the existing literature. High EGFR expression was significantly associated with improved survival in ER-positive/HER2-negative breast cancer in both cohorts (both p &amp;lt; 0.001) and also significantly associated with high level of intratumor heterogeneity and homologous recombination deficits (HRD) (both p &amp;lt; 0.001) in the TCGA cohort. Furthermore, high EGFR expression tumor enriched not only several pro-cancer-related gene sets, but also immune-related gene sets, including Allograft rejection, inflammatory response, IL2/STAT5 signaling, IL6/JAK/STAT3 signaling, coagulation and complement pathway in both cohorts. On the other hand, low EGFR tumor enriched cell proliferation-related gene sets, including E2F targets, G2M checkpoint MYC targets v1 and v2, and DNA repair, which may explain the observed worse survival (all normal enrichment score &amp;lt; -0.50). However, these findings were not observed in TNBC. Interestingly, high EGFR ER-positive/HER2-negative breast cancers were infiltrated with anti-cancer immune cells (CD8+ T cell, CD4+ T cell, and dendritic cell), and associated with high level of cytolytic activity (CYT) (p &amp;lt; 0.001). On the other hand, a lower fraction of immune cells was observed in high EGFR TNBC along with low level of CYT (p &amp;lt; 0.001). In the single cell sequence data, tumor cells have significantly higher EGFR expression compared to immune cells (p &amp;lt; 0.001). High EGFR expression among ER-positive/HER2-negative breast cancer was significantly associated with higher expression of immune checkpoint molecules (PD-1, PD-L1 and PD-L2, CTLA4, IDO1, and BTLA; all p &amp;lt; 0.001), while, on the other hand, there was lower immune checkpoint molecule expression in high EGFR TNBC. Finally, high EGFR metastatic tumor was associated with worse survival, but there was no association with infiltrating immune cells. In conclusion, EGFR expression was found to have different characteristics depending on breast cancer subtype. Especially, high EGFR ER-positive/HER2-negative breast cancer was significantly associated with better survival and immunity in tumor immune microenvironment. Citation Format: Masanori Oshi, Shipra Gandhi, Yoshihisa Tokumaru, Rongrong Wu, Li Yan, Akimitsu Yamada, Takashi Ishikawa, Itaru Endo, Kazuaki Takabe. Conflicting roles of EGFR expression by subtypes in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-03-01.

Whole-exome sequencing identifies somatic mutations and intratumor heterogeneity in inflammatory breast cancer

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Although it is a rare subtype, IBC is responsible for roughly 10% of breast cancer deaths. In order to obtain a better understanding of the genomic landscape and intratumor heterogeneity (ITH) in IBC, we conducted whole-exome sequencing of 16 tissue samples (12 tumor and four normal samples) from six hormone-receptor-positive IBC patients, analyzed somatic mutations and copy number aberrations, and inferred subclonal structures to demonstrate ITH. Our results showed that KMT2C was the most frequently mutated gene (42%, 5/12 samples), followed by HECTD1, LAMA3, FLG2, UGT2B4, STK33, BRCA2, ACP4, PIK3CA, and DNAH8 (all nine genes tied at 33% frequency, 4/12 samples). Our data indicated that PTEN and FBXW7 mutations may be considered driver gene mutations for IBC. We identified various subclonal structures and different levels of ITH between IBC patients, and mutations in the genes EIF4G3, IL12RB2, and PDE4B may potentially generate ITH in IBC.

Intratumor Heterogeneity in Uveal Melanoma BAP-1 Expression.

Simple SummaryUveal melanoma is the most common primary eye malignancy. In this paper, we examine how the expression of the tumor suppressor BRCA1 associated protein 1 (BAP-1) varies across different parts of these tumors. We find that there are considerable differences, but that these do not matter as long as proper rules for interpretation are applied. Further, we show that analysis of BAP-1 expression adds significant prognostic information to both tumor size and gene expression classifications.Malignant tumors are rarely homogenous on the morphological, genome, transcriptome or proteome level. In this study, we investigate the intratumor heterogeneity of BAP-1 expression in uveal melanoma with digital image analysis of 40 tumors. The proportion of BAP-1 positive cells was measured in full tumor sections, hot spots, cold spots and in scleral margins. The mean difference between hot spots and cold spots was 41 percentage points (pp, SD 29). Tumors with gene expression class 1 (associated with low metastatic risk) and 2 (high metastatic risk) had similar intratumor heterogeneity. Similarly, the level of intratumor heterogeneity was comparable in tumors from patients that later developed metastases as in patients that did not. BAP-1 measured in any tumor region added significant prognostic information to both American Joint Committee on Cancer (AJCC) tumor size category (p ≤ 0.001) and gene expression class (p ≤ 0.04). We conclude that there is substantial intratumor heterogeneity in uveal melanoma BAP-1 expression. However, it is of limited prognostic importance. Regardless of region, analysis of BAP-1 expression adds significant prognostic information beyond tumor size and gene expression class.

Evolution of DNA methylome from precancerous lesions to invasive lung adenocarcinomas

The evolution of DNA methylome and methylation intra-tumor heterogeneity (ITH) during early carcinogenesis of lung adenocarcinoma has not been systematically studied. We perform reduced representation bisulfite sequencing of invasive lung adenocarcinoma and its precursors, atypical adenomatous hyperplasia, adenocarcinoma in situ and minimally invasive adenocarcinoma. We observe gradual increase of methylation aberrations and significantly higher level of methylation ITH in later-stage lesions. The phylogenetic patterns inferred from methylation aberrations resemble those based on somatic mutations suggesting parallel methylation and genetic evolution. De-convolution reveal higher ratio of T regulatory cells (Tregs) versus CD8 + T cells in later-stage diseases, implying progressive immunosuppression with neoplastic progression. Furthermore, increased global hypomethylation is associated with higher mutation burden, copy number variation burden and AI burden as well as higher Treg/CD8 ratio, highlighting the potential impact of methylation on chromosomal instability, mutagenesis and tumor immune microenvironment during early carcinogenesis of lung adenocarcinomas.

Abstract PO-106: Genomic heterogeneity and clonal dynamics of resistance evolution and metastatic progression in rectal cancer

Abstract The standard treatment for locally advanced rectal cancer is chemoradiotherapy followed by surgery. However, patient response to pre-operative chemoradiotherapy is highly heterogeneous, ranging from complete tumor regression to no response. Over the course of disease, up to 40% of patients suffer from local relapse or the development of metachronous metastatic disease, compromising survival. It is not yet known how treatment resistance and disease progression in these patients evolve – whether from the selective outgrowth of pre-existing resistant clones during treatment or through the acquisition of additional, resistance conferring genomic alterations. We aim to delineate the genomic evolution and clonal architecture underlying treatment resistance and metastatic progression in rectal cancer based on a cohort of 42 patients, from which 98 tumor samples were collected longitudinally over the course of treatment and metastatic progression. Tumor diagnosis and treatment response assessment are routinely based on the histopathologic evaluation of formalin-fixed paraffin-embedded (FFPE) patient tissues. While formalin fixation and paraffin embedding optimally preserve histomorphology and allow long-term tissue storage at ambient temperature, they, on the other hand, lead to DNA crosslinks posing a challenge for sequencing analyses. To overcome this limitation, we have successfully developed a protocol for dissociation of FFPE tissues into single cells that are suitable for immunolabeling, flow sorting and genomic analysis by whole exome sequencing of pure tumor cell populations, single cell copy number variation (CNV) sequencing and multiplex interphase fluorescence in situ hybridization (miFISH). Our genomic analyses at the population as well as individual cell level revealed divergent aberration patterns and distinct levels of intra-tumor heterogeneity. Major tumor clones were characterized by gains of EGFR (7p11.2), MYC (8q24.21), CDX2 (13q12.2) and ZNF217 (20q13.2) along with losses of SMAD4 (18q21.2) and TP53 (17p13.1). While in some patients the clonal composition remained largely stable throughout treatment, in others major clonal shifts occurred favoring clones with TP53 loss. Our preliminary results indicate different propensities of genetically distinct tumor cell clones in therapy response and metastatic progression. Furthermore, our data show the feasibility of high-resolution clonal reconstruction from whole exome sequencing and single cell CNV sequencing data of FFPE cells. Citation Format: Daniela Hirsch, Judith Lieberich, Kerstin Heselmeyer-Haddad, Yonca Ceribas, Michael Kelly, Keyur Talsania, Yongmei Zhao, Timo Gaiser, Thomas Ried. Genomic heterogeneity and clonal dynamics of resistance evolution and metastatic progression in rectal cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-106.

Abstract 225: Frequent abnormalities in TP53 and increased genetic instability in myxofibrosarcoma

Abstract Myxofibrosarcoma (MFS) is a rare subtype of soft tissue sarcomas (STSs) preferentially affecting the elderly. Histologically, MFS is distinct from other STSs, in that it is characterized by the proliferation of pleomorphic spindle cells with varying degrees of myxoid components. However, the molecular pathogenesis of MSF is poorly understood. We conducted an integrated molecular study involving 70 samples from primary MFS patients, in which samples were analyzed by whole-genome sequencing (WGS, n=5), whole-exome sequencing (WES, n=44), targeted-capture sequencing (TS, n=65), RNA sequencing (n=3), and immunohistochemistry (IHC, n=50). Copy number (CN) alterations were detected by sequencing-based CN analysis. We combined our STS data with those from from the Cancer Genome Atlas and European Genome-phenome Archive cohort, including 116 MSF samples, and compared to the data from other STS samples (n=189). To further investigate the genetic basis of mixed histological components and chronological changes in MFS, we performed WES of multi-regional and/or multi-time-point samples from 8 MSF cases. A total of 9,246 mutations were identified by WES in 130 primary MFS samples with a median of 55.5 mutations/sample. Mutations were dominated by age-related C to T transitions at CpG sites. When combining the results from TS, among 188 primary MFS samples, most frequently mutated and/or CN-altered genes included TP53 (n=82, 43.6%), RB1 (n=31, 16.5%), ATRX (n=29, 15.4%), and CDKN2A (n=19, 10.2%). A fusion gene involving TRIO was newly identified by RNA sequencing (n=1). A similar mutational profile was observed in undifferentiated pleomorphic sarcoma (UPS), in which TP53 (59.1%), ATRX (34.0%), RB1 (22.7%), and CDKN2A (20.5%) were the major mutational targets, suggesting the common molecular pathogenesis between these two subtypes. Combined with frequent positive staining in IHC (n=22, 44.0%), TP53 was affected in 55.3% of the MFS cases (n=104). Five MFS cases evaluated by WGS showed complex structural abnormalities suggestive of increased genetic instability, where a median of 179 structural variations were detected per sample. WES with multi-regional sampling (n=5) disclosed a high level of intratumor heterogeneity, in which less than 29.0% of mutations were shared by different samples taken from the same tumor. An analysis of longitudinal samples (n=6) revealed significantly higher numbers of mutations in relapse samples (1.6 times on average, p = 0.03). In all cases, TP53 lesions were present from at the time of the initial diagnosis, while most of other lesions were subclonal and acquired during the clinical course. In summary, the genetic profile of MFS is characterized by frequent abnormalities in TP53, RB1, CDKN2A and ATRX, and closely related to other STSs, particularly to UPS. Clonal TP53 abnormalities resulted in complex chromosomal structure and a high degree of intratumor heterogeneity. Citation Format: Yasuhide Takeuchi, Annegret Kunitz, Adriane Halik, Hiromichi Suzuki, Kenichi Yoshida, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Nobuyuki Kakiuchi, Yusuke Shiozawa, Akira Yokoyama, Yoshikage Inoue, Tetsuichi Yoshizato, Kosuke Aoki, Yoichi Fujii, Yasuhito Nannya, Hideki Makishima, Satoru Miyano, Hironori Haga, Frederik Damm, Seishi Ogawa. Frequent abnormalities in TP53 and increased genetic instability in myxofibrosarcoma [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 225.

Cooperation among Tumor Cell Subpopulations Leads to Intratumor Heterogeneity

Heterogeneity is a hallmark of all cancers. Tumor heterogeneity is found at different levels — interpatient, intrapatient, and intratumor heterogeneity. All of them pose challenges for clinical treatments. The latter two scenarios can also increase the risk of developing drug resistance. Although the existence of tumor heterogeneity has been known for two centuries, a clear understanding of its origin is still elusive, especially at the level of intratumor heterogeneity (ITH). The coexistence of different subpopulations within a single tumor has been shown to play crucial roles during all stages of carcinogenesis. Here, using concepts from evolutionary game theory and public goods game, often invoked in the context of the tragedy of commons, we explore how the interactions among subclone populations influence the establishment of ITH. By using an evolutionary model, which unifies several experimental results in distinct cancer types, we develop quantitative theoretical models for explaining data from in vitro experiments involving pancreatic cancer as well as in vivo data in glioblastoma multiforme. Such physical and mathematical models complement experimental studies, and could optimistically provide new ideas for the design of efficacious therapies for cancer patients.

Low intratumor heterogeneity correlates with increased response to PD-1 blockade in renal cell carcinoma.

Background:Intratumor heterogeneity (ITH) has been shown to be inversely associated with immune infiltration in several cancers including clear cell renal cell carcinoma (ccRCC), but it remains unclear whether ITH is associated with response to immunotherapy (e.g. PD-1 blockade) in ccRCC.Methods:We quantified ITH using mutant-allele tumor heterogeneity, investigated the association of ITH with immune parameters in patients with ccRCC (n = 336) as well as those with papillary RCC (pRCC, n = 280) from The Cancer Genome Atlas, and validations were conducted in patients with ccRCC from an independent cohort (n = 152). The relationship between ITH and response to anti-PD-1 immunotherapy was explored in patients with metastatic ccRCC from a clinical trial of anti-PD-1 therapy (n = 35), and validated in three equal-size simulated data sets (n = 60) generated by random sampling with replacement based on this clinical trial cohort.Results:In ccRCC, low ITH was associated with better survival, more reductions in tumor burden, and clinical benefit of anti-PD-1 immunotherapy through modulating immune activity involving more neoantigens, elevated expression of HLA class I genes, and higher abundance of dendritic cells. Furthermore, we found that the association between the level of ITH and response to PD-1 blockade was independent of the mutation status of PBRM1 and that integrating both factors performed better than the individual predictors in predicting the benefit of anti-PD-1 immunotherapy in ccRCC patients. In pRCC, increased immune activity was also observed in low- versus high-ITH tumors, including higher neoantigen counts, increased abundance of monocytes, and decreased expression of PD-L1 and PD-L2.Conclusions:ITH may be helpful in the identification of patients who could benefit from PD-1 blockade in ccRCC, and even in pRCC where no genomic metrics has been found to correlate with response to immune checkpoint inhibitors.

SpliceHetero: An information theoretic approach for measuring spliceomic intratumor heterogeneity from bulk tumor RNA-seq.

MotivationIntratumor heterogeneity (ITH) represents the diversity of cell populations that make up cancer tissue. The level of ITH in a tumor is usually measured by a genomic variation profile, such as copy number variation and somatic mutation. However, a recent study has identified ITH at the transcriptome level and suggested that ITH at gene expression levels is useful for predicting prognosis. Measuring ITH levels at the spliceome level is a natural extension. There are serious technical challenges in measuring spliceomic ITH (sITH) from bulk tumor RNA sequencing (RNA-seq) due to the complex splicing patterns.ResultsWe propose an information-theoretic method to measure the sITH of bulk tumors to overcome the above challenges. This method has been extensively tested in experiments using synthetic data, xenograft tumor data, and TCGA pan-cancer data. As a result, we showed that sITH is closely related to cancer progression and clonal heterogeneity, along with clinically significant features such as cancer stage, survival outcome and PAM50 subtype. As far as we know, it is the first study to define ITH at the spliceome level. This method can greatly improve the understanding of cancer spliceome and has great potential as a diagnostic and prognostic tool.

Addressing cellular heterogeneity in tumor and circulation for refined prognostication

Despite pronounced genomic and transcriptomic heterogeneity in non-small-cell lung cancer (NSCLC) not only between tumors, but also within a tumor, validation of clinically relevant gene signatures for prognostication has relied upon single-tissue samples, including 2 commercially available multigene tests (MGTs). Here we report an unanticipated impact of intratumor heterogeneity (ITH) on risk prediction of recurrence in NSCLC, underscoring the need for a better genomic strategy to refine prognostication. By leveraging label-free, inertial-focusing microfluidic approaches in retrieving circulating tumor cells (CTCs) at single-cell resolution, we further identified specific gene signatures with distinct expression profiles in CTCs from patients with differing metastatic potential. Notably, a refined prognostic risk model that reconciles the level of ITH and CTC-derived gene expression data outperformed the initial classifier in predicting recurrence-free survival (RFS). We propose tailored approaches to providing reliable risk estimates while accounting for ITH-driven variance in NSCLC.

Abstract LB-300: The life history of breast cancer in Nigerian women: Evidence for ethnic differences in tumor evolution based on whole-genome sequencing

Abstract Breast cancer is a heterogeneous disease and the incidence to mortality ratio is highest for women of African ancestry. Paucity of data from non-European ancestry groups limits our understanding of the underlying etiological differences or alternative routes to progression that may explain differential outcomes. To examine the contribution of genomic differences, we performed high-depth whole-genome sequencing on 100 breast tumors (90X) and their paired normal samples (30X) from indigenous African women with breast cancer in Southwest Nigeria and performed comparative analysis with 84 TCGA whole genome sequences of breast tumor/normal pairs (46 White, 30 African American and 8 samples representing other ethnicities). High confidence somatic single nucleotide variants (SNVs) were obtained by using MuTect and Strelka. To analyse intra-tumoral heterogeneity (ITH), Battenberg and DPClust were used to call copy number aberrations (CNA) and cluster somatic SNVs based on cancer cell fraction (CCF) respectively. We find that in the HR-/HER2+ subtype, clonal losses of chromosome 14q are highly enriched in Nigerians (41%) but absent in the Whites even though the proportion of this subtype was comparable between the two groups (42% and 33% respectively). This is an interesting observation since not only 14q loss is known to be an event associated with breast cancer aggressiveness, but HR-/HER2+ has also been reported to be enriched within the relatively younger Nigerian patients with breast cancer. This may in part explain inter-ethnic disparity in survival. Also, somatic SNV clustering analysis showed that Nigerian cancers have a higher level of ITH than Whites, which may explain the pronounced aggressiveness of breast cancer in women of African ancestry. In contrast, early drivers (e.g. TP53 and PIK3CA) and whole genome duplication rates were mostly similar between the groups. Our observations suggest differences in the underlying evolutionary trajectories of breast cancer across ethnic backgrounds. These data underscore the need for larger and more in-depth studies of diverse cancer genomes and, if validated, may translate into clinical intervention opportunities tailored to women of African ancestry and accelerate progress in precision cancer care. Note: This abstract was not presented at the meeting. Citation Format: Naser Ansari-Pour, Jason J. Pitt, Stefan Dentro, Dominic Fitzgerald, Yonglan Zheng, Toshio F. Yoshimatsu, Padma Rajagopal, Andreas Gruber, Ayodele Sanni, Olayiwola Oluwasola, Mustapha Ajani, Abayomi Odetunde, Abiodun Popoola, Adeyinka Falusi, Temidayo Ogundiran, John Obafunwa, Oladosu Ojengbede, Nasiru Ibrahim, Kevin P. White, Dezheng Huo, Peter Van Loo, David C. Wedge, Olufunmilayo Olopade. The life history of breast cancer in Nigerian women: Evidence for ethnic differences in tumor evolution based on whole-genome sequencing [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 LB-300.

Abstract 738: Myxofibrosarcoma is characterized by frequent abnormalities in TP53 and increased genetic instability

Abstract Myxofibrosarcoma (MFS) is a rare subtype of soft tissue sarcomas (STSs) preferentially affecting the elderly. Histologically, MFS is distinct from other STSs, in that it is characterized by the proliferation of pleomorphic spindle cells with varying degrees of myxoid components. However, the molecular pathogenesis of MSF is poorly understood. We conducted an integrated molecular study involving 70 samples from primary MFS patients, in which samples were analyzed by whole-genome sequencing (WGS) (n=5), whole-exome sequencing (WES) (n=44), targeted-capture sequencing (n=65), RNA sequencing (n=3), and immunohistochemistry (IHC, n=50). Copy number (CN) alterations were detected by sequence-based CN analysis. The obtained genomic data were combined with those from STS cases from the Cancer Genome Atlas (TCGA) cohort, including 17 MSF samples and compared to the data from other STS samples (n=189). To further investigate the genetic basis of mixed histological components and chronological changes in MFS, we performed WES of multi-regional and/or multi-time-point samples from 8 MSF cases. A total of 4,613 mutations were identified by WES in 61 primary MFS samples with a median of 44.0 mutations/sample. Mutations were dominated by age-related C to T transitions at CpG sites. Among 84 primary MFS samples, most frequently mutated and/or CN-altered genes included TP53 (n=64, 76.2%), RB1 (n=29, 34.5%), CDKN2A (n=25, 30.0%), and ATRX (n=18, 21.4%). A fusion gene involving TRIO was newly identified by RNA sequencing in a single case. A similar mutational profile was observed in undifferentiated pleomorphic sarcoma (UPS), in which TP53 (59.1%), ATRX (34.0%), RB1 (22.7%), and CDKN2A (20.5%)) were the major mutational targets, suggesting the common molecular pathogenesis between these two subtypes. Combined with frequent positive staining in IHC (n=22, 44.0%), TP53 was affected in as many as 83.3% of the MFS cases (n=70). Five MFS cases evaluated by WGS showed complex structural abnormalities suggestive of increased genetic instability, where a median of 179 structural variations were detected per sample. WES with multi-regional sampling (n=5) disclosed a high level of intratumor heterogeneity, in which less than 29.0% of mutations were shared by different samples taken from the same tumor. Finally, an analysis of longitudinal samples (n=6) revealed significantly higher numbers of mutations in relapse samples (1.6 times on average, p = 0.03). In all cases, TP53 lesions were present from at the time of initial diagnosis, while most of the other lesions were subclonal and acquired during the clinical course. In summary, the genetic profile of MFS is characterized by frequent abnormalities in TP53, RB1, CDKN2A and ATRX, and closely related to other STSs, particularly to UPS. Clonal TP53 abnormalities resulted in complex chromosomal structure and a high degree of intratumor heterogeneity. Citation Format: Yasuhide Takeuchi, Annegret Kunitz, Hiromichi Suzuki, Kenichi Yoshida, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Teppei Shimamura, Nobuyuki Kakiuchi, Yusuke Shiozawa, Akira Yokoyama, Tetsuichi Yoshizato, Kosuke Aoki, Yoichi Fujii, Yasuhito Nannya, Hideki Makishima, Satoru Miyano, Hironori Haga, Frederik Damm, Seishi Ogawa. Myxofibrosarcoma is characterized by frequent abnormalities in TP53 and increased genetic instability [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 738.

Abstract 212: High-resolution barcoding in patient-derived xenografts of triple-negative breast cancer reveals reversible chemoresistance conferred by non-mutational mechanisms

Abstract Fifty percent of patients with localized triple negative breast cancer (TNBC) have substantial residual cancer burden following treatment with neoadjuvant chemotherapy (NACT), resulting in a 40-80% risk of recurrence which leads to distant metastases and death for most patients. Intra-tumor heterogeneity (ITH) is a pervasive feature of TNBCs but the relative contributions of heterogeneous tumor cell populations to chemoresistance are not understood. To investigate the clonal dynamics that accompany chemotherapy treatment, we employed orthotopic patient-derived xenograft (PDX) models of treatment-naïve TNBC, thus enabling experimentation with heterogeneous populations of human tumor cells that have undergone minimal manipulation. To monitor the fates of PDX tumor cell lineages undergoing treatment with front-line NACT, we treated multiple treatment-naïve PDX models with Adriamycin combined with Cytoxan (AC). Some PDXs initially exhibited partial sensitivity followed by maintenance of residual tumors that were resistant to chemotherapy. Residual tumors re-grew to regain partial chemo-sensitivity. To conduct barcode-mediated clonal tracking, we introduced a pooled lentiviral barcode library (Cellecta; 50M unique barcodes) into freshly dissociated PDX tumor cells which were orthotopically engrafted into recipient mice. Strikingly, residual tumors maintained the same clonal architecture as untreated tumors. In contrast, only 20% of residual tumor clones repopulated tumors after discontinuation of treatment. Whole-exome sequencing revealed conservation of mutant allele frequencies throughout treatment. Together, these studies demonstrate that re-growth of residual tumors is accompanied by a non-random selection of subclones, that residual tumors surviving AC maintain the same levels of ITH as untreated tumors, and that selection of genomic subclones did not confer the observed chemoresistance. Transcriptomic, proteomic, and histologic profiling revealed that residual tumors exist in a distinct state characterized by alterations in EMT, metabolic, and cell adhesion programs. This state was reverted as tumors re-grew after discontinuation of AC treatment, indicating that the residual state may be a unique therapeutic window. In silico prediction of drug sensitivity revealed candidate drivers of resistance in the residual tumor state, and pharmacologic targeting identified multiple existing therapies that significantly delayed the regrowth of residual tumors. These data suggest that sequential administration of AC followed by these targeted agents could prolong TNBC responses, which may delay time to recurrence for patients with this highly aggressive disease. Citation Format: Gloria Vittone Echeverria, Sahil Seth, Zhongqi Ge, Alessandro Carugo, Christopher Bristow, Prabjhot Mundi, Sabrina Jeter-Jones, Xiaomei Zhang, Xinhui Zhou, Aaron McCoy, Shirong Cai, Yizheng Tu, Yuting Sun, Joseph Marszalek, Andrea Califano, William F. Symmans, Stacy L. Moulder, Jeffery T. Chang, Timothy P. Heffernan, Helen Piwnica-Worms. High-resolution barcoding in patient-derived xenografts of triple-negative breast cancer reveals reversible chemoresistance conferred by non-mutational mechanisms [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 212.

Abstract 3401: Comprehensive analysis of genetic alterations and intratumor heterogeneity in myxofibrosarcoma

Abstract Myxofibrosarcoma (MFS) is a rare subtype of soft tissue sarcomas (STSs) preferentially affecting elderly. Histologically, MFS is distinct from other STSs, in that it is characterized by the proliferation of pleomorphic spindle cells with varying degrees of myxoid components. However, the molecular pathogenesis of MSF is poorly understood. In this study, we conducted an integrated molecular analysis of 44 samples from primary MFS patients, in which samples were analyzed by whole-genome sequencing (WGS) (n=2), whole-exome sequencing (WES) (n=44), RNA sequencing (n=3), DNA methylation array (n=16), and immunohistochemistry (IHC, n=27). Copy number alterations (CNAs) were identified by sequence based copy number analysis. The obtained genomic data were combined with those from STS cases from the Cancer Genome Atlas (TCGA) cohort, including 17 MSF samples and compared to the data from other STS samples (n=189). To further investigate the genetic basis of mixed histological components and chronological changes in MFS, we performed analysis from multi-regional and/or multi-time-point samples from 8 MSF cases. A total of 4,613 mutations were identified by WES in 61 primary MFS samples with a median of 44.0 mutations/sample. Mutations were dominated by age-related C to T transitions at CpG sites. Most frequently mutated genes included TP53 (n=21, 34.4%), ATRX (n=9, 14.8%), and RB1 (n=3, 4.9%). A fusion gene associated with TRIO was detected by RNA sequencing in a single case. Among other STSs, undifferentiated pleomorphic sarcoma (UPS) harbored the most similar genetic abnormalities (most frequently mutated genes included TP53 (40.9%), ATRX (29.5%), and RB1 (11.3%)), suggesting that these two subtypes are genetically closely related. Also combined cases with CNAs (n=35) and strong staining in IHC (n=13), TP53 abnormality was found in most cases (n=56, 91.8%). Two MSF cases evaluated by WGS showed complex structural abnormalities, where 491 and 198 somatic structural variations were detected suggestive of increased genetic instability. Multi-regional sampling (n=5) disclosed a high level of intratumor heterogeneity with less than 29.0% of mutations being shared by different samples. Multi-time points sampling (n=6) revealed that the number of mutations was significantly higher in relapse samples (odds ratio 1.6, p = 0.03). While in all cases, TP53 lesions were observed at the initial time-point, others were subclonal and acquired during the clinical course. Finally, in methylation analysis, 3,817 differentially methylated regions were detected (Stouffer's p &amp;lt; 0.05), based on which MFS were clustered into two distinct subtypes. In summary, the genetic profile of MFS is characterized by frequent abnormalities in TP53, ATRX, and RB1 and closely related to other STSs, especially to UPS. Clonal TP53 abnormalities resulted in complex chromosomal structure and a high degree of intratumor heterogeneity. Citation Format: Yasuhide Takeuchi, Annegret Kunitz, Hiromichi Suzuki, Kenichi Yoshida, Nobuyuki Kakiuchi, Yusuke Shiozawa, Akira Yokoyama, Yoichi Fujii, Tetsuichi Yoshizato, Kosuke Aoki, Keisuke Kataoka, Yasuhito Nannya, Yuichi Shiraishi, Teppei Shimamura, Kenichi Chiba, Hiroko Tanaka, Hideki Makishima, Satoru Miyano, Hironori Haga, Frederik Damm, Seishi Ogawa. Comprehensive analysis of genetic alterations and intratumor heterogeneity in myxofibrosarcoma [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 3401.

Multi-regional sequencing reveals intratumor heterogeneity and positive selection of somatic mtDNA mutations in hepatocellular carcinoma and colorectal cancer.

Recent studies have revealed significant intratumor heterogeneity (ITH) of nuclear genome mutations and highlighted its function in tumor progression and treatment resistance. However, the ITH of somatic mitochondrial DNA (mtDNA) mutations detected in cancers remains unknown. In this study, we performed multiregional mtDNA sequencing of tumor and paratumor tissue samples from 12 hepatocellular carcinoma (HCC) and 13 colorectal cancer (CRC) patients. A substantial level of mtDNA mutations was found in paired non-HCC inflammatory tissues, suggesting that these tissues might not be mtDNA-genetically "normal." Moreover, our data indicated that the ITH of somatic mtDNA mutations was a common feature in HCC and CRC patients. In addition, we found that shared mutations which were observed in at least 2 samples in each patient exhibited a significantly higher heteroplasmic level than mutations that were private to a specific tumor region from both HCC (p = 0.039) and CRC patients (p = 0.001). The heteroplasmic level of shared mutations was positively correlated with intratumoral recurrence of mtDNA mutations. We also found that shared mutations in tumor tissues with a higher degree of pathogenicity risk exhibited a higher heteroplasmic level and intratumoral recurrence in both HCC and CRC patients. These findings suggest that some mtDNA mutations may undergo positive selection during the clonal expansion. Taken together, our analyses identified various levels of ITH of somatic mtDNA mutations in HCC and CRC patients and provided evidence supporting the positive selection working on some somatic mtDNA mutations in tumor tissues.