Considerable Intratumoral Heterogeneity Research Articles

Intratumor Heterogeneity Predicts Prognosis in Lepidic Predominant Lung Adenocarcinoma.

Among the different subtypes of invasive lung adenocarcinoma, lepidic predominant adenocarcinoma (LPA) has been recognized as the lowest-risk subtype with good prognosis. The aim of this study is to provide insight into the heterogeneity within LPA tumors and to better understand the influence of other sub-histologies on survival outcome. Overall, 75 consecutive patients with LPA in pathologic stage I (TNM 8th edition) who underwent resection between 2010 and 2022 were included into this retrospective, single center analysis. The proportions of different growth patterns were reported in 5% increments according to the WHO classification. All tumors exhibited a predominantly lepidic growth pattern (median proportion 70%, IQR 60%-85%). The invasive component included acinar (n = 66, 88%), papillary (n = 41, 55%), micropapillary (n = 14, 19%), and solid growth patterns (n = 4, 5%), with most tumors exhibiting more than one invasive growth pattern. The presence of high-risk growth, that is, micropapillary and solid, was associated with higher T stage (r = 0.423, p = 0.0002). A classification of patients as lepidic/high-risk or lepidic/low-risk based on the presence of micropapillary and solid growth patterns resulted in a significantly worse disease-free survival (p = 0.0169, 5-year DFS: lepidic/high-risk 73% vs. lepidic/low-risk: 95%) for the lepidic/high-risk group, while the groups did not differ in age, gender, smoking status, or extent of resection. Patients with stage I LPA exhibit considerable intratumor heterogeneity regarding growth patterns, which can be used for prognostic stratification. The occurrence of micropapillary and solid growth patterns in LPA is associated with poorer disease-free survival.

Abstract C169: Investigating the effects of ancestry in KRAS mediated lung adenocarcinoma: Exploring intratumoral heterogeneity and mitochondrial dynamics

Abstract Demonstrating a combination of high prevalence, late detection, and low survival, Lung cancer is the leading cause of cancer related deaths worldwide (Sainz de Aja J, 2021) and Lung Adenocarcinoma (LUAD) is its most prevalent pathologic subtype (Zhou, 2021). Emerging from within the distal alveolar epithelium, LUAD presents with vast heterogeneity in histology, mutational spectra, and epigenomic dysregulation (Sainz de Aja J, 2021) (Zhou, 2021). Additionally, LUAD’s considerable intratumoral heterogeneity manifests vast genomic and phenotypic diversity across tumor cells and cellular components of the tumor microenvironment (TME) within the same tumor tissue. LUAD also reveals significant racial disparities in incidence, survival and mortality rates between White and Black men in the United States. The occurrence of LUAD is approximately 68.3 per capita for Black men and 61.5 per capita for White men, reflecting an 11% higher rate in the former. Further, survival outcomes indicate that Black men generally exhibit lower 5-year relative survival rates compared to White men, highlighting greater mortality risk and conspicuously poorer prognosis (Siegel RL et al., 2023). While addressing the social determinants of health is vital for the mitigation of health disparities, understanding the genetic and molecular foundations that underly racial health inequities is essential for developing comprehensive interventions and advancing more precise therapeutic modalities for more diverse patient populations. Single Nucleotide Polymorphisms (SNPs) can influence oncogenic transformation and may be associated with distinct molecular landscapes in tumors and adjacent TME. Furthermore, ancestry may play a pivotal role in shaping individual susceptibility and risk for mitochondrial dysfunction and ensuing oncogenic transformation. To elucidate the biological underpinnings of racial health disparities in LUAD, 10 Black and 10 White male LUAD patients’ tumor and normal corresponding adjacent tissues underwent molecular profiling for KRAS mutation status, Whole Exome Sequencing (WES) to determine ancestry, and analysis of associated LUAD induced Mitochondrial Somatic Nucleotide Variants (mtSNVs). We also employed the 10X Visium HD spatial transcriptomic platform to evaluate spatially resolved molecular and cellular differences in KRAS mediated LUAD between Black and White men. Hypothesis: Thus, we propose that global ancestry is associated with variations in intratumoral heterogeneity, TME composition, and mitochondrial dynamics, indicating a potential role in the observed disparities in susceptibility, malignancy and mortality in LUAD across diverse patient populations. Citation Format: Eun Kyu Sung, Yuxin Jin, Yonatan Amzaleg, John Carpten, William Dean Wallace, Dan Raz, Aaron M. Neely. Investigating the effects of ancestry in KRAS mediated lung adenocarcinoma: Exploring intratumoral heterogeneity and mitochondrial dynamics [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C169.

T-cell receptor architecture and clonal tiding provide insight into the transformation trajectory of peripheral T-cell lymphomas.

While T cell lymphomas are classified as mature neoplasms, emerging evidence indicates that malignant transformation may occur at an earlier stage of T cell maturation. In this study, we determined clonal architectures in a broad range of T cell lymphomas. Our multidimensional profiling indicates that a large part of these lymphomas in fact emerge from an immature lymphoid T cell precursor at a maturation stage prior to V(D)J rearrangement that undergoes branching evolution. Consequently, at single cell resolution we observed considerable clonal tiding under selective therapeutic pressure. T cell receptor next-generation sequencing suggested a highly biased usage of TRBV20-1 gene segments as part of multiple antigen receptor rearrangements per patient. The predominance of TRBV20-1 was found across all major T cell lymphoma subtypes analyzed. This suggested that this particular V gene - independently of complementarity-determining region 3 (CDR3) configuration - may represent a driver of malignant transformation. Together, our data indicate that T cell lymphomas derive from immature lymphoid precursors and display considerable intratumoral heterogeneity that may provide the basis for relapse and resistance in these hard-to-treat cancers.

Unfolding the mysteries of heterogeneity from a high-resolution perspective: integration analysis of single-cell multi-omics and spatial omics revealed functionally heterogeneous cancer cells in ccRCC.

The genomic landscape of clear cell renal cell carcinoma (ccRCC) has a considerable intra-tumor heterogeneity, which is a significant obstacle in the field of precision oncology and plays a pivotal role in metastasis, recurrence, and therapeutic resistance of cancer. The mechanisms of intra-tumor heterogeneity in ccRCC have yet to be fully established. We integrated single-cell RNA sequencing (scRNA-seq) and transposase-accessible chromatin sequencing (scATAC-seq) data from a single-cell multi-omics perspective. Based on consensus non-negative matrix factorization (cNMF) algorithm, functionally heterogeneous cancer cells were classified into metabolism, inflammatory, and EMT meta programs, with spatial transcriptomics sequencing (stRNA-seq) providing spatial information of such disparate meta programs of cancer cells. The bulk RNA sequencing (RNA-seq) data revealed high clinical prognostic values of functionally heterogeneous cancer cells of three meta programs, with transcription factor regulatory network and motif activities revealing the key transcription factors that regulate functionally heterogeneous ccRCC cells. The interactions between varying meta programs and other cell subpopulations in the microenvironment were investigated. Finally, we assessed the sensitivity of cancer cells of disparate meta programs to different anti-cancer agents. Our findings inform on the intra-tumor heterogeneity of ccRCC and its regulatory networks and offers new perspectives to facilitate the designs of rational therapeutic strategies.

Spatial characterization and quantification of CD40 expression across cancer types

BackgroundCD40, a TNF receptor family member, is expressed by a variety of immune cells and is involved in the activation of both adaptive and innate immune responses. Here, we used quantitative immunofluorescence (QIF) to evaluate CD40 expression on the tumor epithelium of solid tumors in large patient cohorts of lung, ovarian, and pancreatic cancers.MethodsTissue samples from nine different solid tumors (bladder, breast, colon, gastric, head and neck, non-small cell lung cancer (NSCLC), ovarian, pancreatic and renal cell carcinoma), constructed in tissue microarray format, were initially assessed for CD40 expression by QIF. CD40 expression was then evaluated on the large available patient cohorts for three of the tumor types demonstrating high CD40 positivity rate; NSCLC, ovarian and pancreatic cancer. The prognostic impact of CD40 expression on tumor cells was also investigated.ResultsCD40 expression on tumor cells was found to be common, with 80% of the NSCLC population, 40% of the ovarian cancer population, and 68% of the pancreatic adenocarcinoma population displaying some degree of CD40 expression on cancer cells. All of three of these cancer types displayed considerable intra-tumoral heterogeneity of CD40 expression, as well as partial correlation between expression of CD40 on tumor cells and on surrounding stromal cells. CD40 was not found to be prognostic for overall survival in NSCLC, ovarian cancer, or pancreatic adenocarcinoma.ConclusionsThe high percentage of tumor cells expressing CD40 in each of these solid tumors should be considered in the development of therapeutic agents designed to target CD40.

Quantitative assessment of Siglec-15 expression in lung, breast, head, and neck squamous cell carcinoma and bladder cancer

AbstractImmune checkpoint blockade with programmed cell death (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors has resulted in significant progress in the treatment of various cancer types. However, not all patients respond to PD-1/PD-L1 blockade, underscoring the importance of identifying new potential targets for immunotherapy. One promising target is the immune system modulator Siglec-15. In this study, we assess Siglec-15 expression in solid tumors, with a focus on lung, breast, head and neck squamous and bladder cancers. Using quantitative immunofluorescence (QIF) with a previously validated antibody, we found increased Siglec-15 expression in both tumor and immune cells in all the four cancer types. Siglec-15 was seen to be predominantly expressed by the stromal immune cells (83% in lung, 70.1% in breast, 95.2% in head and neck squamous cell and 89% in bladder cancers). Considerable intra-tumoral heterogeneity was noted across cancer types. As previously described for non-small cell lung cancer (NSCLC), Siglec-15 expression was seen to be mutually exclusive to PD-L1 in all the four cancer types, although this differential expression was maintained but somewhat diminished in head and neck squamous cell carcinoma (HNSCC). Siglec-15 was not prognostic either for overall survival (OS) or progression-free survival (PFS). In summary, we show broad expression of this potential immune modulatory target in a wide range of cancer types. These data suggest potential future clinical trials in these tumor types.

An In Silico Analysis Identified Members of the Pleckstrin Homology-Like Domain, Family B (PHLDB family) as Potential Prognostic and Predictive Biomarkers of Treatment Response in Breast Cancer Patients.

Breast cancer is the leading cause of morbidity and mortality in women worldwide. This malignant neoplasm can be classified into four clinically relevant subtypes according to the expression of a number of biomarkers. However, these tumors show considerable intratumoral heterogeneity and multidrug resistance. Members of the pleckstrin homology-like domain, family B (PHLDB) play a critical role in the regulation of p53 and AKT signaling pathways, important for cancer and cellular metabolism. The present study was performed to evaluate the expression pattern of PHLDB family members in breast cancer and its potential prognostic and predictive value for therapeutic response using bioinformatics tools. This in silico analysis was performed using several online repositories, including UALCAN, GEPIA2, bc-GenExMiner, KM Plotter, PrognoScan and ROC Plotter. PHLDB family genes were found to be differentially expressed in tumor samples when compared to healthy breast tissue samples. Furthermore, epigenetic regulation may be one of the regulatory mechanisms for the expression of these markers. The PHLDB family of genes proved to be potential markers for predicting the development of lymph node metastasis (p<0.0001) and poor clinical outcome. All members of the PHLDB family were significantly correlated with hormone receptors. High levels of PHLDBs expression were associated with worse overall survival and recurrence-free survival in breast cancer patients. Finally, our data demonstrate that members of the PHLDB family can be promising markers in the stratification of patients who may or may not respond to different available therapies. Our cumulative results demonstrate that PHLDB family members may be promising biomarkers for predicting prognosis and therapeutic response in breast cancer patients.

Editors' Selections from Relevant Scientific Publications.

Editors' Selections from Relevant Scientific Publications.

The differential distributions of ASPM isoforms and their roles in Wnt signaling, cell cycle progression, and pancreatic cancer prognosis

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and treatment‐resistant malignancy. The lack of pathway‐informed biomarkers hampers the development of rational diagnostics or therapies. Recently, the protein abnormal spindle‐like microcephaly‐associated (ASPM) was identified as a novel Wnt and stemness regulator in PDAC, while the pathogenic roles of its protein isoforms remain unclarified. We developed novel isoform‐specific antibodies and genetic knockdown (KD) of putative ASPM isoforms, whereby we uncovered that the levels of ASPM isoform 1 (iI) and ASPM‐iII are variably upregulated in PDAC cells. ASPM isoforms show remarkably different subcellular locations; specifically, ASPM‐iI is exclusively localized to the cortical cytoplasm of PDAC cells, while ASPM‐iII is predominantly expressed in cell nuclei. Mechanistically, ASPM‐iI co‐localizes with disheveled‐2 and active β‐catenin as well as the stemness marker aldehyde dehydrogenase‐1 (ALDH‐1), and its expression is indispensable for the Wnt activity, stemness, and the tumorigenicity of PDAC cells. By contrast, ASPM‐iII selectively regulates the expression level of cyclin E and cell cycle progression in PDAC cells. The expression of ASPM‐iI and ASPM‐iII displays considerable intratumoral heterogeneity in PDAC tissues and only that of ASPM‐iI was prognostically significant; it outperformed ALDH‐1 staining and clinico‐pathological variables in a multivariant analysis. Collectively, the distinct expression patterns and biological functions of ASPM isoforms may illuminate novel molecular mechanisms and prognosticators in PDAC and may pave the way for the development of therapies targeting this novel oncoprotein. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Abstract LB-325: Developmental hierarchy in Langerhans cell histiocytosis

Abstract Langerhans Cell Histiocytosis (LCH) is a neoplastic disease characterized by the accumulation of CD1a+CD207+ cells of unknown origin. Constitutive activation of the ERK signaling pathway is a common feature of LCH, but the pathogenesis of this disease remains unclear. Here we present a comprehensive analysis of the composition of LCH lesions that provides new insight into the development and molecular mechanisms of this disease. We used a multilayered approach employing immunohistochemistry, flow cytometry, single- cell RNA-sequencing, and ATAC- sequencing of LCH biopsies obtained from different patients. Based on the single- cell RNA expression data, we identified different LCH cell populations within LCH lesions that display distinct pathway signatures, including a stem-cell-like, proliferative state. Computational analysis discovered a hierarchy between these cells that suggest a directed developmental program in each tumor. Finally, functional chromatin mapping inferred distinct regulatory signatures discriminating the different cell states. Taken together, our study shows at single-cell resolution that there is considerable intra-tumoral heterogeneity in LCH and provides detailed insight into the molecular framework underlying tumor development. Our transcriptomic and epigenomic analyses have generated a reference map of subpopulations of LCH cells with distinct functional and phenotypic properties. We propose that the lesions are comprised of different LCH cell subsets that arise in an intrinsic developmental process. This work can serve as a template for the analysis of tumorigenesis beyond LCH. Citation Format: Florian Halbritter, Matthias Farlik, Christoph Bock, Caroline Hutter. Developmental hierarchy in Langerhans cell histiocytosis [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-325.

Unravelling the Heterogeneity of Mantle Cell Lymphoma Ecosystem By Single Cell RNA Sequencing

Background: Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma that is incurable. MCL has a complex ecosystem of malignant B-cells and stromal and immune cells that play a supporting role for tumor growth, ultimately leading to the potential re-emergence of the disease. The tumor microenvironment has been reported as a crucial factor in MCL pathogenesis and progression. Thus, if we can identify the tumor microenvironment components and define the characteristics of malignant and non-malignant cells, this will pave the way for studying clonal evolution of MCL in vivo.Methods: Both pre- and post-treatment, fresh tumor biopsy samples of MCL were obtained. The cells were dissociated and re-suspended in PBS with >10% serum. A final concentration of 1,200 cells/uL were used for single cell sorting in the chromium system (10X Genomics, California). We sequenced the mRNA in the NextSeq 500 platform. All analysis was conducted using R-programming language (version 3.4).Results: From four MCL patients (L1-L4), we obtained 9,400 cells. Three of the four samples were collected through apheresis (i.e., L1-L3), and one sample (i.e., L4) from surgical biopsy of the involved lymph node. One patient had known TP53 mutated status (i.e., L1) and another patient had CCND1 translocation (i.e., L2). From the apheresis samples (L1-L3), the proportion of lymphocytes was 87%, 68% and 65%. We identified 10 defined clusters of cells based upon their gene expression from all four samples. Six of the 10 clusters were clonal B-cells with strong expression of CCND1, CD79A and CD79B. We also identified clonal T-cells (both CD8+ and CD4+) and monocyte/macrophage clusters. SOX11 expression was absent in one B-cell clone, indicating this clone may be SOX11-negative MCL. The monocyte-macrophage cluster demonstrated strong BCL2 expression, which was not expressed by the B-cells clones. CD19 expression was ubiquitous among the B-cell clones but weaker as compared with other B-cell markers. When the signaling was compared among the four samples, the chemo-resistant cells (sample L1) demonstrated upregulation of NOTCH1 signaling, DNA-damage repair, interferon-alpha response, MYC targets and the HIF1A pathway. Two cell clones did not express any canonical markers and were not identified as a defined cluster.Conclusions: We identified meaningful sub-populations of MCL that define the tumor microenvironment. There is considerable inter-tumor and intra-tumor heterogeneity of MCL at a single cell resolution, which indicates that developing a uniform treatment regimen may prove to be difficult. Ubiquitous expression of CD79A or CD79B may help guiding precision medicine such as the development of novel CAR-T cell therapy. Longitudinal follow up of the same patients may define clonal evolution of MCL and unravel the spatio-temporal interplay. [Display omitted] DisclosuresWang:AstraZeneca: Consultancy, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MoreHealth: Consultancy; Novartis: Research Funding; Acerta Pharma: Honoraria, Research Funding; Dava Oncology: Honoraria; Juno: Research Funding; Pharmacyclics: Honoraria, Research Funding.

Abstract 1187: Comparison of intra-tumor heterogeneity and clonal evolution across lung cancer subtypes by multi-region whole genome sequencing

Abstract Both lung squamous cell carcinoma (LUSC) and lung cancer with neuroendocrine features (LUNE; i.e., small cell carcinoma (SCLC), large cell carcinoma (LCNEC) and other subtypes) are associated with tobacco smoking and have a very poor prognosis. We sought to characterize their clonal architecture and illustrate how the evolutionary patterns differ across these lung cancer subtypes. We performed multi-region whole genome sequencing of 83 samples from 36 LUSC and 67 samples from 23 LUNE patients that had been resected before systemic therapy. Among somatic copy number alterations, we observed genome doubling intra-tumor heterogeneity (ITH) and massive genomic instability in both subtypes. For somatic nucleotide variants (SNVs), the total mutation burden was higher in LUSC (16.7 average number of mutations/Mb) than LUNE (12.7/Mb), but for SNVs ITH was higher in LUNE. Among driver genes, TP53, CDKN2A, CR1, and NRK in LUSC and TP53, KRAS, KMT2D, and RB1 in LUNE were frequently mutated and were always clonal. Evidence of positive selection was found for TP53, PTEN, KMT2D and KEAP1 in LUSC, and for TP53 and KRAS in LUNE. As expected, the dominant mutation signatures in LUSC clones included signatures 4, 1, and 5, while APOBEC signatures (2 and 13) were predominantly found in sub-clones. In contrast, no evidence of APOBEC signatures were found in LUNE. Among structural variants, LUNE had more events (339.25 average number of events/tumor) than LUSC (206.7/tumor), with considerable ITH in both subtypes, particularly in LUNE. In LUNE, the dominant mechanism responsible for SVs included fork stalling and template switching within the microhomology-mediated break-induced repair (MMBIR). We found recurrent fusion events with hotspots including those previously identified near TTC28/CHEK2 (42% LUSC and 35% LUNE) and LRP1B (42% LUSC). Additional hotspots were found in chromosome bands 20p12.1 (22% LUSC), 1p31.1 (19% LUSC) and 12p11.22 (22% LUNE). We found &amp;gt;10-fold higher transposon insertions (LINE1) in LUSC (71.9 average number of events/tumor) than LUNE (6.3/tumor). Our findings improve our evolutionary understanding of these lung cancer subtypes and warrant further investigations on the mechanisms involved in clonal genomic instability. Citation Format: Tongwu Zhang, Joshua Sampson, Pier Alberto Bertazzi, Angela Pesatori, Dario Consonni, Bin Zhu, Belynda Hicks, Xing Hua, Jianxin Shi, Kevin Brown, Stephen Chanock, Maria Teresa Landi. Comparison of intra-tumor heterogeneity and clonal evolution across lung cancer subtypes by multi-region whole genome sequencing [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 1187.

Spatiotemporal Optoacoustic Mapping of Tumor Hemodynamics in a Clinically Relevant Orthotopic Rabbit Model of Head and Neck Cancer

The purpose of this study was to investigate the usefulness of photoacoustic imaging (PAI) for spatiotemporal mapping of tumor hemodynamics in a rabbit model of head and neck carcinoma. Shope cottontail rabbit papilloma virus associated VX2 carcinomas were established in adult male New Zealand White rabbits (n = 9) by surgical transplantation of tumor tissue in the neck. Noninvasive PAI with co-registered ultrasound (US) was performed to longitudinally monitor tumor growth, oxygen saturation (%sO2), and hemoglobin concentration (HbT). PAI findings were validated with Doppler sonography measures of percent vascularity (PV). Differences in tumor volumes, %sO2, HbT, and PV values over time were analyzed using repeated-measures analysis of variance with multiple comparisons. Two-tailed Spearman correlation analysis was performed to determine the correlation coefficient (r) for comparisons between %sO2, HbT, and tumor volume. US revealed a significant (P < .0001) increase in tumor volume over the 3-week period from 549 ± 260 mm3 on day 7 to 5055 ± 438 mm3 at 21 days postimplantation. Consistent with this aggressive tumor growth, PAI revealed a significant (P < .05) and progressive reduction in %sO2 from day 7 (37.6 ± 7.4%) to day 21 (9.5 ± 2.1%). Corresponding Doppler images also showed a decrease in PV over time. PAI revealed considerable intratumoral spatial heterogeneity with the tumor rim showing two- to three-fold higher %sO2 values compared to the core. Noninvasive PAI based on endogenous contrast provides a label-free method for longitudinal monitoring of temporal changes and spatial heterogeneity in thick head and neck tumors.

A randomized, open-label study of the efficacy and safety of AZD4547 monotherapy versus paclitaxel for the treatment of advanced gastric adenocarcinoma with FGFR2 polysomy or gene amplification

Approximately 5%-10% of gastric cancers have a fibroblast growth factor receptor-2 (FGFR2) gene amplification. AZD4547 is a selective FGFR-1, 2, 3 tyrosine kinase inhibitor with potent preclinical activity in FGFR2 amplified gastric adenocarcinoma SNU16 and SGC083 xenograft models. The randomized phase II SHINE study (NCT01457846) investigated whether AZD4547 improves clinical outcome versus paclitaxel as second-line treatment in patients with advanced gastric adenocarcinoma displaying FGFR2 polysomy or gene amplification detected by fluorescence in situ hybridization. Patients were randomized 3:2 (FGFR2 gene amplification) or 1:1 (FGFR2 polysomy) to AZD4547 or paclitaxel. Patients received AZD4547 80 mg twice daily, orally, on a 2 weeks on/1 week off schedule of a 21-day cycle or intravenous paclitaxel 80 mg/m2 administered weekly on days 1, 8, and 15 of a 28-day cycle. The primary end point was progression-free survival (PFS). Safety outcomes were assessed and an exploratory biomarker analysis was undertaken. Of 71 patients randomized (AZD4547 n = 41, paclitaxel n = 30), 67 received study treatment (AZD4547 n = 40, paclitaxel n = 27). Among all randomized patients, median PFS was 1.8 months with AZD4547 and 3.5 months with paclitaxel (one-sided P = 0.9581); median follow-up duration for PFS was 1.77 and 2.12 months, respectively. The incidence of adverse events was similar in both treatment arms. Exploratory biomarker analyses revealed marked intratumor heterogeneity of FGFR2 amplification and poor concordance between amplification/polysomy and FGFR2 mRNA expression. AZD4547 did not significantly improve PFS versus paclitaxel in gastric cancer FGFR2 amplification/polysomy patients. Considerable intratumor heterogeneity for FGFR2 gene amplification and poor concordance between FGFR2 amplification/polysomy and FGFR2 expression indicates the need for alternative predictive biomarker testing. AZD4547 was generally well tolerated.

Intratumoral Morphological Heterogeneity of Breast Cancer As an Indicator of the Metastatic Potential and Tumor Chemosensitivity

Breast cancer (BC) demonstrates considerable intratumoral morphological heterogeneity. The aim of this work was to evaluate the relationship among different morphological structures, the rate of metastasis, and efficacy of neoadjuvant chemotherapy (NAC) in NAC-treated (n = 427) and NAC-naïve (n = 249) BC patients. We also studied the involvement of an epithelial-mesenchymal transition (EMT) in the development of the intratumoral morphological heterogeneity of BC. We found a significant association between the intratumoral morphological heterogeneity and the rate of BC metastasis and response to NAC, which, in most cases, correlated with the presence of alveolar and trabecular structures. In particular, the rate of lymph node metastasis in tumors containing alveolar and trabecular structures was higher compared to that in tumors lacking such structures. NAC-treated patients with alveolar and trabecular structures had a high distant metastasis rate and a low metastasis-free survival rate. Furthermore, alveolar and trabecular structures were found to be associated with a lack of response to NAC. Interestingly, the association between alveolar structures and a high distant metastasis rate was found only in NAC-unresponsive patients, whereas the association between trabecular structures and an increased distant metastasis was revealed in responders. Alveolar structures were associated with chemoresistance only in patients with lymph node metastases, whereas trabecular structures were associated with chemoresistance only in patients without lymph node metastases. In general, increased intratumoral morphological diversity correlated with considerable chemoresistance and a high metastasis rate of BC. We found variable expressions of epithelial (EPCAM and CDH1) and mesenchymal (ITGA5, ITGB5, CDH2, CDH11, TGFb2, ZEB1, MMP2, DCN, MST1R) markers and, thus, different EMT manifestations in different morphological structures. Therefore, intratumoral morphological heterogeneity of BC may serve as an indicator of the metastatic potential and tumor chemosensitivity.

PD-L1 Expression and Intratumoral Heterogeneity Across Breast Cancer Subtypes and Stages: An Assessment of 245 Primary and 40 Metastatic Tumors.

Tumor expression of programmed cell death ligand 1 (PD-L1) is associated with immune evasion in a variety of malignancies, including a subset of triple-negative breast carcinomas, and may mark cancers as susceptible to PD-1/PD-L1 inhibitor therapies. We herein characterize PD-L1 expression in breast cancers across the full range of histomorphologies and investigate its intratumoral heterogeneity and fidelity across primaries and metastases. A total of 245 primary and 40 metastatic (20 nodal, 20 distant) breast carcinomas were evaluated with PD-L1 immunohistochemistry on tissue microarray. Tumor PD-L1 staining was seen in 12% of all primaries including 32% of triple-negative cancers. Staining was common in ductal cancers with medullary (54%), apocrine (27%), and metaplastic features (40%). However, diffuse (>50%) staining was rare (2% of all cancers and 5% of triple negatives). Immune staining was seen in 29% of all primaries and 61% of triple negatives. Tumor expression of PD-L1 was conserved in 94% of matched primary/metastasis pairs, while immune staining showed fidelity in 71%; the remaining cases acquired PD-L1 immune cell expression in the metastasis. Only half of cases with positive tumor staining showed concordance across all analyzed cores. These data demonstrate that PD-L1 expression is prevalent among high-grade, hormone receptor-negative breast cancers with a range of histomorphologies and shows fidelity between primary and metastatic sites in treatment-naive cancers, although acquisition of immune PD-L1 staining in metastases is not uncommon. There is considerable intratumoral heterogeneity in PD-L1 expression, undermining the suitability of core biopsy in the determination of PD-L1 status. Clinical trials are needed to determine PD-L1 staining thresholds required for therapeutic response, as diffuse staining is rare.

Intratumoral Genetic Heterogeneity in Rectal Cancer: Are Single Biopsies representative of the entirety of the tumor?

Demonstrate intratumoral genetic heterogeneity in rectal cancer. Several clinical management decisions in rectal cancer may be influenced by pretreatment biopsy information. However, in the setting of significant intratumoral heterogeneity, biopsies may not be representative of the entirety of the tumor and limit the reliability of the information provided from them for clinical decision management. Three fragments from a single rectal adenocarcinoma were chosen for whole-exome sequencing followed by mutation detection analysis. About 25 Gb of unambiguously mapped sequences were generated for each sample resulting in a median fold-coverage of 35x. Captured sequences mapped to the reference human genome were then used for the detection of somatic point mutations. Overall, 193 unique somatic point mutations were identified. Only 53 (27%) of these were shared by all three fragments, including known genes involved in early phases of the adenoma-carcinoma sequence (such as, APC). Approximately, 115 (59%) mutations were exclusively present in only one of the fragments, including mutations in "driver" genes (DNAH12). Jaccard distances showed a median distance of 0.603 for pair-wise comparison of fragments indicating significant heterogeneity between them. Considerable intratumoral heterogeneity is present among naive rectal cancers. The majority of point mutations detected in different fragments from rectal cancers are frequently unique to a single fragment. These findings support that gene mutations found on single pretreatment biopsies will not necessarily be representative of mutations present in the entirety of the tumor and therefore may limit the utility of the biological information provided by single biopsy fragments for clinical management decisions.

Cell Culture System for Analysis of Genetic Heterogeneity Within Hepatocellular Carcinomas and Response to Pharmacologic Agents

No targeted therapies have been found to be effective against hepatocellular carcinoma (HCC), possibly due to the large degree of intratumor heterogeneity. We performed genetic analyses of different regions of HCCs to evaluate levels of intratumor heterogeneity and associate alterations with responses to different pharmacologic agents. We obtained samples of HCCs (associated with hepatitis B virus infection) from 10 patients undergoing curative resection, before adjuvant therapy, at hospitals in China. We collected 4-9 spatially distinct samples from each tumor (55 regions total), performed histologic analyses, isolated cancer cells, and carried them low-passage culture. We performed whole-exome sequencing, copy-number analysis, and high-throughput screening of the cultured primary cancer cells. We tested responses of an additional 105 liver cancer cell lines to a fibroblast growth factor receptor (FGFR) 4 inhibitor. We identified a total of 3670 non-silent mutations (3192 missense, 94 splice-site variants, and 222 insertions or deletions) in the tumor samples. We observed considerable intratumor heterogeneity and branched evolution in all 10 tumors; the mean percentage of heterogeneous mutations in each tumor was 39.7% (range, 12.9%-68.5%). We found significant mutation shifts toward C>T and C>G substitutions in branches of phylogenetic trees among samples from each tumor (P<.0001). Of note, 14 of the 26 oncogenic alterations (53.8%) varied among subclones that mapped to different branches. Genetic alterations that can be targeted by existing pharmacologic agents (such as those in FGF19, DDR2, PDGFRA, and TOP1) were identified in intratumor subregions from 4HCCs and were associated with sensitivity to these agents. However, cells from the remaining subregions, which did not have these alterations, were not sensitive to these drugs. High-throughput screening identified pharmacologic agents to which these cells were sensitive, however. Overexpression of FGF19 correlated with sensitivity of cells to an inhibitor of FGFR 4; this observation wasvalidated in 105 liver cancer cell lines (P=.0024). By analyzing genetic alterations in different tumor regions of 10 HCCs, we observed extensive intratumor heterogeneity. Our patient-derived cell line-based model, integrating genetic and pharmacologic data from multiregional cancer samples, provides a platform to elucidate how intratumor heterogeneity affects sensitivity to different therapeutic agents.

Phenotypic Transition as a Survival Strategy of Glioma.

Malignant glioma is characterized by rapid proliferation, invasion into surrounding central nervous system tissues, and aberrant vascularization. There is increasing evidence that shows gliomas are more complex than previously thought, as each tumor comprises considerable intratumoral heterogeneity with mixtures of genetically and phenotypically distinct subclones. Heterogeneity within and across tumors is recognized as a critical factor that limits therapeutic progress for malignant glioma. Recent genotyping and expression profiling of gliomas has allowed for the creation of classification schemes that assign tumors to subtypes based on similarity to defined expression signatures. Also, malignant gliomas frequently shift their biological features upon recurrence and progression. The ability of glioma cells to resist adverse conditions such as hypoxia and metabolic stress is necessary for sustained tumor growth and strongly influences tumor behaviors. In general, glioma cells are in one of two phenotypic categories: higher proliferative activity with angiogenesis, or higher migratory activity with attenuated proliferative ability. Further, they switch phenotypic categories depending on the situation. To date, a multidimensional approach has been employed to clarify the mechanisms of phenotypic shift of glioma. Various molecular and signaling pathways are involved in phenotypic shifts of glioma, possibly with crosstalk between them. In this review, we discuss molecular and phenotypic heterogeneity of glioma cells and mechanisms of phenotypic shifts in regard to the glioma proliferation, angiogenesis, and invasion. A better understanding of the molecular mechanisms that underlie phenotypic shifts of glioma may provide new insights into targeted therapeutic strategies.

P78: Intratumor morphological heterogeneity in breast cancer and distant metastasis: Expression analysis of genes involved in cell motility and pre-metastatic niche formation

Background Breast cancer, particularly invasive carcinoma of no special type (IC NST), demonstrates considerable intratumor morphological heterogeneity. Five types of morphological structures representing different architectural arrangements of tumor cells – tubular, alveolar, trabecular, solid structures, and discrete groups have been described in IC NST. Previous studies reported the contribution of intratumor morphological heterogeneity of IC NST to chemotherapy efficiency and lymph node metastasis (Zavyalova et al., 2013; Denisov et al., 2014); however, its role in distant metastasis remains unidentified. Aim: to study the contribution of intratumor morphological heterogeneity of IC NST to distant metastasis and to identify gene expression features of metastatic behavior of different morphological structures. Materials and methods 358 IC NST patients (age range 29–90, mean age 49.8 ± 9.5, T1-4N0-3M0-1) treated with neoadjuvant chemotherapy (NAC) have been enrolled in this study. Chi-square test and Kaplan–Meier analysis were used to estimate the association between the presence of certain morphological structures in breast tumors and the frequency of distant metastasis and metastasis-free survival. qRT-PCR was applied for measurement of the expression levels of genes involved in cell motility (CDH1, CDH2, CDH3, CTNNA1, CTNNB1, ITGA6, ITGAV, ITGB1, ITGB3, ITGB4, SNAIL, MMP14, ROCK2, L1CAM, MMP2, MMP9, PDPN) and pre-metastatic niche formation (TNFa, TGFb, VEGFa, LOX, M-CSF, GM-CSF, HIF1A, SDF2) in different morphological structures isolated from breast tumors ( n = 4) by laser microdissection. Results Patients with alveolar structures in breast tumors more frequently displayed distant metastasis than cases without this morphological variant (71.9% vs. 56.5%; p = 0.004). The association between alveolar structures and high frequency of hematogenous metastasis was found only in patients with poor response to NAC ( p = 0.003), but not in cases with good chemotherapy efficiency ( p = 0.377). Increased distant metastasis was also shown in patients with trabecular structures as compared to cases without this morphological type (88.3% vs. 70.0%; p = 0.0001). Kaplan–Meier analysis demonstrated a significantly higher probability of developing metastasis in patients with alveolar or trabecular structures in breast tumors ( p = 0.011). No significant association between other morphological structures and distant metastasis was found. Expression analysis showed the presence of cell motility phenotype in all morphological structures. In particular, we found changes in cell adhesion gene expression, which declined in the row: solid–alveolar–trabecular structures–discrete groups of tumor cells ( p p Conclusion Intratumor morphological heterogeneity of IC NST contributes to distant metastasis probably by variations in expression of genes involved in cell motility and pre-metastatic niche formation between different morphological structures. The study was supported by the Russian Scientific Foundation (Grant #14-15-00318 ).