Physical Forces and Transient Nuclear Envelope Rupture during Metastasis: The Key for Success?

During metastasis, invading tumor cells and circulating tumor cells (CTC) face multiple mechanical challenges during migration through narrow pores and cell squeezing. However, little is known on the importance and consequences of mechanical stress for tumor progression and success in invading a new organ. Recently, several studies have shown that cell constriction can lead to nuclear envelope rupture (NER) during interphase. This loss of proper nuclear compartmentalization has a profound effect on the genome, being a key driver for the genome evolution needed for tumor progression. More than just being a source of genomic alterations, the transient nuclear envelope collapse can also support metastatic growth by several mechanisms involving the innate immune response cGAS/STING pathway. In this review we will describe the importance of the underestimated role of cellular squeezing in the progression of tumorigenesis. We will describe the complexity and difficulty for tumor cells to reach the metastatic site, detail the genomic aberration diversity due to NER, and highlight the importance of the activation of the innate immune pathway on cell survival. Cellular adaptation and nuclear deformation can be the key to the metastasis success in many unsuspected aspects.

Understanding CLL biology through mouse models of human genetics

Rapid advances in large-scale next-generation sequencing studies of human samples have progressively defined the highly heterogeneous genetic landscape of chronic lymphocytic leukemia (CLL). At the same time, the numerous challenges posed by the difficulties in rapid manipulation of primary B cells and the paucity of CLL cell lines have limited the ability to interrogate the function of the discovered putative disease “drivers,” defined in human sequencing studies through statistical inference. Mouse models represent a powerful tool to study mechanisms of normal and malignant B-cell biology and for preclinical testing of novel therapeutics. Advances in genetic engineering technologies, including the introduction of conditional knockin/knockout strategies, have opened new opportunities to model genetic lesions in a B-cell–restricted context. These new studies build on the experience of generating the MDR mice, the first example of a genetically faithful CLL model, which recapitulates the most common genomic aberration of human CLL: del(13q). In this review, we describe the application of mouse models to the studies of CLL pathogenesis and disease transformation from an indolent to a high-grade malignancy (ie, Richter syndrome [RS]) and treatment, with a focus on newly developed genetically inspired mouse lines modeling recurrent CLL genetic events. We discuss how these novel mouse models, analyzed using new genomic technologies, allow the dissection of mechanisms of disease evolution and response to therapy with greater depth than previously possible and provide important insight into human CLL and RS pathogenesis and therapeutic vulnerabilities. These models thereby provide valuable platforms for functional genomic analyses and treatment studies.

Specific T Cell Receptor Gene Repertoire Profiles in Subgroups of CLL Patients with Distinct Genomic Aberrations

Chronic lymphocytic leukemia (CLL) B cells engage in multifaceted bi-directional interactions with bystander cells, including T cells. Immunogenetic studies in CLL revealed clonal expansions of T cells and shared T cell clonotypes between different patients, strongly implying clonal selection by antigens. Although the exact nature of these antigens remains largely elusive, evidence exists that the clonotypic B cell receptor immunoglobulin (BcR IG) may serve as a source of antigenic epitopes for T cells. That said, recurrent genomic aberrations associated with distinct abnormal expression profiles could represent an alternative, non mutually exclusive, source of potent immunogenic onco-antigens that might shape the T cell repertoire in CLL. On these grounds, here we interrogated the T cell receptor (TR) gene repertoire of CLL patients with different genomic aberration profiles with the aim to identify unique signatures that would allude to distinct antigen selection pressures. The study group included 46 patients with CLL, sampled before treatment initiation, who were categorized in 5 subgroups defined by a unique genomic aberration, as follows: +12, n=18; del(11q), n=10; del(13q), n=7; del(17p)/TP53mut, n=6; NOTCH1mut, n=5. Confounding effects of multiple aberrations have been minimized, as we previously established through comprehensive characterization (including FISH, SNP arrays and gene panels) that the analyzed patients carried only one of the above aberrations. Starting material was RNA extracted from blood mononuclear cells. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end next generation sequencing (NGS). Raw NGS reads (n=13,213,563| median: 294,757/sample) were processed through a purpose-built bioinformatics pipeline. Only productive rearrangements (n=9,249,546 | median=199,184/sample) were taken into consideration for the computation of clonotypes i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 (CDR3) sequence. Overall, 513,984 distinct clonotypes (median=10,304 clonotypes/sample) were assessed. The main measure of clonality employed in this study was the median cumulative frequency of the 10 most expanded T cell clonotypes/sample (MCF-10). For comparisons of the clonality profiles, a group of 17 aged-matched healthy individuals were used as controls. All patients displayed oligoclonal T cell expansions with the following MCF-10 values: del(11q): 21.6%, +12: 25%, del(13q): 20.6%, NOTCH1mut: 9.1%, del(17p)/TP53mut: 12.9%; the difference between the del(11q) and +12 groups versus the NOTCH1mut group was statistically significant (p<0.05). The MCF-10 value of the control group was estimated at 17.5%, supporting the notion of age-related decrease in TR repertoire diversity. However, the del(11q), +12 and del(13q) CLL groups displayed elevated clonality, reaching statistical significance (p<0.002) in the case of +12. TRBV gene repertoire analysis revealed that the TRBV12-3 gene predominated in most groups, except for the del(17p)/TP53mut, where the predominant gene was TRBV10-3. Clonotype comparisons disclosed the presence of shared TR clonotypes both within a particular group but also between groups. Overall, 446/513,984 clonotypes were found to be shared by at least two patients across all groups; the vast majority (392/446, 88%) of shared clonotypes appeared to be CLL-biased since they did not match entries in public databases of TR clonotypes from various contexts. Subgroup-specific clonotypes were identified for all aberrations examined; these emerged as unique to the particular subgroups, as revealed by extensive comparisons against both public databases but also a large TR clonotype database from CLL available to us from our previous studies. In conclusion, recurrent genomic aberrations, especially large chromosomal abnormalities, display an oligoclonal TR gene repertoire. The distinct immunogenetic profile of each group examined here and, most importantly, the existence of subgroup-specific clonotypes, suggest that abnormal protein expression and gene dosage effects likely represent a relevant source of CLL-specific selecting antigens. Disclosures Scarfo: Janssen: Honoraria, Other: Travel grants; Astra Zeneca: Honoraria; Abbvie: Honoraria. Anagnostopoulos: Abbvie: Other: clinical trials; Sanofi: Other: clinical trials ; Ocopeptides: Other: clinical trials ; GSK: Other: clinical trials; Incyte: Other: clinical trials ; Takeda: Other: clinical trials ; Amgen: Other: clinical trials ; Janssen: Other: clinical trials; novartis: Other: clinical trials; Celgene: Other: clinical trials; Roche: Other: clinical trials; Astellas: Other: clinical trials . Ghia: AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; ArQule/MSD: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Celgene/Juno/BMS: Consultancy, Honoraria; Gilead: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Sunesis: Research Funding. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Rosenquist: Roche: Honoraria; Janssen: Honoraria; Illumina: Honoraria; AstraZeneca: Honoraria; Abbvie: Honoraria. Stamatopoulos: Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Baliakas: Janssen: Honoraria; Gilead: Honoraria, Research Funding; Abbvie: Honoraria. Chatzidimitriou: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

Immune cell deconvolution of bulk DNA methylation data reveals an association with methylation class, key somatic alterations, and cell state in glial/glioneuronal tumors

It is recognized that the tumor microenvironment (TME) plays a critical role in the biology of cancer. To better understand the role of immune cell components in CNS tumors, we applied a deconvolution approach to bulk DNA methylation array data in a large set of newly profiled samples (n = 741) as well as samples from external data sources (n = 3311) of methylation-defined glial and glioneuronal tumors. Using the cell-type proportion data as input, we used dimensionality reduction to visualize sample-wise patterns that emerge from the cell type proportion estimations. In IDH-wildtype glioblastomas (n = 2,072), we identified distinct tumor clusters based on immune cell proportion and demonstrated an association with oncogenic alterations such as EGFR amplification and CDKN2A/B homozygous deletion. We also investigated the immune cluster-specific distribution of four malignant cellular states (AC-like, OPC-like, MES-like and NPC-like) in the IDH-wildtype cohort. We identified two major immune-based subgroups of IDH-mutant gliomas, which largely aligned with 1p/19q co-deletion status. Non-codeleted gliomas showed distinct proportions of a key genomic aberration (CDKN2A/B loss) among immune cell-based groups. We also observed significant positive correlations between monocyte proportion and expression of PD-L1 and PD-L2 (R = 0.54 and 0.68, respectively). Overall, the findings highlight specific roles of the TME in biology and classification of CNS tumors, where specific immune cell admixtures correlate with tumor types and genomic alterations.

Targeting RB1 Loss in Cancers

Retinoblastoma protein 1 (RB1) is encoded by a tumor suppressor gene that was discovered more than 30 years ago. Almost all mitogenic signals promote cell cycle progression by braking on the function of RB1 protein through mono- and subsequent hyper-phosphorylation mediated by cyclin-CDK complexes. The loss of RB1 function drives tumorigenesis in limited types of malignancies including retinoblastoma and small cell lung cancer. In a majority of human cancers, RB1 function is suppressed during tumor progression through various mechanisms. The latter gives rise to the acquisition of various phenotypes that confer malignant progression. The RB1-targeted molecules involved in such phenotypic changes are good quarries for cancer therapy. Indeed, a variety of novel therapies have been proposed to target RB1 loss. In particular, the inhibition of a number of mitotic kinases appeared to be synthetic lethal with RB1 deficiency. A recent study focusing on a neighboring gene that is often collaterally deleted together with RB1 revealed a pharmacologically targetable vulnerability in RB1-deficient cancers. Here we summarize current understanding on possible therapeutic approaches targeting functional or genomic aberration of RB1 in cancers.

Impact of genomic aberrations and additional therapies on survival outcomes of patients with operable non-small cell lung cancer (NSCLC) from the NEOSTAR study.

8542 Background: The NEOSTAR study compared nivolumab (N) vs. nivolumab plus ipilimumab (NI) with major pathological response (MPR; ≤10% viable tumor) as primary outcome. We report updated rates of treatment failure (TF), including in patients whose tumors harbored genomic aberrations, and outcomes of additional treatments. Methods: Patients (pts) with stage I-IIIA resectable NSCLC (AJCC 7th) were randomized to either neoadjuvant N or NI followed by surgery (n = 44). TF was defined as radiographic and/or biopsy-proven recurrence from primary lung cancer and/or death (treatment or cancer-related). Additional systemic therapy at recurrence included immuno-oncology (IO)-based therapy (IO or chemo-IO), targeted therapy (TT), or chemotherapy. Disease control rate (DCR) was defined as the proportion of pts with radiographic objective responses and stable disease at first restaging. Cox proportional hazards model was used to associate baseline characteristics and time to TF. Results: A total of 44 randomized pts were evaluated, the median follow-up was 35 months (mts) as of February 4, 2021. Among the 12 TF pts (12/44, 27%), 42% (5/12) did not undergo surgery on trial, 9 (9/44, 20%) experienced recurrence and 6 (6/44, 14%) died (1 non-cancer-related, 5 cancer-related). TF was less likely in smokers vs. never smokers (hazard ratio = 0.20, 95% confidence interval = 0.06-0.65, p = 0.007). Among pts with pathological specimen resected on trial, MPR was achieved in 40% (12/30) of non-TF pts. Only 1 (1/7, 14%) TF pt achieved MPR, but died of a non-cancer related cause. TF-free survival rate at 2 years was 92% in MPR and 78% in non-MPR pts. Eight (8/9, 89%) pts had tumors with canonical oncodriver aberrations (5 EGFR mutations, 1 with STK11+ KRAS Q61H mutations, 1 ALK translocation and 1 RET fusions). Of the 9 recurrences, 44% (4/9) were treated with IO therapy, and all 7 pts with targetable aberrations were treated with TT (3 after retreatment with IO therapies). Of the 4 pts retreated with IO therapy, duration between end of neoadjuvant and retreatment were 20, 17, 23, and 19 mts. Duration from retreatment until progression (PD) were 1, 1, and 2 mts, respectively. Last pt was treated without PD for 2 mts but switched to TT due to discovery of genomic aberration. IO retreatment achieved 25% DCR (1/4). In comparison, the DCR for TT treated pts was 71% (5/7, p = 0.242). Median time to treatment was 21 mts, and median time to PD was not reached. Among 32 non-TF pts, 12 had genomic analysis and 7 aberrations were found in 6 pts (2 STK11, 2 ERBB2, 1 STK11 + 1 KRAS G12C, and 1 KRAS G12C mutation). Conclusions: A 27% TF rate was observed after neoadjuvant IO. TF was less likely to occur in smokers and MPR pts, and 42% of TF pts did not undergo curative-intent surgery on trial. Genomic aberrations were common in pts with recurrence (89%), and treatment with TT achieved 71% DCR vs. 25% DCR with IO-based retreatment. Clinical trial information: NCT: 03158129.

Metastatic and recurrent adrenocortical cancer is not defined by its genomic landscape

Background Adrenocortical carcinoma (ACC) is a rare, often-aggressive neoplasm of the adrenal cortex, with a 14–17 month median overall survival. We asked whether tumors from patients with advanced or metastatic ACC would offer clues as to putative genes that might have critical roles in disease progression or in more aggressive disease biology. Methods We conducted comprehensive genomic and expression analyses of ACCs from 43 patients, 30 female, and 42 from metastatic sites, including deep sequencing, copy number analysis, mRNA expression and microRNA arrays. Results Copy number gains and losses were similar to that previously reported for ACC. We identified a median mutation rate of 3.38 per megabase (Mb). The mutational signature was characterized by a predominance of C > T, C > A and T > C transitions. Only cancer genes TP53 (26%) and beta-catenin (CTNNB1, 14%) were mutated in more than 10% of samples. The TCGA-identified putative cancer genes MEN1 and PRKAR1A were found in low frequency—4.7 and 2.3%, respectively. The majority of the mutations were in genes not implicated in the etiology or maintenance of cancer. Specifically, amongst the 38 genes that were mutated in more than 9% of samples, only four were represented in Tier 1 of the 576 COSMIC Cancer Gene Census (CCGC). Thus, 82% of genes found to have mutations likely have no role in the etiology or biology of ACC; while the role of the other 18%, if any, remains to be proven. Finally, the transcript length for the 38 most frequently mutated genes in ACC is statistically longer than the average of all coding genes, raising the question of whether transcript length in part determined mutation probability. Conclusions We conclude that the mutational and expression profiles of advanced and metastatic tumors are very similar to those from newly diagnosed patients—with very little in the way of genomic aberration to explain differences in biology. With relatively low mutation rates, few major oncogenic drivers, and loss of function mutations in several epigenetic regulators, an epigenetic basis for ACC may be postulated and serve as the basis for future studies.

Comprehensive profiling of MDM2/TP53 genomic aberration in Chinese patients with diverse malignancies.

e13508 Background: Murine double minute-2 (MDM2) has increasing clinic relevance as preclinical and early clinical evidences have showed that the MDM2 inhibitors are promising alternative treatments for patients with MDM2-amplified (-amp) and TP53-wild-type (-WT) tumors by preventing TP53 protein degradation. However, exploration into genomic landscape of MDM2/TP53 across various solid tumors in Chinese patients is still limited. Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor samples were collected from 10010 Chinese patients with solid tumors and subjected to a clinical-grade next-generation sequencing (NGS)-based 450 gene panel testing carried out by a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Genomic alterations (GAs) including single base substitutions, short and long insertion/deletions (Indels), copy number variations, gene fusions, and rearrangements were analyzed. Tumor mutational burden (TMB) was measured by a NGS algorithm. Results: Amplification of MDM2 occurred in 3.3% of Chinese patients. MDM2-amp was detected most frequently in soft tissue sarcoma (STS, 11.4 %), melanoma (10.3 %) and osteosarcoma (6.3 %). Notably, MDM2-amp was revealed to be mutually exclusive with TP53-mut in most tumor types other than esophageal carcinoma (93.9 %), head and neck carcinoma (68.4%) and ovarian carcinoma (50 %). Tumors with highest frequencies of MDM2-amp/ TP53-WT were melanoma (100%), osteosarcoma (100%) and urothelial carcinoma (100 %). The most common co-altered genes accompanied with MDM2-amp were FRS2 (84%), CDK4 (38%), EGFR (26%), TP53 (17%) and GLI1 (14%). 82.8% patients who had one or more co-alterations potentially targetable with either FDA-approved or investigational agent. Moreover, TMB of MDM2-amp tumors were significantly lower than that of MDM2-WT and MDM2-mutation (-mut) tumors (median TMB: 3.1 vs. 4.6 vs. 10.8, p < 0.001 respectively). Conclusions: MDM2 amplification was found in 3.3% of 10010 Chinese cancer patients, 83.4% of whom harbored wild-type TP53 that were potentially responsive to MDM2 inhibitors. In addition, 82.8% of the patients with MDM2-amp harbored genomic co-alterations and were potentially therapy targetable. This study provided insights into identifying patients who might potentially benefit from anti-MDM2 therapies.

Recurrent or metastatic salivary gland tumor (MSGT) patients treated with selinexor, a first in class selective exportin-1 (XPO1) inhibitor.

6586 Background: MSGT are rare with limited systemic treatments. This single institution, prospective study in recurrent or metastatic (RM) MSGT involved 2 phases: genomic profiling followed by treatment with either genomically-matched or unmatched therapy. Here we present the results of the unmatched arm for patients (pts) treated with S an oral selective inhibitor of XPO1 that leads to activation of tumor suppressor proteins and retention of oncoprotein mRNAs in the cell nucleus, inducing cancer cell apoptosis. Methods: Patients (pts) with RM-MSGT had archived paraffin embedded tumor samples profiled with targeted next generation sequencing, immunohistochemistry for androgen receptor (AR) and fluorescent in-situ hybridization for HER-2 and ALK. If no actionable mutations were identified or if no matched agents were available, pts with progressive disease could receive S (60mg given twice weekly Q28 days). The study had a simon-2 stage design; 1 partial response in the first 18 pts treated with S, would trigger an additional 7pts to receive S in stage 2. Results: Between July 2014 and April 2019 85 pts were enrolled on study: 73 had sequencing which identified 41 with no actionable mutations and 32 with actionable mutations. 18 pts (10F/8M, median age 61 years [40-79]) were treated with S and included adenoid cystic (n = 8), salivary duct (n = 4), acinic cell (n = 2) and other subtypes (n = 4). Of these 18, 4 pts had actionable aberrations: AR amplification (n = 2), mutations in SMARCB1 (n = 1) and CDKN2A (n = 1). 13pts were treatment naïve, 3pts and 2pts received 1 and 2 or more lines of treatment respectively prior to enrollment: androgen deprivation therapy (n = 2), chemotherapy (n = 3), early phase clinical trials (n = 3). The median number of cycles of S received were 3 (range: 1-19). The best response by RECIST was SD in 13pts (72%) (SD > 6 months (range: 6-18 months) in 5pts (28%); tumor reduction measured in 7pts (39%)), no PRs, PD in 3pts (17%), and 2pts (11%) were not evaluable for response due to insufficient duration of treatment coming off early due to toxicity. The median PFS (95% CI) was 7.6 (3.5-NA) months and the median OS (95% CI) was 15.4 (7.3-NA) months. The most common drug-related toxicities were grade 1-2 fatigue 14pts (78%), nausea 13pts (72%) and dysguesia 10pts (56%). 5 (28%) pts had a dose reduction and 6 (33%) in total had a dose interruption due to toxicity. Conclusions: Single agent antitumor activity was limited and the side effect profile was tolerable. No specific genomic aberration was associated with response to S. Clinical trial information: NCT02069730 .

Genomic aberration of chromatin regulatory BAF complex as predictive biomarker for immunotherapy in gastrointestinal adenocarcinoma.

4035 Background: SNF/SWI, a large ATP-dependent chromatin remodeling complex, is required for transcriptional activation of genes normally repressed by chromatin, and critical to tumor initiation and progression. Here, we analyzed the predictive utility of the mutations of the SNF/SWI members involved in BAF and PBAF complexes, and sought to explore the potential mechanisms. Methods: Clinical, genomic, transcriptional, and immunohistochemical data from immunotherapeutic cohort (MSKCC, n=185), Cancer Cell Line Encyclopedia (CCLE, n=92), The Cancer Genome Atlas (TCGA, n=925), and 3D Medicines database (3DMed, n=1812) were analyzed to explore the predictive effect of genomic aberration of BAF complex on the benefit from immunotherapy in patients with gastrointestinal adenocarcinoma. Results: In the MSKCC cohort involving 185 patients with gastrointestinal adenocarcinoma, the mutation of any member involved in BAF complex ( ARID1A, ARID1B, SMARCA4, SMARCB1, and SMARCD1) was significantly associated with prolonged OS of ICI treatment (HR 0.53, 95%CI 0.31-0.90, P=0.019), instead of the mutations of PBAF members including PBRM1 and ARID2. In addition, BAF mutation was not linked with better prognosis in TCGA database, indicating its predictive, not prognostic efficacy of immunotherapy. BAF-mutated samples exhibited higher tumour mutational burden (TMB, P<0.05, Table), and increased mRNA expression of immune-related genes including chemokines and granzyme A. In the 3DMed cohort where tumour samples received both genomic sequencing and PD-L1 immunohistochemical staining, BAF mutation was associated with higher PD-L1 positive rate in tumour cells (P<0.05, Table). Conclusions: Genomic aberration of members in chromatin regulatory BAF complex may serve as a predictive, not prognostic biomarker of ICI benefit in patients with gastrointestinal adenocarcinoma, partially underlying the mechanisms including higher mutational burden, transcription of immune-related genes, and protein-level PD-L1 expression. [Table: see text]