Cancer tumor cell dormancy is a significant clinical problem in breast cancer (BrCa). We used a 3D in vitro model of the endosteal bone niche (EN), consisting of endothelial, bone marrow stromal cells and fetal osteoblasts in a 3D collagen matrix (GELFOAM™), to identify genes required for dormancy. Human triple-negative MDA-MB-231 BrCa cells, but not the bone-tropic metastatic variant, BoM1833, established dormancy in 3D-EN cultures in a p38-MAPK-dependent manner, whereas both cell types proliferated on 2D plastic or in 3D collagen alone. “Dormancy-reactivation suppressor genes” (DRSG) were identified using a genomic shRNA screen in MDA-MB-231 cells for gene knockdowns that induced proliferation in the 3D-EN. DSRG candidates enriched for genes controlling stem cell biology, neurogenesis, MYC targets, ribosomal structure and translational control. Several potential DRSG were confirmed using independent shRNAs, including BHLHE41, HBP1 and WNT3. Overexpression of the WNT3/a antagonists, secreted frizzled-related protein 2 or 4 (SFRP2/4), induced MDA-MB-231 proliferation in the EN. In contrast, overexpression of SFRP3, known not to antagonize WNT3/a, did not induce proliferation. Decreased WNT3 or BHLHE41 expression was found in clinical BrCa metastases compared to primary-site lesions, and the loss of WNT3 or BHLHE41 or gain of SFRP1, 2 and 4 in the context of TP53 loss/mutation correlated with decreased progression-free and overall survival.
Recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) patients overall have a poor prognosis. However, human papillomavirus (HPV)-associated R/M oropharyngeal squamous cell carcinoma (OPSCC) is associated with a better prognosis compared to HPV−negative disease. Immune checkpoint blockade (ICB) is the standard of care for R/M HNSCC. However, whether HPV and its surrogate marker, p16, portend an improved response to ICB remains controversial. We queried the Caris Life Sciences CODEai database for p16+ and p16− HNSCC patients using p16 as a surrogate for HPV. A total of 2905 HNSCC (OPSCC, n = 948) cases were identified. Of those tested for both HPV directly and p16, 32% (251/791) were p16+ and 28% (91/326) were HPV+. The most common mutation in the OPSCC cohort was TP53 (33%), followed by PIK3CA (17%) and KMT2D (10.6%). TP53 mutations were more common in p16− (49%) versus the p16+ group (10%, p < 0.0005). Real-world overall survival (rwOS) was longer in p16+ compared to p16− OPSCC patients, 33.3 vs. 19.1 months (HR = 0.597, p = 0.001), as well as non-oropharyngeal (non-OP) HNSCC patients (34 vs. 17 months, HR 0.551, p = 0.0001). There was no difference in the time on treatment (TOT) (4.2 vs. 2.8 months, HR 0.796, p = 0.221) in ICB-treated p16+ vs. p16− OPSCC groups. However, p16+ non-OP HNSCC patients treated with ICB had higher TOT compared to the p16− group (4.3 vs. 3.3 months, HR 0.632, p = 0.016), suggesting that p16 may be used as a prognostic biomarker in non-OP HNSCC, and further investigation through prospective clinical trials is warranted.
8535 Background: Large-cell neuroendocrine carcinoma (LCNEC) is a rare type of lung cancer with a poor prognosis. Due to its rarity, molecular characterization of LCNEC is not well elucidated. We aim to understand the genomic and immunologic landscape of LCNEC to identify molecular alterations and relevant biological pathways with potential therapeutic value. Methods: Comprehensive profiling including whole exome sequencing (WES), next-generation sequencing (NGS), whole transcriptome sequencing (WTS), and immunohistochemistry (IHC) for PD-L1 was performed (Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous mutations. LCNEC was categorized as small cell lung cancer (SCLC)-like LCNEC ( TP53/ RB1 co-mutated) and non-small-cell lung cancer (NSCLC)-like LCNEC (wild type for one or both of TP53/ RB1). Molecular features of LCNEC were compared among the subgroups and with those of SCLC using the χ2 test with Benjamini & Hochberg correction. Results: A total of 467 cases of LCNEC were included. Commonly altered genes (≥ 5%) included TP53 (79.1%), RB1 (36.8%), SMARCA4 (10.4%), ARID1A (10.3%), KRAS (9.7%), KEAP1 (9.2%), KMT2D (8.7%), STK11 (8.4%), NF1 (7.1%), PTEN (6.1%), and CDKN2A (5.9%) . The prevalence of potentially actionable mutations was as follows: EGFR exon 19 deletion (0.48%), EGFR L858R (0.48%), ALK fusion (1.7%), KRAS G12C (2.9%). EGFR exon 19 deletion, EGFR L858R, and ALK fusion were exclusive to NSCLC-like LCNEC tumors. RET fusion, NTRK fusion and BRAFV600E were not detected. Copy number alterations (CNAs) were found in MYC (8.8%), ZNF703 (4.1%), FOXA1 (4.0%), FGFR1 (4.0%), ATK2 (3.9%), CCNE1 (3.7%), FGF19 (3.4%), TNFRSF14 (3.4%), and CCND1 (2.7%). Over-expression of cMET was noted in 10% and PD-L1 expression (by 22C3 pharmDx) of > 1% was noted in 21.5% of samples. WTS detected cMET exon 14 skipping mutations in 2.4% of samples. High tumor mutation burden (TMB; ≥ 10 Mut/MB) was seen in 40.6%. Among the 467 cases of LCNEC, 112 (24%) were SCLC-like LCNEC and 335 (76%) NSCLC-like LCNEC. Mutations in KRAS (12%), STK11 (11%), CDKN2A (9%), and SMARCA4 (14%) were more common in NSCLC-like LCNEC, compared with SCLC-like LCNEC (p value < 0.05). 442 cases of SCLC were compared with LCNEC tumors. SLFN11:SLFN12 fusion events, detected by WTS, were exclusively seen in SCLC and were not seen in any of the LCNEC cases. Gene expression profiles revealed that 1) B cell infiltration was higher in SCLC-like LCNEC, compared with SCLC, and 2) NK and T cell infiltration was lower, but B-cell infiltration was higher in NSCLC-like LCNEC, compared with SCLC. Conclusions: LCNEC displays a broad pattern of genomic alterations that overlap in the SCLC-like subset with the classic alterations in SCLC. The distinct genomic alterations and transcriptomic profiles present opportunities for therapeutic targeting and inform a future framework for development of therapeutics for LCNEC.
Human prostate cancer confined to the gland is indolent (low-risk), but tumors outside the capsule are aggressive (high-risk). Extracapsular extension requires invasion within and through a smooth muscle-structured environment. Because integrins respond to biomechanical cues, we used a gene editing approach to determine if a specific region of laminin-binding a6b1 integrin was required for smooth muscle invasion both in vitro and in vivo. Human tissue specimens showed prostate cancer invasion through smooth muscle and tumor coexpression of a6 integrin and E-cadherin in a cell-cell location and a6 integrin in a cell-extracellular matrix (ECM) distribution. Prostate cancer cells expressing a6 integrin (DU145 a6WT) produced a 3D invasive network on laminin-containing Matrigel and invaded into smooth muscle both in vitro and in vivo. In contrast, cells without a6 integrin (DU145 a6KO) and cells expressing an integrin mutant (DU145 a6AA) did not produce invasive networks, could not invade muscle both in vitro and in vivo, and surprisingly formed 3D cohesive clusters. Using electric cell-substrate impedance testing, cohesive clusters had up to a 30-fold increase in normalized resistance at 400 Hz (cell-cell impedance) as compared with the DU145 a6WT cells. In contrast, measurements at 40,000 Hz (cell-ECM coverage) showed that DU145 a6AA cells were twofold decreased in normalized resistance and were defective in restoring resistance after a 1 mmol/L S1P challenge as compared with the DU145 a6WT cells. The results suggest that gene editing of a specific a6 integrin extracellular region, not required for normal tissue function, can generate a new biophysical cancer phenotype unable to invade the muscle, presenting a new therapeutic strategy for metastasis prevention in prostate cancer. Significance: This study shows an innovative strategy to block prostate cancer metastasis and invasion in the muscle through gene editing of a specific a6 integrin extracellular region.
465 Background: UTUC is a rare genitourinary malignancy and a number of studies, limited by small sample sizes, have attempted to characterize its mutational landscape. Because immunotherapy is commonly used for this disease type, we evaluated the prevalence of microsatellite instability and characterized the mutational landscapes of UTUC in a large contemporary patient cohort. Methods: UTUC tumor samples were analyzed using next generation sequencing (NGS) (NextSeq, 592 gene panel) or whole exome sequencing (WES) (NovaSeq) (Caris Life Sciences, Phoenix, AZ). Mismatch repair status (deficient [dMMR] or proficient [pMMR]) and microsatellite instability status (MSI-high or stable [MSS]) were detected by immunohistochemistry (IHC), fragment analysis, and NGS. Tumor mutational burden (TMB) was measured by counting all somatic mutations found per tumor (high cutoff ≥ 10 mutations per MB). PD-L1 expression was tested by IHC using PD-L1 antibody clones 22c3 (Agilent; positive cutoff CPS ≥ 10) and SP142 (Ventana; positive cutoff ≥ 5% IC). Pathogenic fusion events were detected using whole transcriptome sequencing (NovaSeq). Statistical significance was determined using the Chi-square test and adjusted for multiple comparison. Results: 538 patients with included – median (range) age 71.5 (30-89) years and 37.5% female/62.5% male. Prevalence of dMMR/MSI-H was 3.9% (21/538) and TMB-high was 22.7% (96/423). Significant molecular differences were not detected in primary vs metastatic disease or in male vs female cases. dMMR/MSI-H tumors had higher frequency of TMB-high compared to MSS tumors (100% vs. 19%, p = 0.00003). dMMR/MSI-H tumors also had a higher frequency than MSS tumors for mutations in genes involved in chromatin remodeling (ASXL 82.4%, CREBBP 60%, SMARCA4 40%, KMT2D 95%, ARIDIA 100%, KMT2A 20%, KMT2C 35.3%, NSD1 20%), DNA-damage repair (FANCG 10%, ATM 45%, ATRX 40%) and other biological pathways (RNF43 10%, PTCH1 21.4%, ERBB3 30%, CDKN2A 25%, TSC2 15%, FLNC 15%, HNF1A 20%, CIC 15%, DNMT3A 17.6%); all adjusted p < 0.05. Pathogenic fusions were detected in 3.8% (17/443) cases, with FGFR3 fusion being the most common, occurring in 2.7% (12/443) cases. PD-L1 positivity was identified in 33.2% (133/400) cases tested by 22c3 antibody and 28.4% (89/313) cases tested by SP142 antibody. No difference was seen in PD-L1 positivity between MSI-H/dMMR vs. MSS tumors. Conclusions: In the largest analysis to date, we found a 3.9% prevalence of dMMR/MSI-high rate in UTUC. All dMMR/MSI-H tumors displayed TMB-high. PD-L1 positivity was comparable between dMMR/MSI-H and MSS tumors. dMMR/MSI-H tumors had a significantly higher rate of mutations in genes involved in chromatin remodeling and DDR biological pathways. These results could inform design of targeted therapy trials in UTUC.
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