Immune checkpoint inhibitors (ICI) are the current standard-of-care first-line treatment for advanced non-small cell lung cancer (NSCLC) without actionable genomic alterations. Yet some patients do not achieve clinical benefit, underscoring the need for predictive biomarkers. High burden of somatic copy number alterations (SCNAs), including chromosomal arm- and whole-chromosome-level changes, has been linked to reduced benefit from ICI. However, its predictive value in chemotherapy or combination regimens remains largely unexplored. Here, we evaluated SCNA burden across multiple first-line treatment strategies in a cohort of patients with NSCLC. We assembled a cohort of 258 patients with advanced NSCLC treated with first-line ICI (n = 86), chemotherapy (ChT; n = 107), or a combination of both (ICI + ChT; n = 65). Tumor DNA was analyzed using shallow whole-genome sequencing (sWGS). SCNA burden was quantified as the fraction of the genome altered at the chromosomal arm and whole-chromosome level (FGAa+c). We performed Kaplan-Meier and Cox regression analyses to assess its value across treatment groups. Additionally, we examined associations between recurrent SCNAs, clinical efficacy, and tumor microenvironment (TME) features using data from The Cancer Genome Atlas (TCGA). High SCNA burden (FGAa+c > 4.25) was significantly associated with diminished progression-free survival (PFS) in patients treated with ICI (p-val = 0.0097). In contrast, FGAa+c did not associate with PFS following ChT (p-val = 0.9566) or ICI + ChT (p-val = 0.6472). Specific SCNAs in chromosome 9p were independently associated with reduced ICI benefit (p-val = 0.0469), even after adjusting for FGAa+c (p-val = 0.0229). This SCNA was negatively associated with immune infiltration in the TME and was linked to differences in the immune cell type composition. Our findings suggest that FGAa+c may help identify patients with differential benefit from ICI monotherapy in advanced NSCLC, without evidence of a prognostic role in other treatment modalities. These findings warrant prospective studies to validate its clinical utility in guiding treatment selection.

Multiple endocrine neoplasia type 4 (MEN4) is caused by a germline CDKN1B deleterious variant. CDKN1B encodes p27Kip1, a cyclin-dependent kinase inhibitor that acts as tumor-suppressor. Clinical presentation of MEN4 is similar to multiple endocrine neoplasia type 1 (MEN1) but the diagnosis of MEN4 can only be established once a germline CDKN1B pathogenic variant has been confirmed. We describe a unique case presenting with two -rare endocrine conditions. A 59-year-old female patient was diagnosed with medullary thyroid cancer (MTC) without evidence of a germline pathogenic variant in the RET proto-oncogene. Five years later, she developed Cushing’s disease. A heterozygous germline variant was identified in the CDKN1B gene, specifically c.536del (p.Prol179GlnfsTer46), corresponding to a single-nucleotide deletion at position 536. This variant induces a frameshift, leading to an alternative stop codon. Immunostaining of the pituitary and thyroid tumors revealed a weak nuclear expression of p27/Kip1 without significant differences of expression between tumor and non-tumoral tissues. The NGS panel (Oncomine Comprehensive Assay v3) performed in both MTC and pituitary tissues identified the germline CDKN1B variant, as well as a pathogenic missense somatic variant c.182 A > G, p.(Gln61Arg) in HRAS in the MTC, without any RET somatic pathogenic variant. Evaluation of loss of heterozygosity (LOH) in both MTC and pituitary tissues showed compatibility with copy-neutral LOH, although further evidence is required for definitive confirmation. In conclusion, we report a clinical case of MTC coexisting with MEN4 due to a novel CDKN1B germline heterozygote frameshift variant.

e20672 Background: This study compares the concordance and clinical utility of ctDNA-based plasma testing with Next-Generation Sequencing (NGS) performed on tumor tissue, providing insights into their respective roles in personalized cancer care. Methods: We retrospectively analyzed genomic data from 112 patients (p) diagnosed with stage III-IV lung cancer and with tissue biopsies deemed suitable for tissue-based NGS (Oncomine Precision Assay[OPA]) who underwent concurrent plasma-based NGS (VHIO360, an ISO15189 accredited ctDNA panel using Guardant360 technology), within a 40-day window, between July 2022 and April 2024. Liquid biopsy specimens were always obtained at the time of diagnosis or disease progression. Concordance between techniques was evaluated with overlap coefficient and Jaccard index. Results: Among the 112 p, 62(55.3%) were male. All had stage III (9.8%) or IV (90.2%) disease. 93 (82%) with lung adenocarcinoma, 6 (5.3%) with large cell neuroendocrine carcinoma, 2 (1.8%) with squamous carcinoma, and 2 (1.8%) with small cell carcinoma. Tumor tissue had inadequate DNA/RNA quality for analysis in 5 p (4.5%). Liquid biopsy found no mutations in 16 p (14.3%). A total of 71 p showed concordant pathogenic mutations (mut) in both tissue and plasma samples. Gene fusions were detected in 8 tissue samples and 6 in plasma samples. ESCAT I-II genomic alterations (alt) were identified in 59 p (52.7%); 57 p detected by tissue and 39 p through plasma. The overlap coefficient for ESCAT I-II alt was 94.9%, whereas Jaccard index was 62.7%. Particularly, Jaccard index was 100% for EGFR uncommon mut, ERBB2 , RET and ROS1 fusions and 50% for EGFR exon 20 insertions (ins), ALK fusion and MET amplifications (Table 1). Regarding TP53 mut, 80 patients ≥ 1pathogenic TP53 mut detected by at least one of both panels. Among these, 41.3% (33/80) were associated with ESCAT I-II alt, and 58.7% (47/80) were not. Conclusions: ctDNA and tissue NGS presented similar ESCAT I-II genomic alterations identification, particularly when adequate tissue samples are available. However, in certain cases, clinically meaningful mut were detected by only one method, demonstrating the utility of complementary testing, specially for EGFR exon 20 ins. Additionally, some relevant alt, such as concomitant mut in TP53 , were identified differently by the two approaches, further emphasizing their complementary roles. Genomic alt in tissue-NGS and plasma-NGS. Gene alt OPA VHIO 360 Same alt in both OPA only VHIO360 only Overlap coefficient (%) Jaccard Index (%) EGFR (L858R, deletion exon 19)* (exon 20 ins)* (G719, L861Q, S768I)* 2514 1924 1914 600 010 100100100 7650100 ALK fusions* 5 4 3 2 1 75 50 KRAS G12C* 12 9 8 4 0 88.9 61.5 RET fusions* 1 1 1 0 0 100 100 ROS1 fusions* 1 1 1 0 0 100 100 BRAF (V600E)* 1 0 0 1 0 0 0 MET Mut exon 14 skipping Amplifications 42 01 01 41 00 0100 050 ERBB2* 1 1 1 0 0 100 100 TP53 mut 45 70 33 12 37 73.3 41.3 *Genomic alterations level I/II according to ESCAT. Mosele, M.F. et al. Ann Oncol.2022; 35: 588-606.

Sarcomas characterized by BCOR gene alterations, are a distinct clinico-pathological group of high-grade tumors, that represent 5% of small round cell tumors without EWSR or FUS fusion. Diverse genetic alterations characterize this group, including BCOR-CCNB3 gene fusion being the most common alteration and less frequently internal tandem duplications (ITDs). We present a compelling case of a 3-year-old girl diagnosed with a high-grade nasoethmoidal sarcoma exhibiting BCOR-ITD. The diagnostic process illustrates the histological and immunophenotypic spectrum, requiring an extensive immunohistochemical panel and diverse molecular tests for accurate classification. Additionally, this case highlights the challenges in detecting BCOR-ITDs using different NGS panels, advocating for alternative molecular approaches. Our patient after 10 months since diagnosis is alive with progressive disease. This emphasizes the urgency for ongoing research to refine diagnostic methods and develop effective therapeutic strategies for these rare and aggressive tumors.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Background: Loss of expression of BRAC-1 associated protein 1 (BAP1) was reported in 69% of biphasic mesotheliomas and 15% of sarcomatoid mesotheliomas (Cigognetti M et al.Modern Pathology 2015; 28:1043-1057).However, available data on the BAP1 expression in carcinomas and other tumors were limited.Because differentiating a mesothelioma from an adenocarcinoma and other tumors is clinically significant, in this study we focused on the evaluation of BAP1 expression in a large series of carcinomas and tumors from various organs.Design: Immunohistochemical analysis of BAP1 (sc-28383; Santa Cruz Biotechnology, Inc.) was performed on 1233 cases of tumor and normal tissues on tissue microarray (TMA) sections, including mesothelioma (N=18), lung neuroendocrine carcinoma (CA) (N=24), lung adenocarcinoma (ADC) (N=88), lung squamous cell CA (N=66), papillary thyroid CA (N=47), ENT squamous cell CA (N=28), breast fibroadenoma (N=20), pancreatic neuroendocrine tumor (N=33), pancreatic ADC (N=43), adrenal pheochromocytoma (N=14), endometrial CA FIGO II (N=59), ovary papillary serous CA (N=41), clear cell CA of uterus and ovary (N=22), melanoma (N=32), skin neuroendocrine CA (N=27), invasive urothelial CA (N=43), bladder small cell CA (N=24), prostatic ADC (N=38), germ cell tumors (N=60), colonic ADC (N=164), angiosarcoma (N=12), papillary renal cell carcinoma (RCC) (N=33), clear cell RCC, low grade (N=79), clear cell RCC, high grade (N=51), hepatocellular CA (N=47), liver metastatic neuroendocrine CA (N=18), glioblastoma multiforme (N=23), mantle cell lymphoma (N=13) and hairy cell leukemia (N=1).Samples from normal tissue included pancreas (N=13), rectum/appendix/colon (N=39) and ilium/duodenum/stomach (N=13).Nuclear expression for BAP1 was regarded as positive.Loss of expression of BAP1 was recorded when internal positive controls were present.Results: Loss of expression of BAP1 was noted in 17% of mesothelioma cases (3 of 18).Intact BAP1 expression was present in all other tumors and normal tissues. Conclusions:Our data demonstrated that BAP1 expression was not identified in carcinomas and other tumors from various organs, which further validated the previous report that loss of expression of BAP1 was highly specific for a malignant mesothelioma.However, the loss of expression of BAP1 was only seen in a minority of mesotheliomas.The low diagnostic sensitivity in this study is most likely due to the small number of cases, including some with sarcomatoid features.

In this review, we highlight the role of intratumoral heterogeneity, focusing on the clinical and biological ramifications this phenomenon poses. Intratumoral heterogeneity arises through complex genetic, epigenetic, and protein modifications that drive phenotypic selection in response to environmental pressures. Functionally, heterogeneity provides tumors with significant adaptability. This ranges from mutual beneficial cooperation between cells, which nurture features such as growth and metastasis, to the narrow escape and survival of clonal cell populations that have adapted to thrive under specific conditions such as hypoxia or chemotherapy. These dynamic intercellular interplays are guided by a Darwinian selection landscape between clonal tumor cell populations and the tumor microenvironment. Understanding the involved drivers and functional consequences of such tumor heterogeneity is challenging but also promises to provide novel insight needed to confront the problem of therapeutic resistance in tumors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Introduction: Tumors are complex systems where cell-cell communication between intra-tumoral and extra-tumoral cells play a crucial role. Extracellular vesicles (EVs), membrane-limited vesicles secreted by normal and malignant cells, have been proposed to act as mediators of intercellular communication in physiological and pathological scenarios. It is now well established that integrins are constantly endocytosed and recycled back to the plasma membrane through multiple routes. Accumulating evidence clearly indicates that endocytosis and recycling of integrins plays a crucial role during cancer progression, invasion, and metastasis. Here we describe a fundamental link between endocytosis mediated integrin trafficking and the integrin-mediated uptake of exosomes. Objectives: As the integrins are crucial for the role of exosomes in cancer progression and particularly in metastasis we were aiming at delineating the role of ITGB3 in exosome-mediated intercellular communication in the triple negative breast cancer cell line MB.MDA.231. Methodology: Using stable shRNA of ITGB3 (shITGB3) in the triple-negative breast cancer cell line MDA.MB.231, EVs were isolated by ultracentrifugation and characterized by Western blot analysis to determine the protein composition and by NTA and CryoEM to measure the amount of EVs. An LC-MS/MS-based survey was performed, to determine the differences in the protein composition of EVs after knock-down of ITGB3. Uptake of fluorescently labeled exosomes into recipient cells was measured by flow cytometry. The colony forming capacity induced by conditioned medium was used to assess the functional impact on the role of ITGB3 in intercellular communication. Results: Analysis of the EVs fraction of shITGB3 cell revealed a striking decrease of classical markers of exosomes as judged by western blot analysis of proteins such as TSG101, CD81, but not others like Flotillin-1. LC-MS/MS analysis of shITGB3-MDA.MB.231 derived-exosomes furthermore revealed clear defects in protein complexes required for exosome formation, namely the ESCRT complex and the Syndecan-syntenin-ALIX complex. Both complexes are required for the formation of endosomal intraluminal vesicles that get released as exosomes. On the other hand, NTA analysis and CryoEM showed the overall amount of EVs was increased. In line with this, the uptake of fluorescently labeled EVs was strongly impaired in shITGB3 cells. By the use of small molecule inhibitors, we can furthermore demonstrate that this defect linked to endocytosis of EVs. Conclusions: Here we demonstrate that ITGB3 plays a crucial role in controlling the composition of different vesicle populations in the extracellular space, linked to the endocytosis of integrins and interference with this mechanism results in defects in the uptake of EVs and impedes the generation of exosomes. The functional effects are discussed Citation Format: Pedro Fuentes Varela, Marta Sesé, Pedro Jesús Guijarro, Marta Emperador, Héctor Peinado, Stefan Hümmer, Santiago Ramon y Cajal. Exosome mediated intercellular communication in the triple negative breast cancer cell line MB.MDA.231 relies on ITGB3 [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 1897.

Precision medicine has revolutionized the understanding and treatment of cancer by identifying subsets of patients who are amenable to specific treatments according to their molecular characteristics, as exemplified by epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC). Although tissue biopsy is the gold standard for determining molecular alterations in tumors, its limitations have prompted the development of new techniques for studying tumor biomarkers in liquid biopsies, such as mutation analysis in cell-free DNA (cfDNA). cfDNA analysis can accurately determine tumor progression and prognosis and more effectively identify appropriate targeted therapies. However, cfDNA is vulnerable, particularly during plasma sample shipping. We compared the cell- and DNA-stabilizing properties of cell-free DNA blood collection tubes (BCTs) with those of the traditional shipping method (frozen plasma) for EGFR mutation testing using the cobas® EGFR Mutation Test v2 in a prospective cohort of 49 patients from three different Spanish hospitals. In total, 98 NSCLC samples, two from each patient, were studied; five of the 49 cases were considered invalid by cobas® with one of the two shipping methods analyzed. After excluding these samples, we analyzed 88 samples from 44 patients. Considering the current methodology (frozen plasma) for sending samples as the gold standard, we evaluated the sensitivity and specificity of cfDNA BCT shipment. The global agreement between the two methods was 95.4%, with 100% sensitivity and 94.6% specificity for the cfDNA BCTs. cfDNA BCTs had a positive predictive value of 81.8% and negative predictive value of 100%. cfDNA BCTs have the same sensitivity for EGFR mutation analysis in liquid biopsy as the current methodology and very high specificity. They also have some additional advantages in terms of collection and further shipment. Therefore, cfDNA BCTs can be perfectly incorporated into the routine practice for EGFR mutation determination. Roche Farma S.A., Spain.

Introduction The VEGF family has been identified as abnormal in preeclampsia (PE). Hypertensive disorders of pregnancy (HDP) are major contributors to maternal and neonatal morbidity and mortality worldwide; likewise, umbilical cord anatomical abnormalities (UCAA) are linked to poor neonatal outcomes. Based on the relationship described between PE and UCAA and the role of the VEGF family in PE, this study explored VEGF expression in placental and UC tissued from patients with PE and with UCAA. Methods We performed an observational, analytical study on placentas, comparing protein and mRNA expression in four groups: patients with PE, patients with UC abnormalities, patients with both, and patients with none of them. Using immunohistochemistry, we studied VEGF A, VEGF R1 (FLT1), MMP1, and PLGF. With quantitative reverse transcription polymerase chain reaction we described mRNA expression of PLGF, VEGF and sFLT1, and sFLT1/PLGF ratio. Results Forty newborns were included. Sixty-seven percent of mothers and 45% of newborns developed no complications. Immunohistochemistry was performed on UC and placental disc paraffin-embedded tissue; in the latter, the mRNA of the VEGF family was also measured. Statistically significant differences were observed among different expressions in both HDP and UCAA groups. Interestingly, the UCAA group exhibited lower levels of sFLT1 and VEGF-A in comparison with other groups, with significant P-value for sFLT1 (P=0000.1). Conclusion The origin of UCAA abnormalities and their relation with HDP are still unknown. VEGF family alterations could be involved in both. This study provides the first approach related to molecules linked to UCAA.

Breast cancer is the most prevalent malignancy in women and there is an urgent need for new therapeutic drugs targeting aggressive and metastatic subtypes, such as hormone-refractory triple-negative breast cancer (TNBC). Control of protein synthesis is vital to cell growth and tumour progression and permits increased resistance to therapy and cellular stress. Hypoxic cancer cells attain invasive and metastatic properties and chemotherapy resistance, but the regulation and role of protein synthesis in this setting is poorly understood. We performed a polysomal RNA-Seq screen in non-malignant breast epithelial (MCF10A) and TNBC (MDA-MB-231) cells exposed to normoxic or hypoxic conditions and/or treated with an mTOR pathway inhibitor. Analysis of both the transcriptome and the translatome identified mRNA transcripts translationally activated or repressed by hypoxia in an mTOR-dependent or -independent manner. Integrin beta 3 (ITGB3) was translationally activated in hypoxia and its knockdown increased apoptosis and reduced survival and migration, particularly under hypoxic conditions. Moreover, ITGB3 was required for sustained TGF-β pathway activation and for the induction of Snail and associated epithelial-mesenchymal transition markers. ITGB3 downregulation significantly reduced lung metastasis and improved overall survival in mice. Collectively, these data suggest that ITGB3 is translationally activated in hypoxia and regulates malignant features, including epithelial-mesenchymal transition and cell migration, through the TGF-β pathway, revealing a novel angle for the treatment of therapy-resistant hypoxic tumours.

Gap junctions allow intercellular communication. Their structural subunits are four-transmembrane proteins named connexins (Cxs), which can be post-transcriptionally regulated by developmental and cellular signalling cues. Cx translation and mRNA stability is regulated by miRNAs and RNA-binding proteins (RBPs) such as human antigen R (HuR). In addition, several Cxs have also been suggested to contain 5' internal ribosome entry site (IRES) elements that are thought to allow cap-independent translation in situations such as mitosis, stress and senescence. Furthermore, several recent reports have documented internal translation of Cx mRNAs that result in N-terminally truncated protein isoforms that may have unique gap junction-independent functions [Ul-Hussain et al. (2008) BMC Mol. Biol. 9, 52; Smyth and Shaw (2013) Cell Rep. 5, 611-618; Salat-Canela et al. (2014) Cell Commun. Signal. 12, 31; Ul-Hussain et al. (2014) J. Biol. Chem. 289, 20979-20990]. This review covers the emerging field of the post-transcriptional regulation of Cxs, with particular focus on the translational control of Cx 43 and its possible functional consequences.