Incidental germline findings during molecular profiling of tumor tissues for precision oncology: molecular survey and methodological obstacles

Background A fraction of patients referred for complex molecular profiling of biopsied tumors may harbor germline variants in genes associated with the development of hereditary cancer syndromes (HCS). Neither the bioinformatic analysis nor the reporting of such incidental germline findings are standardized. Methods Data from Next-Generation Sequencing (NGS) of biopsied tumor samples referred for complex molecular profiling were analyzed for germline variants in HCS-associated genes. Analysis of variant origin was performed employing bioinformatic algorithms followed by manual curation. When possible, the origin of the variant was validated by Sanger sequencing of the sample of normal tissue. The variants’ pathogenicity was assessed according to ACMG/AMP. Results Tumors were sampled from 183 patients (Males: 75 [41.0%]; Females: 108 [59.0%]; mean [SD] age, 57.7 [13.3] years) and analysed by targeted NGS. The most common tumor types were colorectal (19%), pancreatic (13%), and lung cancer (10%). A total of 56 sequence variants in genes associated with HCS were detected in 40 patients. Of them, 17 variants found in 14 patients were predicted to be of germline origin, with 6 variants interpreted as pathogenic (PV) or likely pathogenic (LPV), and 9 as variants of uncertain significance (VUS). For the 41 out of 42 (97%) missense variants in HCS-associated genes, the results of computational prediction of variant origin were concordant with that of experimental examination. We estimate that Sanger sequencing of a sample of normal tissue would be required for ~ 1–7% of the total assessed cases with PV or LPV, when necessity to follow with genetic counselling referral in ~ 2–15% of total assessed cases (PV, LPV or VUS found in HCS genes). Conclusion Incidental findings of pathogenic germline variants are common in data from cancer patients referred for complex molecular profiling. We propose an algorithm for the management of patients with newly detected variants in genes associated with HCS.

Treatments after Immune Checkpoint Inhibitors in Patients with dMMR/MSI Metastatic Colorectal Cancer

Background: Several studies reported improved outcomes with conventional treatments (CT, i.e., chemotherapy ± targeted therapy) administered after immune checkpoints inhibitors (ICI) in certain tumor types. No data are available concerning patients (pts) with metastatic colorectal cancer (mCRC) harboring mismatch repair deficiency/microsatellite instability (dMMR/MSI). We aimed to assess the outcomes of dMMR/MSI mCRC pts receiving CT after ICI failure. Methods: We conducted a retrospective multicenter study investigating the outcomes of all dMMR/MSI mCRC pts who received post-ICI CT between 2015 and 2020. Results: 31 pts (male 61%, median age 56 years) were included. ICI was an anti-PD(L)1 monotherapy in 71% of pts, and 61% received >2 lines before post-ICI CT. The overall response rate and disease control rate were 13% and 45%, with a median progression-free survival (PFS) and overall survival of 2.9 and 7.4 months, respectively. No association of the outcomes with either ICI efficacy or anti-angiogenic agents was observed. Prolonged PFS (range 16.1–21.3 months) was observed in 4 pts (13%). Conclusions: Although conducted on a limited number of patients, our results do not support an association of previous ICI treatment with an enhanced efficacy of CT in dMMR/MSI mCRC. However, prolonged disease control was observed in several cases, suggesting that some pts might derive an unexpected benefit from post-ICI treatments.

Challenges in the Intraoperative Consultation of Low-Grade Epilepsy-Associated Neuroepithelial Tumors by Cytomorphology in Squash Preparations

<b><i>Introduction:</i></b> Low-grade epilepsy-associated neuroepithelial tumors (LEATs) create a diagnostic challenge in daily practice and intraoperative pathological consultation (IC) in particular. Squash smears are extremely useful in IC for accurate diagnosis; however, the knowledge on cytopathologic features of LEATs is based on individual case reports. Here, we discuss the 3 most common and well-established entities of LEATs: ganglioglioma (GG), dysembryoplastic neuroepithelial tumor (DNT), and papillary glioneuronal tumor (PGNT). <b><i>Methods:</i></b> Thirty patients who underwent surgery for GG, DNT, and PGNT between 2001 and 2021 were collected. Squash smears prepared during intraoperative consultation were reviewed by 1 cytopathologist and an experienced neuropathologist. <b><i>Results:</i></b> Among the 30 tumors, 16 (53.3%) were GG, 11 (36.6%) DNT, and 3 (10%) PGNT. Cytomorphologically, all of the 3 tumor types share 2 common features such as dual cell population and vasculocentric pattern. GG smears were characteristically composed of dysplastic ganglion cells and piloid-like astrocytes on a complex architectural background of thin- to thick-walled vessels. DNT, on the other hand, showed oligodendroglial-like cells in a myxoid thin fibrillary background associated with a delicate capillary network. Common cytological features of PGNT were hyperchromatic cells with narrow cytoplasm surrounding hyalinized vessels forming a pseudopapillary pattern and bland cells with neuroendocrine nuclei dispersed in a neuropil background. <b><i>Conclusion:</i></b> A higher diagnostic accuracy can be obtained when squash smears are applied with frozen sections. However, it is important to integrate clinical and radiologic features of the patient as well as to know the cytopathologic features of the LEAT spectrum in the context of differential diagnosis to prevent misinterpretation in the IC.

Oncogenic role of a developmentally regulated NTRK2 splice variant

Temporally-regulated alternative splicing choices are vital for proper development yet the wrong splice choice may be detrimental. Here we highlight a novel role for the neurotrophin receptor splice variant TrkB.T1 in neurodevelopment, embryogenesis, transformation, and oncogenesis across multiple tumor types in both humans and mice. TrkB.T1 is the predominant NTRK2 isoform across embryonic organogenesis and forced over-expression of this embryonic pattern causes multiple solid and nonsolid tumors in mice in the context of tumor suppressor loss. TrkB.T1 also emerges the predominant NTRK isoform expressed in a wide range of adult and pediatric tumors, including those harboring TRK fusions. Affinity purification-mass spectrometry (AP-MS) proteomic analysis reveals TrkB.T1 has distinct interactors with known developmental and oncogenic signaling pathways such as Wnt, TGF-β, Hedgehog, and Ras. From alterations in splicing factors to changes in gene expression, the discovery of isoform specific oncogenes with embryonic ancestry has the potential to shape the way we think about developmental systems and oncology.

Genomic studies controvert the existence of the CUX1 p75 isoform

CUX1, encoding a homeodomain-containing transcription factor, is recurrently deleted or mutated in multiple tumor types. In myeloid neoplasms, CUX1 deletion or mutation carries a poor prognosis. We have previously established that CUX1 functions as a tumor suppressor in hematopoietic cells across multiple organisms. Others, however, have described oncogenic functions of CUX1 in solid tumors, often attributed to truncated CUX1 isoforms, p75 and p110, generated by an alternative transcriptional start site or post-translational cleavage, respectively. Given the clinical relevance, it is imperative to clarify these discrepant activities. Herein, we sought to determine the CUX1 isoforms expressed in hematopoietic cells and find that they express the full-length p200 isoform. Through the course of this analysis, we found no evidence of the p75 alternative transcript in any cell type examined. Using an array of orthogonal approaches, including biochemistry, proteomics, CRISPR/Cas9 genomic editing, and analysis of functional genomics datasets across a spectrum of normal and malignant tissue types, we found no data to support the existence of the CUX1 p75 isoform as previously described. Based on these results, prior studies of p75 require reevaluation, including the interpretation of oncogenic roles attributed to CUX1.

Recurrent Spliceosome Mutations in Cancer: Mechanisms and Consequences of Aberrant Splice Site Selection

Splicing alterations have been widely documented in tumors where the proliferation and dissemination of cancer cells is supported by the expression of aberrant isoform variants. Splicing is catalyzed by the spliceosome, a ribonucleoprotein complex that orchestrates the complex process of intron removal and exon ligation. In recent years, recurrent hotspot mutations in the spliceosome components U1 snRNA, SF3B1, and U2AF1 have been identified across different tumor types. Such mutations in principle are highly detrimental for cells as all three spliceosome components are crucial for accurate splice site selection: the U1 snRNA is essential for 3′ splice site recognition, and SF3B1 and U2AF1 are important for 5′ splice site selection. Nonetheless, they appear to be selected to promote specific types of cancers. Here, we review the current molecular understanding of these mutations in cancer, focusing on how they influence splice site selection and impact on cancer development.

A Pan-Cancer Analysis of Tumor-Infiltrating B Cell Repertoires

Tumor-infiltrating B cells can play an important role in anti-tumor responses but their presence is not well understood. In this study, we extracted the B cell receptor repertoires from 9522 tumor and adjacent non-tumor samples across 28 tumor types in the Cancer Genome Atlas project and performed diversity and network analysis. We identified differences in diversity and network statistics across tumor types and subtypes and observed a trend towards increased clonality in primary tumors compared to adjacent non-tumor tissues. We also found significant associations between the repertoire features and mutation load, tumor stage, and age. Our V-gene usage analysis identified similar V-gene usage patterns in colorectal and endometrial cancers. Lastly, we evaluated the prognostic value of the repertoire features and identified significant associations with survival in seven tumor types. This study warrants further research into better understanding the role of tumor-infiltrating B cells across a wide range of tumor types.

Prediction of pan-solid tumor pembrolizumab benefit by integrating tumor mutation and gene expression profiling

Pembrolizumab is approved in many advanced solid tumor types, however predictive biomarkers and the proportion of pembrolizumab-benefiting patients vary. Biomarkers beyond PD-L1 immunohistochemistry, microsatellite instability (MSI) status, and tumor mutation burden (TMB) may improve benefit prediction. Here, leveraging treatment data (time to next treatment [TTNT]) and comprehensive genomic and quantitative transcriptomic profiling on routine tumor tissue from 708 patients (24 tumor types) collected in an ongoing observational trial (NCT03061305), we report a multivariate, integrative predictor of pan-solid tumor pembrolizumab benefit. The Immune Response Score (IRS) model, which includes TMB and quantitative PD-1, PD-L2, ADAM12 and CD4 RNA expression, was confirmed as predictive through comparison of pembrolizumab TTNT with previous chemotherapy TTNT in a subset of 166 patients treated with both. Applying IRS to the entire NCT03061305 cohort (n=25,770 patients), 13.2-30.7% of patients (2.2-9.6% of patients outside of pembrolizumab approved tumor types [including TMB-High and MSI-High]) are predicted to benefit substantially from pembrolizumab. Hence, if prospectively validated, the IRS model may improve pembrolizumab benefit prediction across approved tumor types including patients outside of currently approved indications.

Neomorphic DNA-binding enables tumor-specific therapeutic gene expression in fusion-addicted childhood sarcoma

Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer types including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity. Here, we show in the EwS model that - capitalizing on neomorphic DNA-binding preferences - the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes. We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to the otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor types typically considered to be immunologically cold. Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties.

Glutamine-Derived Aspartate Biosynthesis in Cancer Cells: Role of Mitochondrial Transporters and New Therapeutic Perspectives

Aspartate has a central role in cancer cell metabolism. Aspartate cytosolic availability is crucial for protein and nucleotide biosynthesis as well as for redox homeostasis. Since tumor cells display poor aspartate uptake from the external environment, most of the cellular pool of aspartate derives from mitochondrial catabolism of glutamine. At least four transporters are involved in this metabolic pathway: the glutamine (SLC1A5_var), the aspartate/glutamate (AGC), the aspartate/phosphate (uncoupling protein 2, UCP2), and the glutamate (GC) carriers, the last three belonging to the mitochondrial carrier family (MCF). The loss of one of these transporters causes a paucity of cytosolic aspartate and an arrest of cell proliferation in many different cancer types. The aim of this review is to clarify why different cancers have varying dependencies on metabolite transporters to support cytosolic glutamine-derived aspartate availability. Dissecting the precise metabolic routes that glutamine undergoes in specific tumor types is of upmost importance as it promises to unveil the best metabolic target for therapeutic intervention.