Background Distinguishing separate primary lung cancers (SPLCs) from intrapulmonary metastases (IPMs) is essential for staging and treatment, yet histology alone may be inconclusive. Molecular profiling can enhance diagnostic accuracy, but most supporting evidence to date stems from North America and Asia. Methods We retrospectively analyzed 49 patients (111 tumour nodules) with multiple lung cancers (MLCs) surgically resected at a Swiss tertiary centre (2015–2023). Tumours were classified histologically using three algorithms (Martini and Melamed, comprehensive histologic assessment [CHA], and CHA with low-grade lepidic features) and compared with molecular classification using a 52-gene targeted next-generation sequencing (NGS) panel integrating somatic mutation and copy number alteration (CNA) analysis to assess clonal relatedness. Results Histologic algorithms showed high internal concordance (up to 96.1%) but only modest agreement with molecular classification (69–75%). NGS enabled definitive classification in 85.5% of tumour pairs. CNA analysis contributed to clonality assessment in 28% of tumours and was the sole discriminatory feature in two mutation-negative cases. Discordance between CHA and molecular classification was frequent when tumours harboured common driver mutations (e.g., KRAS p.G12C, EGFR p.L858R). Overall, 31% of comparisons showed disagreement between CHA-based histologic assessment and molecular profiling. Conclusions In this first Swiss cohort, targeted NGS with CNA analysis improved discrimination between SPLCs and IPMs, but the standard routine NGS panel size was limited when tumours shared common driver mutations, particularly KRAS p.G12C and EGFR p.L858R. These findings underscore the potential need for broader genomic approaches, alongside histology, to achieve accurate classification and guide precise staging and treatment.

Primary intestinal T-cell lymphomas (ITCLs), comprising enteropathy-associated T-cell lymphoma (EATL) and monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), are rare aggressive tumors. The role of DNA mismatch repair (MMR) deficiency (dMMR) and microsatellite instability (MSI) in the development of ITCLs remains largely unexplored. Here, we investigated the incidence, molecular mechanisms and clinical relevance of dMMR/MSI in 86 ITCLs (30 EATLs, 56 MEITLs) using whole-exome sequencing, PCR-based MSI testing, DNA methylation profiling, and immunohistochemistry for MLH1, MSH2, MSH6 and PMS2. MMR deficiency was detected in 3 of 53 MEITLs (6%) but in none of the EATLs. dMMR MEITLs showed the highest tumor mutational burden (8.3-17.1 mutations/Mb), compared to median TMBs of 1.9 in MEITL and 2.4 in EATL. The complete loss of MLH1/PMS2 expression in two MSI-high tumors and isolated PMS2 loss in the third case were all associated with biallelic deletions of the affected loci. Notably, MLH1 deletions significantly co‑occurred with SETD2 deletions (p = 0.001), the latter representing a major driver of MEITL tumorigenesis. dMMR MEITLs lacked distinctive clinicopathologic features. These findings indicate that dMMR/MSI occurs in a subset of MEITLs probably during tumor progression, rather than being an initiating driver event, and provide a biological rationale to explore the efficacy of immune checkpoint inhibitors in some of this unfavorable subtype of ITCL.

Transdifferentiation from follicular lymphoma (FL) to histiocytic/dendritic cell sarcoma (HDS) is rare and requires molecular confirmation of shared clonal origin. Targetable mutations such as BRAF V600E may offer therapeutic opportunities in such aggressive neoplasms. We report an exceptional case of untreated localised FL transdifferentiated to an HDS after 18 years. Shared BCL2 rearrangement and mutation profile confirmed a clonal link, while the HDS acquired an additional BRAF V600E mutation. Treatment with BRAF/MEK inhibitors yielded a sustained 18-month clinical response. The disease later relapsed as high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-MYC/BCL2), still harbouring the BRAF mutation. Complete remission was achieved with Rituximab, Cyclophosphamide, Hydroxydaunorubicin, Oncovin and Prednisone, but the double-hit lymphoma relapsed 14 months later.This case illustrates sequential transformation from FL to BRAF-mutated HDS with excellent response to BRAF/MEK inhibition, followed by evolution into HGBCL-MYC/BCL2 responding transiently to immunochemotherapy, emphasising the value of repeated histological and molecular reassessment in FL evolution.

Enteropathy-associated intestinal T-cell lymphoma (EATL) and monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) have distinctive clinical context, morphology, and immunophenotype. To characterize their genetic and molecular specificities, we compared 30 EATLs and 52 MEITLs by whole-exome, RNA and miRNA sequencing and DNA methylation profiling. Highly recurrent SETD2 loss-of-function alterations and frequent mutations of H3-3A/B consistently altering H3R2, implying deregulation of histone marks, were selectively found in MEITL. EATL instead harbored frequent mutations in TET2, ARID1A, and KMT2D. Highly prevalent JAK-STAT pathway mutations preferentially affected JAK3 and STAT5B in MEITL, and JAK1 and STAT3 in EATL. Half of EATLs contained disruptive mutations in HLA class I genes, impacting class I molecule expression. EATL containing more abundant macrophages was enriched in inflammatory response signatures, with upregulation of CD274, CXCL13, and IDO1 transcripts, suggesting an immunosuppressive tumor microenvironment. CpGs hypomethylated in MEITL compared to EATL were enriched in promoter regions. Unsupervised analyses of mutations, transcription, and methylation profiles concordantly segregated EATLs from MEITLs. In summary, the distinctive genetic, epigenetic, and expression footprints of EATL and MEITL established by this study expand disease-defining features, have diagnostic implications, and provide a rationale for targeted therapies.

Sarcomas are traditionally considered “cold” tumors with poor response to immunotherapy. However, evidence accumulating over the last years shows that immune checkpoint inhibitors (ICIs) may have a role in selected sarcoma patients according to predictive markers. Here, we report the case of a woman diagnosed with a primary cardiac undifferentiated sarcoma. Following failure of standard first line chemotherapy, high-throughput sequencing (HTS) revealed a high tumor mutational burden (TMB), pathogenic mutations in FAT1 and NOTCH2 and a microsatellite instability (MSI)-associated signature. Immunohistochemistry confirmed mismatch repair-deficiency (MMRd) and abundant CD8+ tumor-infiltrating lymphocytes (TILs), in the absence of tertiary lymphoid structures. The patient was, therefore, treated with the ICI pembrolizumab, reaching a complete response that continues to persist at last follow-up, more than seven years from initial diagnosis and nearly six years from initiation of ICI treatment. This case illustrates the importance of performing HTS in rare sarcomas given the availability of efficient therapies, such as those for tumors displaying high TMB or MMRd/MSI. In agreement with other reports, it supports the contention that MMRd/MSI status and high numbers of TILs are valuable predictive markers of response to immunotherapy in sarcomas.

ABSTRACT:The RHOA G17V mutation is highly recurrent in T follicular helper (TFH) cell lymphoma of the angioimmunoblastic type (AITL; 60%-70% of cases) and frequently associated with mutations in other T-cell receptor signaling genes, including CD28. Here, we sought to elucidate how RHOA and CD28 variants may work in concert to sustain T-cell activation by generating stable Jurkat T-cell lines expressing wild-type (wt) RHOA or RHOA G17V with wt CD28 or CD28 T195P. Concomitant expression of RHOA G17V and CD28 T195P induced significantly higher levels of interleukin-2 (IL-2) production and NFAT nuclear factor of activated T cells (NFAT) and activator protein 1 (AP1) transcriptional activities than either variant alone upon T-cell activation with agonistic anti-CD3 and anti-CD28 antibodies. We identified the histone acetyltransferase p300 as a major interacting partner of RHOA G17V in our model and human primary T cells. p300 inhibition abolished the increased IL-2 secretion induced by CD3/CD28 stimulation in cells expressing RHOA G17V and/or CD28 T195P. Chromatin immunoprecipitations and immunofluorescence staining revealed an increase of p300-specific H3K18ac and H3K27ac marks at the IL-2 promoter and across whole genome, respectively, in cells expressing RHOA G17V. Finally, immunofluorescence staining of tumor samples from 4 patients with AITL carrying RHOA G17V variant and 4 carrying wt RHOA showed that neoplastic TFH cells with RHOA G17V have increased H3K18ac and H3K27ac levels compared with non-neoplastic T cells. Collectively, these findings uncover a new mechanism of action by which RHOA G17V potentiates CD28 T195P-induced NFAT and AP1 transcriptional activities by enhancing p300 histone acetyltransferase activity and expand the notion that epigenetic deregulation contributes to the pathogenesis of TFH lymphomas.

Whereas immunotherapies have revolutionized the treatment of different solid and hematologic cancers, their efficacy in nodal peripheral T-cell lymphomas (PTCL) is limited, due to a lack of understanding of the immune response they trigger. To fully characterize the immune tumor microenvironment (TME) of PTCL, we performed spectral flow cytometry analyses on 11 angioimmunoblastic T-cell lymphomas (AITL), 7 PTCL, not otherwise specified (PTCL, NOS) lymph node samples, and 10 non-tumoral control samples. The PTCL TME contained a larger proportion of regulatory T cells and exhausted CD8+ T cells, with enriched expression of druggable immune checkpoints. Interestingly, CD39 expression was up-regulated at the surface of most immune cells, and a multi-immunofluorescence analysis on a retrospective cohort of 43 AITL patients demonstrated a significant association between high CD39 expression by T cells and poor patient prognosis. Together, our study unravels the complex TME of nodal PTCL, identifies targetable immune checkpoints, and highlights CD39 as a novel prognostic factor.

Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is a heterogeneous group of malignancies with poor outcome. Here, we identify a subgroup, PTCL-NOSSMARCB1-, which is characterized by the lack of the SMARCB1 protein and occurs more frequently in young patients. Human and murine PTCL-NOSSMARCB1- show similar DNA methylation profiles, with hypermethylation of T-cell-related genes and hypomethylation of genes involved in myeloid development. Single-cell analyses of human and murine tumors revealed a rich and complex network of interactions between tumor cells and an immunosuppressive and exhausted tumor microenvironment (TME). In a drug screen, we identified histone deacetylase inhibitors (HDACi) as a class of drugs effective against PTCL-NOSSmarcb1-. In vivo treatment of mouse tumors with SAHA, a pan-HDACi, triggered remodeling of the TME, promoting replenishment of lymphoid compartments and reversal of the exhaustion phenotype. These results provide a rationale for further exploration of HDACi combination therapies targeting PTCL-NOSSMARCB1- within the TME. Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is a heterogeneous and aggressive type of T-cell lymphoma. Here, the authors perform single-cell analyses of human and murine PTCL-NOS tumors, and identify a subtype defined by the loss of SMARCB1 that could be targeted with HDAC-inhibitor combination therapies.

Primary bone lymphoma (PBL) is rare and mostly represented by diffuse large B-cell lymphomas (DLBCL). Follicular lymphoma (FL), albeit commonly disseminating to the bone marrow, rarely presents primarily as bone lesions. Here, we studied 16 patients (12 men:4 women, median age 60 years) who presented with bone pain and/or skeletal radiologic abnormalities revealing bone FL. Lesions were multifocal in 11 patients (spine ± appendicular skeleton), and unifocal in 5 patients (femoral, tibial, or vertebral). An infiltrate of centrocytes and centroblasts (CD20+ CD5- CD10+ BCL2+ BCL6+) with abundant reactive T cells and an increased reticulin fibrosis massively replaced the marrow spaces between preserved bone trabeculae. The pattern was diffuse ± nodular, often with paratrabecular reinforcement and/or peripheral paratrabecular extension. Ki-67 was usually <15%. Two cases had necrosis. BCL2 rearrangement was demonstrated in 14 of 14 evaluable cases (with concomitant BCL6 rearrangement in one). High-throughput sequencing revealed BCL2, KMT2D, and TNFRSF14 to be the most frequently mutated genes. After staging, 5 qualified for PBL (3 limited stage) and 11 had stage IV systemic FL. All patients received rituximab ± polychemotherapy as firstline treatment, and 7 received local therapy (6 radiotherapy and 2 surgery). Three patients experienced transformation to DLBCL. At the last follow-up (15/16, median 48 months), 11 patients achieved complete remission, including all cases with PBL and most patients with limited extraosseous disease (3-year progression-free survival 71%). One patient died of unrelated cause (3-year overall survival 91%). FL may manifest as a localized or polyostotic bone disease. A minority represent PBL, whereas most reveal systemic disease.

Somatic variant testing through next-generation sequencing (NGS) is well integrated into Swiss molecular pathology laboratories and has become a standard diagnostic method for numerous indications in cancer patient care. Currently, there is a wide variation in reporting practices within our country, and as patients move between different hospitals, it is increasingly necessary to standardize NGS reports to ease their reinterpretation. Additionally, as many different stakeholders—oncologists, hematologists, geneticists, pathologists, and patients—have access to the NGS report, it needs to contain comprehensive and detailed information in order to answer the questions of experts and avoid misinterpretation by non-experts. In 2017, the Swiss Institute of Bioinformatics conducted a survey to assess the differences in NGS reporting practices across ten pathology institutes in Switzerland. The survey examined 68 reporting items and identified 48 discrepancies. Based on these findings, the Swiss Society of Molecular Pathology initiated a Delphi method to reach a consensus on a set of recommendations for NGS reporting. Reports should include clinical information about the patient and the diagnosis, technical details about the sample and the test performed, and a list of all clinically relevant variants and variants of uncertain significance. In the absence of a consensus on an actionability scheme, the five-class pathogenicity scheme proposed by the ACMG/AMP guideline must be included in the reports. The Swiss Society of Molecular Pathology recognizes the importance of including clinical actionability in the report and calls on the European community of molecular pathologists and oncologists to reach a consensus on this issue.

Results of colorectal cell lines classification into BM1 and BM2.

Figure S1. Principal component analysis and hierarchical clustering of the 218 BRAF mutant patient cohort;Figure S2: Gene-wise and patient-wise biclustering heatmap of the 218 BRAF mutant patient cohort and comparison of BRAF mutant subtypes obtained with and without adjusting for microsatellite instability status;Figure S3: Geneset enrichment analysis (GSEA) of BM subtypes for the four most enriched signature of the Hallmark collection and gene overlap between signatures and BM-specific genes;Figure S4: Patient-level single sample geneset enrichment analysis (ssGSEA) between BM subtypes, double BRAF wild-type KRAS wild-type (WT2) and KRAS mutant patients for the hallmark signature collection;Figure S5: Patient-averaged single sample geneset enrichment analysis (ssGSEA) between BM subtypes, double BRAF wild-type KRAS wild-type (WT2) and KRAS mutant patients for the hallmark signature collection;Figure S6: Gene overlap between selected sets of gene signatures used in this study;Figure S7: Immune landscape of the BM subtypes;Figure S8: Methylation analysis of BM subtypes using TCGA data;Figure S9: Survival and clinical characteristics of the BRAF mutant patients enrolled in the survival analysis;Figure S10: Performance of the generalized model used to classify patients into BM1 and BM2 subtypes;Figure S11: Drug responses of colorectal cancer cell lines classified into BM1 and BM2.

Supplementary Figures and Tables

Results of differential drug responses of BM1 and BM2 classified colorectal cell lines.

Contains Supplemental Table 1 showing the target sequences used for the 5 genes and Supplementary Figure Legends