CD56-positive diffuse large B-cell lymphoma: comprehensive analysis of clinical, pathological, and molecular characteristics with literature review.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. The expression of CD56 in DLBCL is highly unusual. Little is known about its incidence and clinical importance. So far, no genetic profiling was performed in CD56 positive DLBCL. PATIENTS AND METHODS: Tissue microarrays have been constructed, sectioned, and stained by H&E and immunohistochemistry for 229 patients with DLBCL diagnosed 2008-2017. For CD56 positive cases, clinical data was collected including age at diagnosis, stage of the disease, International Prognostic Index (IPI) score, treatment scheme and number of chemotherapy cycles, radiation therapy, treatment outcome, and possible relapse of the disease. Overall survival (OS) and progression-free survival (PFS) were calculated. For four patients, RNA was extracted and targeted RNA (cDNA) sequencing of 125 genes was performed with the Archer FusionPlex Lymphoma kit. RESULTS: CD56 expression was found in 7 cases (3%). The intensity of expression varied from weak to moderate focal, to very intensive and diffuse. All patients had de novo DLBCL. The median age at the time of diagnosis was 54.5 years. Five of them were women and 2 males. According to the Hans algorithm, 6 patients had the germinal centre B cells (GBC) type and one non-GBC (activated B-cell [ABC]) type, double expressor. Genetic profiling of four patients according to Schmitz's classification showed that 1 case was of the BN2 subtype, 1 of EZB subtype, 2 were unclassified. The six treated patients reached a complete response and did not experience progression of the disease during the median follow-up period of 80.5 months. CONCLUSIONS: We report on one of the largest series of CD56+DLBCL with detailed clinicopathological data and for the first time described genetical findings in a limited number of patients. Our results show that CD56 expression is rare, but seems to be present in prognostic favourable subtypes of DLBCL not otherwise specified (NOS) as tested by immunohistochemical or genetic profiling.

Correlation of treatment outcome in sanger/RT­qPCR KIT/PDGFRA wild­type metastatic gastrointestinal stromal tumors with next­generation sequencing results: A single­center report.

In patients with gastrointestinal stromal tumors (GIST), it has become mandatory to determine the driver mutation in order to predict the response to standard treatment with tyrosine kinase inhibitors (TKI). A total of 10­15% of all GIST lack activating mutations in KIT proto­oncogene, receptor tyrosine kinase (KIT)/platelet­derived growth factor receptor alpha (PDGFRA) and have been classified as KIT/PDGFRA wild­type (WT) GIST. They are characterized by poor response to TKI. From a group of 119 metastatic GIST patients, 17 patients with KIT/PDGFRA/BRAF WT GIST as determined by reverse transcription­quantitative (RT­q) PCR and Sanger sequencing were profiled by a targeted next­generation sequencing (NGS) approach and their treatment outcome was assessed. In the present study, 41.2% of patients as KIT/PDGFRA/BRAF WT GIST examined with RT­qPCR and Sanger sequencing were confirmed to be carriers of pathogenic KIT/PDGFRA mutations by NGS and were responsive to TKI. The percentage of genuinely KIT/PDGFRA WT GIST in the present study thereby dropped from the initial 14.3% detected with the RT­qPCR and Sanger sequencing to 7.6% after NGS. Their outcome was universally poor. The reliability of RT­qPCR and direct Sanger sequencing results in this setting is therefore insufficient and it is recommended that NGS becomes a requirement for treatment decision at least in KIT/PDGFRA/BRAF WT GIST as determined by RT­qPCR and Sanger sequencing.

BAP1-defficient breast cancer in a patient with BAP1 cancer syndrome.

BAP1 cancer syndrome is a rare and highly penetrant hereditary cancer predisposition. Uveal melanoma, mesothelioma, renal cell carcinoma (RCC) and cutaneous melanoma are considered BAP1 cancer syndrome core cancers, whereas association with breast cancer has previously been suggested but not confirmed so far. In view of BAP1 immunomodulatory functions, BAP1 alterations could prove useful as possible biomarkers of response to immunotherapy in patients with BAP1-associated cancers. We present a case of a patient with BAP1 cancer syndrome who developed a metastatic breast cancer with loss of BAP1 demonstrated on immunohistochemistry. She carried a germline BAP1 likely pathogenic variant (c.898_899delAG p.(Arg300Glyfs*6)). In addition, tumor tissue sequencing identified a concurrent somatic variant in BAP1 (partial deletion of exon 12) and a low tumor mutational burden. As her triple negative tumor was shown to be PD-L1 positive, the patient was treated with combination of atezolizumab and nab-paclitaxel. She had a complete and sustained response to immunotherapy even after discontinuation of nab-paclitaxel. This case strengthens the evidence for including breast cancer in the BAP1 cancer syndrome tumor spectrum with implications for future cancer prevention programs. It also indicates immune checkpoint inhibitors might prove to be an effective treatment for BAP1-deficient breast cancer.

Mutational burden, MHC-I expression and immune infiltration as limiting factors for in situ vaccination by TNFα and IL-12 gene electrotransfer.

In situ vaccination is a promising immunotherapeutic approach, where various local ablative therapies are used to induce an immune response against tumor antigens that are released from the therapy-killed tumor cells. We recently proposed using intratumoral gene electrotransfer for concomitant transfection of a cytotoxic cytokine tumor necrosis factor-α (TNFα) to induce in situ vaccination, and an immunostimulatory cytokine interleukin 12 (IL-12) to boost the primed immune response. Here, our aim was to test the local and systemic effectiveness of the approach in tree syngeneic mouse tumor models and associate it with tumor immune profiles, characterized by tumor mutational burden, immune infiltration and expression of PD-L1 and MHC-I on tumor cells. While none of the tested characteristic proved predictive for local effectiveness, high tumor mutational burden, immune infiltration and MHC-I expression were associated with higher abscopal effectiveness. Hence, we have confirmed that both the abundance and presentation of tumor antigens as well as the absence of immunosuppressive mechanisms are important for effective in situ vaccination. These findings provide important indications for future development of in situ vaccination based treatments, and for the selection of tumor types that will most likely benefit from it.

Mutational spectrum and classification of novel mutations in patients with metastatic gastrointestinal stromal tumours.

In total, ~85% of malignant gastrointestinal stromal tumours (GISTs) harbour activating mutations in one of the genes KIT or PDGFRA, while 10­15% of all GISTs have no detectable KIT or PDGFRA mutations, but could have alterations in genes of the succinate dehydrogenase complex or in BRAF, PIK3CA or rarely RAS family genes. The clinical benefit of tyrosine kinase inhibitors, such as imatinib, depends on the GIST genotype, therefore molecular characterization of GIST has a crucial role in overall management of GIST. The aim of the present study was to molecularly characterize a cohort of 70 patients with metastatic GISTs from the Slovenian Cancer Registry (National Cancer Registry) treated between January 2002 and December 2011. Exons 9, 11, 13 and 17 of the KIT gene and exons 12, 14 and 18 of the PDGFRA gene were analysed by direct Sanger sequencing. All KIT/PDGFRA wild­type GISTs were tested for the presence of mutations in hot spot regions of KRAS, NRAS, BRAF, PIK3CA and AKT1 genes. Novel variants were characterized and classified using Cancer Genome Interpreter and according to The American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines. In total, 60 (85.7%) patients had mutations in KIT and 2 (2.9%) in PDGFRA. Whereas, 8 (11.4%) patients with GIST had no mutation in either of the analysed genes. The majority of GIST cases (n=52) had a mutation in KIT exon 11, where 40 different mutations were detected. Eight of the variants were novel: c.1652_1672del, c.1653_1660delinsAA, c.1665_1672delinsCC, c.1668_1686del, c.1676_1720del, c.1715_1756dup, c.1721_1765dup, and c.1722_1766dup. Mutation frequencies of KIT and PDGFRA genes observed in Slovenian patients are comparable with those in other European populations. In the present group of patients analysed, the most frequently mutated region was exon 11 in the KIT gene, responsible for coding juxtamembrane domain of KIT protein. In this region, eight novel mutations were identified and classified as likely pathogenic driver variants. In addition, the present study identified 6 patients with secondary KIT mutation and 1 patient with double mutant GIST, who had two different mutations in PDGFRA exon 14.

A Novel Germline MLH1 In-Frame Deletion in a Slovenian Lynch Syndrome Family Associated with Uncommon Isolated PMS2 Loss in Tumor Tissue.

The diagnostics of Lynch syndrome (LS) is focused on the detection of DNA mismatch repair (MMR) system deficiency. MMR deficiency can be detected on tumor tissue by microsatellite instability (MSI) using molecular genetic test or by loss of expression of one of the four proteins (MLH1, MSH2, MSH6, and PMS2) involved in the MMR system using immunohistochemistry (IHC) staining. According to the National Comprehensive Cancer Network (NCCN) guidelines, definitive diagnosis of LS requires the identification of the germline pathogenic variant in one of the MMR genes. In the report, we are presenting interesting novel MLH1 in-frame deletion LRG_216t1:c.2236_2247delCTGCCTGATCTA p.(Leu746_Leu749del) associated with LS. The variant appears to be associated with uncommon isolated loss of PMS2 immunohistochemistry protein staining (expression) in tumor tissue instead of MLH1 and PMS2 protein loss, which is commonly seen with pathogenic variants in MLH1. The variant was classified as likely pathogenic, based on segregation analysis and molecular characterization of blood and tumor samples. According to the American College of Medical Genetics (ACMG) guidelines, the following evidence categories of PM1, PM2, PM4, and PP1 moderate have been used for classification of the novel variant. By detecting and classifying the novel MLH1 variant as likely pathogenic, we confirmed the LS in this family.

Three Types of Functional Regulatory T Cells Control T Cell Responses at the Human Maternal-Fetal Interface.

During pregnancy, maternal regulatory T cells (Tregs) are important in establishing immune tolerance to invading fetal extravillous trophoblasts (EVTs). CD25HIFOXP3+ Tregs are found at high levels in decidual tissues and have been shown to suppress fetus-specific and nonspecific responses. However, limited data are available on additional decidual Treg types and the mechanisms by which they are induced. This study investigated three distinct decidual CD4+ Treg types in healthy pregnancies with a regulatory phenotype and the ability to suppress T cell responses: CD25HIFOXP3+, PD1HIIL-10+, and TIGIT+FOXP3dim. Moreover, co-culture of HLA-G+ EVTs or decidual macrophages with blood CD4+ T cells directly increased the proportions of CD25HIFOXP3+ Tregs compared to T cells cultured alone. EVTs also increased PD1HI Tregs that could be inhibited by HLA-C and CD3 antibodies, suggesting an antigen-specific induction. The presence of distinct Treg types may allow for the modulation of a variety of inflammatory responses in the placenta.

Two Novel NF1 Pathogenic Variants Causing the Creation of a New Splice Site in Patients With Neurofibromatosis Type I.

Neurofibromatosis type I (NF1) is one of the most common autosomal dominant disorders, since the estimated incidence is one in 3,500 births. In this study, we present bioinformatical and functional characterization of two novel splicing NF1 variants, detected in NF1 patients. Patient 1, carrying NF1:c.122A>T, which introduces a new exonic 5' donor splice site, was diagnosed with hormone-positive, Her-2-negative breast cancer at the age of 47. She had an atypical presentation of NF1, with few café-au-lait spots and no Lisch nodules. Patient developed a hemothorax due to subclavian artery rupture, which has previously been described as an extremely rare complication of NF1. Patient 2, carrying NF1:c.7395-17T>G that creates a new intronic 3' acceptor splice site, had quite a typical clinical presentation of NF1: formations on her tongue in the region of her left metacarpal bones and on her left foot, plexiform neurofibroma in her pelvis, several café-au-lait spots, and axillary freckling. She was also diagnosed with cognitive impairment. In the report, we are presenting two novel variants which were successfully classified based on NGS and mRNA analysis. Based on results of mRNA analysis, both variants were classified as likely pathogenic according to ACMG guidelines applying evidence categories PS3, PM2, PP3, and PP1 supporting. By characterizing those two novel NF1 splicing variants, we have confirmed the neurofibromatosis type I phenotype in the two probands.