Genetic mutations and phenotype characteristics in peripheral vascular malformations: A systematic review.

Vascular malformations (VMs) are clinically diverse with regard to the vessel type, anatomical location, tissue involvement and size. Consequently, symptoms and disease impact differ significantly. Diverse causative mutations in more and more genes are discovered and play a major role in the development of VMs. However, the relationship between the underlying causative mutations and the highly variable phenotype of VMs is not yet fully understood. In this systematic review, we aimed to provide an overview of known causative mutations in genes in VMs and discuss associations between the causative mutations and clinical phenotypes. PubMed and EMBASE libraries were systematically searched on November 9th, 2022 for randomized controlled trials and observational studies reporting causative mutations in at least five patients with peripheral venous, lymphatic, arteriovenous and combined malformations. Study quality was assessed with the Newcastle-Ottawa Scale. Data were extracted on patient and VM characteristics, molecular sequencing method and results of molecular analysis. In total, 5667 articles were found of which 69 studies were included, reporting molecular analysis in a total of 4261 patients and 1686 (40%) patients with peripheral VMs a causative mutation was detected. In conclusion, this systematic review provides a comprehensive overview of causative germline and somatic mutations in various genes and associated phenotypes in peripheral VMs. With these findings, we attempt to better understand how the underlying causative mutations in various genes contribute to the highly variable clinical characteristics of VMs. Our study shows that some causative mutations lead to a uniform phenotype, while other causal variants lead to more varying phenotypes. By contrast, distinct causative mutations may lead to similar phenotypes and result in almost indistinguishable VMs. VMs are currently classified based on clinical and histopathology features, however, the findings of this systematic review suggest a larger role for genotype in current diagnostics and classification.

Presence of a 34-gene signature is a favorable prognostic marker in squamous non-small cell lung carcinoma.

BACKGROUND: The tumor immune microenvironment is a heterogeneous entity. Gene expression analysis allows us to perform comprehensive immunoprofiling and may assist in dissecting the different components of the immune infiltrate. As gene expression analysis also provides information regarding tumor cells, differences in interactions between the immune system and specific tumor characteristics can also be explored. This study aims to gain further insights in the composition of the tumor immune infiltrate and to correlate these components to histology and overall survival in non-small cell lung cancer (NSCLC). METHODS: Archival tissues from 530 early stage, resected NSCLC patients with annotated tumor and patient characteristics were analyzed using the NanoString nCounter Analysis system. RESULTS: Unsupervised clustering of the samples was mainly driven by the overall level of inflammation, which was not correlated with survival in this patient set. Adenocarcinoma (AD) showed a significantly higher degree of immune infiltration compared to squamous cell carcinoma (SCC). A 34-gene signature, which did not correlate with the overall level of immune infiltration, was identified and showed an OS benefit in SCC. Strikingly, this benefit was not observed in AD. This difference in OS in SCC specifically was confirmed in two independent NSCLC cohorts. The highest correlation between expression of the 34-gene signature and specific immune cell populations was observed for NK cells, but although a plausible mechanism for NK cell intervention in tumor growth could be established in SCC over AD, this could not be translated back to immunohistochemistry, which showed that NK cell infiltration is scarce irrespective of histology. CONCLUSIONS: These findings suggest that the ability of immune cell infiltration and the interaction between tumor and immune cells may be different between AD and SCC histology and that a subgroup of SCC tumors seems more susceptible to Natural Killer cell recognition and killing, whereas this may not occur in AD tumors. A highly sensitive technique like NanoString was able to detect this subgroup based on a 34-gene signature, but further research will be needed to assist in explaining the biological rationale of such low-level expression signatures.

Clinical value of ctDNA in upper-GI cancers: A systematic review and meta-analysis.

BACKGROUND: The recent expanding technical possibilities to detect tumor derived mutations in blood, so-called circulating tumor DNA (ctDNA), has rapidly increased the interest in liquid biopsies. This review and meta-analysis explores the clinical value of ctDNA in malignancies of the upper gastro-intestinal tract. METHODS: PubMed, Cochrane and Embase databases were searched to identify studies reporting the diagnostic, prognostic or predictive value of ctDNA in patients with esophageal, gastric and pancreatic cancer, until January 2017. The diagnostic accuracy and, using random-effect pair-wise meta-analyses, the prognostic value of ctDNA was assessed. RESULTS: A total of 34 studies met the inclusion criteria. For esophageal and gastric cancer, amplification of oncogenes in blood, such as HER2 and MYC, can be relevant for diagnostic purposes, and to predict treatment response in certain patient subpopulations. Given the limited number of studies assessing the role of ctDNA in esophageal and gastric cancer, the meta-analysis estimated the diagnostic accuracy and predictive value of ctDNA in pancreatic cancer only (n=10). The pooled sensitivity and specificity of ctDNA as a diagnostic tool in pancreatic cancer were 28% and 95%, respectively. Patients with pancreatic cancer and detectable ctDNA demonstrated a worse overall survival compared to patients with undetectable ctDNA (HR 1.92, 95% confidence interval (CI) 1.15-3.22, p=0.01). CONCLUSION: The presence of ctDNA is significantly associated with a poor prognosis in patients with pancreatic cancer. The use of ctDNA in clinical practice is promising, although standardization of sequencing techniques and further development of high-sensitive detection methods is needed.

The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC.

BACKGROUND: Constitutive Wnt activation is essential for colorectal cancer (CRC) initiation but also underlies the cancer stem cell phenotype, metastasis and chemosensitivity. Importantly Wnt activity is still modulated as evidenced by higher Wnt activity at the invasive front of clonal tumours termed the ß-catenin paradox. SMAD4 and p53 mutation status and the bone morphogenetic protein (BMP) pathway are known to affect Wnt activity. The combination of SMAD4 loss, p53 mutations and BMP signalling may integrate to influence Wnt signalling and explain the ß-catenin paradox. METHODS: We analysed the expression patterns of SMAD4, p53 and ß-catenin at the invasive front of CRCs using immunohistochemistry. We activated BMP signalling in CRC cells in vitro and measured BMP/Wnt activity using luciferase reporters. MTT assays were performed to study the effect of BMP signalling on CRC chemosensitivity. RESULTS: Eighty-four percent of CRCs with high nuclear ß-catenin staining are SMAD4 negative and/or p53 aberrant. BMP signalling inhibits Wnt signalling in CRC only when p53 and SMAD4 are unaffected. In the absence of SMAD4, BMP signalling activates Wnt signalling. When p53 is lost or mutated, BMP signalling no longer influences Wnt signalling. The cytotoxic effects of 5-FU are influenced in a similar manner. CONCLUSIONS: The BMP signalling pathway differentially modulates Wnt signalling dependent on the SMAD4 and p53 status. The use of BMPs in cancer therapy, as has been proposed by previous studies, should be targeted to individual cancers based on the mutational status of p53 and SMAD4.

Radiation-induced melanoma-associated leucoderma, systemic antimelanoma immunity and disease-free survival in a patient with advanced-stage melanoma: a case report and immunological analysis.

BACKGROUND: Melanoma is an immunogenic tumour. The development of skin depigmentation or melanoma-associated leucoderma (MAL) has been associated with favourable clinical outcome in patients with metastatic melanoma, especially after immunotherapy. Evidence for clinically meaningful enhancement of melanoma-directed autoimmunity, as indicated by MAL, after radiotherapy without immunotherapy has not yet been published. OBJECTIVES: We investigated whether a patient with stage IV melanoma, who developed leucoderma in the irradiated skin areas following radiotherapy and experienced exceptional disease-free survival of 3 years despite brain metastasis, possessed antimelanoma immunity that could be linked to the favourable disease course. METHODS: A detailed immunological analysis was performed consisting of immunohistochemistry of several melanoma tissues, and analyses of T cells isolated from the blood and MAL skin tissue for melanocyte/melanoma specificity and functionality, as well as the presence of a melanoma-specific antibody response. RESULTS: Immunological analyses showed the presence of CD8+ T cells and antibody responses directed against melanocyte differentiation antigens expressed in the primary tumour, lymph node and brain metastasis, indicating adequate tumour recognition by activated T cells. CONCLUSION: The immune responses found in this patient, probably enhanced by radiotherapy, are thought to have contributed to his favourable clinical course. Radiotherapy may act as local immunotherapy in patients with melanoma by destroying melanocytes, leading to the induction, or enhancement, of already existent antimelanoma immunity. As in patients treated with immunotherapy, this may lead to MAL, also at distant sites from the treated area. This patient is a clear example of the positive prognostic value of MAL, which is possibly induced by radiotherapy, for patients with melanoma.

Comparison of human B cell antigen receptor complexes: membrane-expressed forms of immunoglobulin (Ig)M, IgD, and IgG are associated with structurally related heterodimers.

We have recently reported that on human B lymphocytes, membrane IgM (mIgM) associates with a heterodimer of 47- and 37-kD polypeptides, the 47-kD subunit being encoded by the mb-1 gene. We show here that expression of mb-1, both at the mRNA and the protein level, is not restricted to IgM+ B cells but can also be found in IgM- pre-B cells and mIgM-IgG+ B cells. Membrane forms of IgD and IgG, isolated from freshly isolated human B cells and B cell lines, are expressed together with heterodimeric protein structures biochemically similar to the mIgM-associated polypeptides, and these were shown to comprise the products of the mb-1 and B29 genes, or homologous genes. Finally, all three classes of antigen receptors are linked to protein kinases, capable of phosphorylating the Ig-associated heterodimers. Our findings provide insight in the structural organization of the different antigen receptors on human B cells and have implications for their function.

De novo gene mutations in normal human memory B cells.

In the past years, the genomes of thousands of tumors have been elucidated. To date however, our knowledge on somatic gene alterations in normal cells is very limited. In this study, we demonstrate that tetanus-specific human memory B lymphocytes carry a substantial number of somatic mutations in the coding regions of the genome. Interestingly, we observed a statistically significant correlation between the number of exome mutations and those present in the immunoglobulin heavy variable regions. Our findings indicate that the majority of these genomic mutations arise in an antigen-dependent fashion, most likely during clonal expansion in germinal centers. The knowledge that normal B cells accumulate genomic alterations outside the immunoglobulin loci during development is relevant for our understanding of the process of lymphomagenesis.

Molecular pathways in follicular lymphoma.

Follicular lymphoma (FL) is one of the most common B-cell non-Hodgkin's lymphomas. The initiating genetic event found in approximately 90% of FL is the t(14;18), causing constitutive expression of the antiapoptotic BCL-2 protein. The exact secondary alterations leading to full FL development are still poorly defined. In this review, we address (i) the genetic pathways associated with tumorigenesis and progression of FL, (ii) the role of micro-environmental factors with emphasis on B-cell receptor ligands and (iii) lymphoma models in mice and what they teach us about lymphomagenesis in man.

Functional and phenotypic evidence for a selective loss of memory T cells in asymptomatic human immunodeficiency virus-infected men.

In addition to a well-documented depletion of CD4+ T helper cells in later stages of human immunodeficiency virus (HIV) infection, evidence has been provided for a specific unresponsiveness to triggering either by specific antigen in the context of autologous major histocompatibility molecules (self + X) or anti-CD3 monoclonal antibodies (MAb) in both CD4 and CD8 cells from asymptomatic HIV-infected individuals. In the present study we analyzed this unresponsiveness using mitogenic antibodies to distinct T cell membrane receptors. T cells from HIV-infected men who had normal numbers of CD4+ T cells responded poorly to activation signals via the CD3 membrane antigen in both accessory cell-dependent as well as accessory cell-independent culture systems. A similar low response was observed in an anti-CD2-driven system. In contrast, proliferation induced by anti-CD3, anti-CD2, or the phorbol ester Phorbol myristate acetate could be normally enhanced by anti-CD28 MAb. We demonstrated that this unresponsiveness is not due to a failure to induce early events required for activation, such as increased intracellular concentration of free calcium and activation of protein kinase C, but is caused by an imbalance between naive and memory T cells. In HIV-infected asymptomatic men, CD29+ memory T cells are selectively depleted which results in a poor responsiveness to self + X. These findings provide new insights that may have implications for our understanding of the immunopathogenesis of AIDS.

High incidence of Kaposi sarcoma-associated herpesvirus infection in HIV-related solid immunoblastic/plasmablastic diffuse large B-cell lymphoma.

Kaposi sarcoma-associated herpesvirus (KSHV) is known to be associated with two distinct lymphoproliferative disorders: primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD)/MCD-associated plasmablastic lymphoma. We here report a high incidence of KSHV infection in solid HIV-associated immunoblastic/plasmablastic non-Hodgkin's lymphomas (NHLs), in patients lacking effusions and without evidence of (prior) MCD. Within a cohort of 99 HIV-related NHLs, 10 cases were found to be KSHV positive on the basis of immunostaining for KSHV LNA-1 as well as KSHV-specific polymerase chain reaction. All but one of the tumors coexpressed Epstein-Barr virus. Interestingly, all KSHV-positive cases belonged to a distinctive subgroup of 26 diffuse large B-cell lymphomas characterized by the expression of CD138 (syndecan-1) and plasmablastic/immunoblastic morphology. These KSHV-positive lymphomas were preceded by Kaposi sarcoma in 60% of the patients and involved the gastrointestinal tract in 80%. Our results indicate that KSHV infection is not restricted to PEL and MCD; it is also common (38%) in HIV-related solid immunoblastic/plasmablastic lymphomas.

A multidimensional network approach reveals microRNAs as determinants of the mesenchymal colorectal cancer subtype.

Colorectal cancer (CRC) is a heterogeneous disease posing a challenge for accurate classification and treatment of this malignancy. There is no common genetic molecular feature that would allow for the identification of patients at risk for developing recurrences and thus selecting patients who would benefit from more stringent therapies still poses a major clinical challenge. Recently, an international multicenter consortium (CRC Subtyping Consortium) was established aiming at the classification of CRC patients in biologically homogeneous CRC subtypes. Four consensus molecular subtypes (CMSs) were identified, of which the mesenchymal CMS4 presented with worse prognosis signifying the importance of identifying these patients. Despite the large number of samples analyzed and their clear association with unifying biological programs and clinical features, single-driver mutations could not be identified and patients are heterogeneous with regard to currently used clinical markers. We therefore set out to define the regulatory mechanisms underlying the distinct gene expression profiles using a network-based approach involving multiple molecular modalities such as gene expression, methylation levels and microRNA (miR) expression. The miR-200 family presented as the most powerful determinant of CMS4-specific gene expression, tuning the majority of genes differentially expressed in the poor prognosis subtype, including genes associated with the epithelial-mesenchymal transition program. Furthermore, our data show that two epigenetic marks, namely the methylation of the two miR-200 promoter regions, can identify tumors belonging to the mesenchymal subtype and is predictive of disease-free survival in CRC patients. Importantly, epigenetic silencing of the miR-200 family is also detected in epithelial CRC cell lines that belong to the mesenchymal CMS. We thus show that determining regulatory networks is a powerful strategy to define drivers of distinct cancer subtypes, which possess the ability to identify subtype affiliation and to shed light on biological behavior.

Immunoglobulin diversification in B cell malignancies: internal splicing of heavy chain variable region as a by-product of somatic hypermutation.

In this study we describe alternative splicing of somatically mutated immunoglobulin (Ig) variable heavy chain (V(H)) genes in three distinct primary B cell non-Hodgkin's lymphomas (B-NHL). In two V4-34 expressing lymphomas, ie a post-germinal center type B cell chronic lymphocytic leukemia (B-CLL) and a follicular lymphoma (FL), internally spliced V(H) gene transcripts were found in which a sequence stretch of 116 bp between the framework region 1 (FR1) and complementarity determining region 2 (CDR2) had been deleted. We provide evidence that for this alternative IgV(H) mRNA processing a known cryptic 5' splice donor site and a previously unidentified cryptic 3' splice acceptor site were used. Site-directed mutagenesis showed that the cryptic 3' splice acceptor site had been activated by specific somatic point mutations. The B-CLL further harbored a triplication of the rearranged JH3 gene segment including the putative N region and part of the JH3-JH4 intron sequence. This triplication probably took place via a repeated mechanism of DNA double strand break followed by homologous recombination, a mechanism which was recently proposed also involved in the somatic hypermutation process and is compatible with the post-germinal center derivation of this B-CLL. Finally, in a V4-34 expressing diffuse large B cell lymphoma, we observed alternative IgV(H) mRNA processing using the same cryptic 5' splice donor site and the normal splice acceptor site of the CH1-C(mu) exon. The significance of alternative IgV(H) processing in B cell malignancies and as a potential mechanism of somatic Ig diversification is discussed.

Biased Iglambda expression in hypermutated IgD multiple myelomas does not result from receptor revision.

Normal IgM(-)IgD(+) CD38(+) B cells and IgM(-)IgD(+) multiple myelomas (MM) are characterized by Cmu deletion, biased Iglambda expression and hypermutated IgV regions. The predominant Iglambda usage has been proposed as resulting from secondary Ig gene rearrangements during extensive clonal expansion in the germinal center environment. Here, four cases of IgDlambda MM were studied to address the question of light chain receptor revision in a 'single cell' model. Detailed analyses of both IGK and IGL alleles of each case were performed by Southern blotting, (RT-) PCR, and sequencing. The expressed IgV genes were extensively mutated and Cmu deletion was confirmed in two cases. In addition, in the four MM a total of six non-functional deletional IGK rearrangements were identified, which proved to be unmutated. We conclude that IgD myelomas indeed originate from (post) germinal center B cells in which, in spite of the fact that they are hypermutated, there is no evidence of receptor revision.

Quality control in diagnostic molecular pathology in the Netherlands; proficiency testing for patient identification in tissue samples.

AIMS: To describe the evolution of proficiency testing for molecular diagnostic pathology with respect to determining unambiguously the patient identity of tissue samples by microsatellite analysis. METHOD: Four rounds of quality control exchanges of samples from different patients were sent with the purpose of identifying the correct origin of these samples. The samples were either paraffin wax embedded sections on glass, sections in tubes, or isolated DNA. Blinded samples were distributed to all participating laboratories. No restrictions to the method and short tandem repeat markers used for identification were imposed. RESULTS: In four subsequent rounds the number of participating laboratories increased from three to 10. The numbers of samples tested increased in time from five to 12. The microsatellite markers used by the different laboratories showed little overlap. In the first three rounds, in which isolated DNA was provided, all samples were accurately classified irrespective of the microsatellite markers used. In the last round, which also included paraffin wax embedded sections, a small number of laboratories experienced problems, either with amplification or incorrect classification of a few samples. CONCLUSION: Proficiency testing was useful, and showed country wide high quality and correct identification of (patient) samples with molecular techniques for diagnostic purposes.

[Mix-up of patient specimen: DNA-microsatellite analysis as a fast identification method]. / Verwisseling van patiëntenmateriaal: DNA-microsatellietanalyse als snelle identificatiemethode.

In a man aged 56 years with dysphagia, an oesophageal biopsy was found to contain an adenocarcinoma. In view of the discrepancy between the clinical and the histological pictures, the biopsy was repeated; in a second biopsy no carcinoma was demonstrable. DNA microsatellite analysis proved that the first biopsy originated from another person. The scheduled oesophageal resection was cancelled and the patient was reassured. A woman aged 77 years underwent gastrectomy because of biopsy samples with adenocarcinoma. However, no tumour was found in the resected stomach. DNA microsatellite analysis showed that the biopsy samples indeed originated from the patient. Unfortunately, mix-up of patient specimens occasionally occurs. Especially a discrepancy between the clinical picture and the histological diagnosis must raise the suspicion of a specimen mix-up. In such cases, DNA microsatellite analysis can give a rapid and reliable answer whether a mix-up has indeed taken place.

Acute sinusitis and blindness as the first presentation of chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is the most frequent form of leukaemia among adults in the Western world, presenting at a median age of 65 years. The diagnosis is usually made incidentally during routine blood examination while the disease is still in its early phase. We report a case of blindness of 24 hours due to acute sinusitis based on CLL localisation in a patient with undiagnosed CLL. Emergency endoscopic sinus surgery and intra- and extra-ocular orbital decompression were performed. The sinusitis resolved after surgery and intravenous antibiotics. Her vision improved within 24 hours and eventually recovered completely after six months. Her CLL remained in an indolent state, needing no active treatment. This case illustrates that blindness from a lymphoproliferative disorder may be treated with emergency endoscopic sinus surgery instead of conventional chemotherapy in order to salvage the vision first, even if the vision is lost for more than 24 hours.

Chlamydia psittaci-negative ocular adnexal marginal zone B-cell lymphomas have biased VH4-34 immunoglobulin gene expression and proliferate in a distinct inflammatory environment.

Ocular adnexal marginal zone B-cell lymphomas (OAMZLs) arise in the connective tissues of the orbit or in the mucosa-associated lymphoid tissue of the conjunctiva. Here, we present the immunological and genetic analyses of 20 primary Chlamydia psittaci (Cp)-negative OAMZLs. Analysis of the immunoglobulin variable heavy chain (IgV(H)) gene usage demonstrated a significant preference for V(H)4-34. A combined analysis across all previously published OAMZLs confirmed that this is a general feature of OAMZL, in particular of the Cp-negative group. Our series of OAMZLs did not express the characteristic rheumatoid factor V(H)DJ(H) rearrangements that were previously found in salivary gland- and gastric-marginal zone B-cell lymphomas (MZBCLs). We did not detect the MZBCL-specific chromosomal translocations, t(11;18) API2-MALT1 (mucosa-associated lymphoid tissue1) and t(14;18) IgH/MALT1. Two cases contained a premature stop codon in the A20 gene (TNFAIP3) and one case harbored the activating MYD88 hotspot mutation L265P. Variable nuclear expression of BCL10, NFκB1 (p50) and NFκB2 (p52) suggests that other additional genetic abnormalities affecting the NFκB pathway exist within this group of lymphomas. OAMZL showed variable expression of the chemokine receptor CXCR3 and integrin α4ß7 by the tumor B cells, and low interferon-γ and interlukin-4 mRNA levels in the tissue, indicative of an inflammatory environment with features in between those previously found in cutaneous and other extranodal MZBCL. The strongly biased usage of V(H)4-34 in Cp-negative OAMZLs suggests involvement of a particular stimulatory (auto-) antigen in their development.