Somatic Gene Mutations Research Articles

Abstract A018: Somatic mutations in the PIK3CA gene and its prognostic implications among Ethiopian Breast cancer patients

Abstract Purpose: The phosphatidylinositol-4, 5-bisphosphate 3-kinase (PIK3CA), catalytic subunit alpha (c) is frequently mutated in breast cancer (BC). Although the biological impact of PIK3CA mutations in BC has been studied in various parts of the world, limited data is available from sub-Saharan Africa. In this study, the frequency and the prognostic role of PIK3CA mutations were investigated among BC patients from Ethiopia. Objective: The aim of the study was to determine the frequency of PIK3CA mutations and its prognostic role in patients with breast cancer in Ethiopia. Methods: Formalin-fixed paraffin-embedded blocks and fresh frozen BC tissue samples were collected from patients newly diagnosed with BC. Histopathological evaluation was carried out using immunohistochemistry (IHC). PIK3CA mutation profiling was carried out using TaqMan® Mutation detection assay. The association between PIK3CA mutation status and clinical and histopathological features was evaluated. A survival regression (Cox’s model) was employed to assess the effect of PIK3CA mutation and other variables on survival. Results: The prevalence of the PIK3CA mutations was 20.4%. Mutation of the PIK3CA gene was mainly observed among younger (24%), early pT stage (19.4%), and G1/G2 tumors (22.3%). The proportion of mutation was 24.4%, 27.3% and 20% in ER+, PR+ and HER2- tumors, respectively. A marked difference was observed in the PIK3CA mutation frequency across the four major tumor subgroups. However, most of the mutated tumors were found among HR+/HER2− (24.1%) followed by HR+/HER2+ tumors (27.8%). In his study, only TNBC was negatively associated with the proportion of mutation in the PIK3CA gene (p-value= 0.02) in comparison to HR+/HER2- tumors. In multivariate Cox’s regression analysis, the advanced pT stage and HR−/HER2- IHC group independently predicted worse overall survival. However, PIK3CA mutation status was not associated with survival. Conclusions: The PIK3CA mutation was relatively high in HR+ tumors. Mutation rates of PIK3CA in Ethiopian BC tumors are similar to those reported for other populations. This finding highlighted that molecular aberrations including mutations in a specific could give insight on prognosis and clinical outcome. Citation Format: Zelalem Desalegn Woldesonbet. Somatic mutations in the PIK3CA gene and its prognostic implications among Ethiopian Breast cancer patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A018.

The “Culture” of Organs: A Holistic Theory on the Origins of the Cancer Tissue Environment

For over a century, the somatic gene mutation theory of cancer has been a scientific orthodoxy. The recent failures of causal explanations using this theory and the lack of significant progress in addressing the cancer problem medically have led to a new competition of ideas about just what cancer is. This essay presents an alternative view of cancer as a developmental process gone wrong. More specifically, cancer is a breakdown in the autopoietic process of organ maintenance and the multicellular coordination of tissues. Breast cancer is viewed through a systems science perspective as an example of the importance of framing one’s theoretical assumptions before making empirical judgments. Finally, a new understanding of the histoarchitecture of the interstitium is presented as a first principle of cancer: a process of cells coming from cells, invading the space between cells.

VCAM-1+ Mesenchymal Stem/Stromal Cells Reveal Preferable Efficacy Upon an Experimental Autoimmune Encephalomyelitis Mouse Model of Multiple Sclerosis Over the VCAM-1− Counterpart

Despite the considerable progress in mesenchymal stem/stromal cells (MSCs)-based novel intervention of multiple sclerosis (MS), yet the disease-modifying effect of VCAM-1− MSCs and novel VCAM-1+ counterpart is largely obscure. In this study, we took advantage of the EAE mouse model and VCAM-1+ human umbilical cord-derived MSCs (hUC-MSCs) for the evaluation of the therapeutic effect of systematic MSCs infusion. On the one hand, we compared the protective effect of VCAM-1− and VCAM-1+ hUC-MSCs against the clinical symptoms, demyelination, active glia cells and neuroinflammation in EAE mice by conducting multifaceted detections upon spinal cord and brain tissues. On the other hand, we conducted RNA-sequencing (RNA-SEQ) and multidimensional bioinformatics analyses for the evaluation of the transcriptomic features of spinal cord tissue in EAE mice after systematic hUC-MSCs infusion. Compared to those with VCAM-1− hUC-MSCs injection, VCAM-1+ mice showed further remission in clinical manifestations, and in particular, the inflammatory infiltration and active glial cells. Mice in all groups revealed conservations in overall gene expression profiling and somatic mutation spectrum. The differentially expressed genes (DEGs) between EAE mice and those with hUC-MSCs infusion were mainly involved in neuroinflammation and inflammatory response. Our findings indicated the feasibility of VCAM-1+ hUC-MSCs for multiple sclerosis treatment, which would supply new references for the development of novel VCAM-1+ MSCs-based cytotherapy in future.

Low-Frequency PPM1D Gene Mutations Associated with Inferior Treatment Response to CD19 Targeted CAR-T Cell Therapy in Mantle Cell Lymphoma

Background/Objectives: Mantle cell lymphoma (MCL) represents a rare B-cell lymphoma subtype with rather high relapse rates. Somatic mutations in the PPM1D gene were shown to be associated with adverse outcomes in patients with diffuse large B-cell lymphoma (DLBCL) who received CD19 CAR-T-cell therapy with tisa-cel, which may also apply to mantle cell lymphoma receiving brexu-cel CAR-T-cells. Methods: In this study, we determined the prevalence of PPM1D mutations in peripheral blood cells of MCL patients before CAR-T-cell infusion and analyzed the impact of low-frequency PPM1D mutations on efficacy and safety aspects of brexu-cel CAR-T-cell treatment in the first 16 r/r MCL patients enrolled at Inselspital Bern. Results: The prevalence of low-frequency PPM1D gene mutations was 25%, with variant allele frequencies (VAF) of 0.011 to 0.099. Clinical response was analyzed in the PPM1D mutated (PPM1Dmut) vs. PPM1D wild-type (PPM1Dwt) groups with median progression-free survival of 1 versus 32 months (p = 0.07) and median overall survival of 1.5 vs. 27 months (p = 0.001). Conclusions: Our data suggest that low-frequency PPM1D gene mutations in peripheral blood cells may predict inferior outcomes in patients with mantle cell lymphoma treated with CAR-T-cell therapy.

Current Therapeutic Opportunities for Estrogen Receptor Mutant Breast Cancer

Estrogen receptor α (ERα) drives two out of three breast cancers and therefore ERα is a major therapeutic target for ER-positive breast cancer patients. Drugs that inhibit ERα activity or block estrogen synthesis in the body are currently being used in the clinic to treat ER-positive breast cancer and have been quite successful in controlling breast cancer progression for the majority of patients. However, ER-positive breast cancer often becomes resistant to these endocrine therapies, leading to endocrine-resistant metastatic breast cancer, a very aggressive cancer that leads to death. Recent large-scale genomic studies have revealed a series of activating somatic mutations in the ERα gene (ESR1) in endocrine-resistant metastatic breast cancer patients. Of these, Y537S and D538G mutations are found at a much higher rate in patients with metastatic breast cancer. Remarkably, these mutations produce an ERα with much higher transcriptional activity than wild type in the absence of estradiol, and traditional endocrine therapy has poor efficacy against ER mutants. Therefore, the development of new drugs that target ER mutants is an unmet clinical need for endocrine-resistant metastatic breast cancer. This review summarizes the recent preclinical and clinical trials targeting estrogen receptor mutant breast cancer.

A four-hit mechanism is sufficient for meningioma development.

Meningiomas are central nervous system tumors whose incidence increases with age. Benign meningioma pathogenesis involves germline or somatic mutation of target genes, such as NF2, leading to clonal expansion. We used an established cancer epidemiology model to investigate the number of rate-limiting steps sufficient for benign meningioma development. Incidence data was obtained from the Surveillance, Epidemiology and End Results Program (SEER) for nonmalignant meningioma from 2004 to 2020. Age-adjusted incidence rates per 100,000 person-years were divided into 5-year bands. This was repeated for vestibular schwannomas as a negative control. The Armitage-Doll methodology was applied. Mathematical solutions correcting for volatile tumor microenvironments were applied to fit higher-order models using polynomial regression when appropriate. A 75:25 training:test split was utilized for validation. 222,509 cases of benign meningiomas were identified. We noted strong linear relationships between log-transformed incidence and age across the cohort and multiple subpopulations: male, white, black, Hispanic, Asian/Pacific Islander, and American Indian subpopulations all demonstrated R2 = 0.99. Slopes were between 3.1 and 3.4, suggesting a four-step process for benign meningioma development. Female patients exhibited nonlinear deviations, but the corrected model demonstrated R2 = 0.99 with a four-hit pathway. This model performed robustly on test data with R2 = 0.99. Vestibular schwannomas demonstrated a slope of 2.1 with R2 = 0.99, suggesting a separate three-step process. Four mutations are uniquely required for the development of benign meningiomas. Correcting for volatile tumor microenvironments reliably accounted for nonlinear deviations in behavior. Further studies are warranted to elucidate genomic findings suggestive of key mutations in this pathway.

VEXAS Syndrome: A Comprehensive Review of Current Therapeutic Strategies and Emerging Treatments.

VEXAS syndrome is a recently identified autoinflammatory disorder resulting from somatic mutations in the UBA1 gene, leading to a complex spectrum of severe inflammatory and hematologic manifestations. The absence of established treatment guidelines and the variability in clinical presentation make its management particularly challenging. Current therapeutic approaches are often based on limited evidence, and their effectiveness remains inconsistent. This review seeks to consolidate the existing knowledge on therapeutic strategies for VEXAS syndrome, offering a critical evaluation of their efficacy and addressing the gaps in the current literature. As the clinical recognition of VEXAS grows, there is an urgent need to explore more targeted, effective treatments that can address both the inflammatory and hematologic aspects of the disease. By providing a comprehensive analysis of the current therapeutic landscape, this review aims to guide clinicians and researchers toward developing more effective, long-term management strategies for this life-threatening condition.

Personalized neoantigen-based therapeutic cancer vaccines

Abstract. Tumor neoantigens are new unknown proteins or peptides encoded by somatic gene mutations, also known as tumor-specific antigens (TSAs). They are located on the surface of tumor cells and are only expressed in tumor cells. Cancer vaccines targeting neoantigens can not only effectively induce immune response, but also have the advantages of high safety and fewer side effects, so it has gradually become a research hotspot in tumor therapy. During the current clinical trials, neoantigen-based personalised tumour therapeutic vaccines have shown excellent experimental results and have also proved to be positively helpful for tumour cancer treatment. However, considering that the heterogeneity of tumours increases the uncertainty of the efficacy of disease vaccines, and the time-consuming and complicated preparation process of personalized vaccines, the research and development of personalized oncology therapeutic vaccines based on neoantigens still need different explorations and attempts.

Methodology and clinical utility of longitudinal UBA1 tracking in VEXAS syndrome.

Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) is a haemato-inflammatory syndrome genetically defined by somatic mutations in the X-linked UBA1 gene, typically Val/Thr/Leu substitutions at the Met41 hotspot. Clinical manifestations are heterogeneous and refractory to most haemato-rheumatological treatments. To date, no guidelines exist for the management of VEXAS, and scarce is the evidence on methodology and clinical significance of longitudinal UBA1 clonal burden evaluation upon therapy. Here, we validated a method to quantify UBA1 clonal burden and explored its applicability in patients with VEXAS. Given the different treatment interactions, droplet digital polymerase chain reaction (ddPCR) may allow for informed therapeutic decisions and implementation of personalized strategies.

PATH-61. GENOMIC SIGNATURE IN NON-SMALL CELL LUNG CANCER PATIENTS METASTATIC TO BRAIN-A SINGLE INSTITUTION EXPERIENCE

Abstract BACKGROUND Lung cancer is leading cause of cancer-related death in US, with 10-30 % of NSCLC patients developing brain metastasis, which challenges curative treatment and life expectancy. Somatic mutations in KRAS and EGFR genes have been commonly implicated with brain metastasis. Other studies have shown higher amplification frequencies in MYC, YAP1, and MMP13 and deletions in CDKN2A/B. METHODS We conducted a retrospective study on observational cohort of NSCLC patients from January 2022 to July 2023, using IRB-approved data in patients with confirmed brain metastasis (CBS). Beyond demographic information, data on genomic signatures was obtained from maximum parallel/next-generation sequencing on tumor biopsy samples. Comparative analysis was conducted to predict commonalities in brain metastasis gene signatures. RESULTS The study involved 143 newly diagnosed lung cancer patients, with 29 having CBS. Genomic data was available in 24 patients. Most common abnormality was noted in TP53 gene in about 47% of patients, followed by LRP1B (12%). Mutations in KRAS, EGFR, NF1, MET, SMARCA4, KEAP1, and STK11 genes were seen in two patients each. Interestingly, 3 of 4 patients with LRP1B had brain metastasis. Actionable mutations/rearrangements were significantly lower compared to general prevalence of these mutations (KRAS gene 8% and EGFR gene 5%). CONCLUSIONS Lung cancer with brain metastasis has an abysmal prognosis due to poor CNS penetration of chemotherapeutic agents. Surgical resection and RT remain cornerstones of treatment, but certain TKI has shown some potential. Our study uncovered a notably higher prevalence of TP53 mutation among patients with brain metastasis. While patient cohort was small, those harboring an LRP1B mutation exhibited significantly higher incidence of brain metastasis. Contrary to prevalent epidemiological data, our patient population demonstrated a lower frequency of targetable mutations (specifically in EGFR and KRAS genes). We hypothesize that this observation is due to a markedly higher smoking prevalence in our region.

P04 A vexing case of polymyalgia rheumatica

Abstract Introduction VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome is a recently described autoinflammatory condition characterised by somatic mutations in the UBA1 gene, resulting in significant morbidity. Disease presentation can mimic several rheumatological conditions, delaying diagnosis and appropriate management. This case report presents the clinical course of a patient with a complex history of musculoskeletal symptoms and systemic inflammation, first diagnosed with polymyalgia rheumatica (PMR), who was later identified as having VEXAS syndrome almost six years after first referral to the rheumatology clinic. It illustrates the importance of considering rare autoinflammatory conditions in patients with atypical presentations of common rheumatological disorders. Case description A 76-year-old gentleman was referred from the haematology clinic where he had undergone investigation for pancytopenia and raised inflammatory markers. Initial investigations under the haematology team revealed normal CT chest, abdomen and pelvis, unremarkable bone marrow biopsy and cytogenetics. He presented with muscle stiffness and proximal limb girdle pain. Additional symptoms included fatigue and a diffuse non-specific erythematous rash. There was no significant past medical history and he was previously very fit and active. Rheumatology investigations were normal, apart from persistently elevated inflammatory markers (ESR 86 mm/h and CRP 70 mg/L). A diagnosis of polymyalgia rheumatica was made and prednisolone 15 mg was started. Although he initially had a good response to this, he continued to experience significant inflammatory and constitutional symptoms. These included small joint polyarthritis, night sweats and weight loss. He was pancytopenic with associated reduced exercise tolerance, requiring transfusions and erythropoietin. To exclude other differentials, a repeat bone marrow biopsy was performed. This revealed dysplasia likely secondary to underlying inflammation, although no clonal abnormalities or vacuoles were detected. MRI right hand showed synovitis, and he was therefore treated for myalgic-onset rheumatoid arthritis. Prednisolone dose was increased temporarily and methotrexate was added, resulting in temporary improvement of his ESR 16 mm/h and CRP 25 mg/L. The patient continued to have ongoing constitutional symptoms and developed new ataxia. Further investigations, including MRI head, gastroscopy and Whipple’s PCR were all normal. Repeat CT chest, abdomen and pelvis and PET-CT were unremarkable. His symptoms and inflammatory markers were responsive to steroids. Given his symptoms and macrocytosis, he was tested for ubiquitin-activating enzyme UBA1 gene which was positive, confirming the diagnosis of VEXAS syndrome. Methotrexate was stopped due to worsening cytopenia, prednisolone continued and tocilizumab started. He showed significant improvement in pancytopenia and symptoms following initiation of tocilizumab. Discussion VEXAS is characterised by somatic mutations in the UBA1 gene; its pathogenesis leads to malfunctioning methionine-41, an enzyme that initiates ubiquitylation. This leads to increased activation of innate immune pathways and induces systemic inflammation, manifesting in a variable clinical presentation. This variability makes the diagnosis challenging and leads to a median diagnostic delay of 5.5 years, similar to our patient’s six-year delay. Our patient was initially managed as PMR due to his clinical presentation and lack of an obvious alternative diagnosis. This misdiagnosis is not unique. Often, patients later diagnosed with VEXAS are first managed as either inflammatory conditions, notably polychondritis, vasculitis or Sweet’s syndrome, or myelodysplastic syndrome (MDS). VEXAS is hallmarked by haematological dysfunction as much as it is by inflammatory features. In fact, our patient’s first signs included a mild cytopenia which later developed into a progressive macrocytic anaemia. This is classical of VEXAS. Other haematological manifestations include thrombotic events and MDS, with minimal response to EPO-stimulating agents as seen here. His ongoing deterioration triggered an alternative diagnosis to be sought. VEXAS is unusual as an autoinflammatory disease: its causative mechanism is a somatic mutation acquired later in life. Its diagnosis can be confirmed by inactivation of the x-linked UBA1 gene. This case highlights the need for UBA1 testing when dealing with an unexplained inflammatory phenotype, especially in men with cytopenia, multiorgan autoinflammation or MDS-like features. VEXAS presents a management challenge. The term ‘haeamatoinflammatory’ has been created to reflect its unique nature, warranting input from both specialist teams as well as the wider MDT. Key learning points • VEXAS should be considered as a differential when dealing with unexplained inflammatory symptoms, especially in men, multiorgan autoinflammation, vasculitis and/or MDS-like features. • VEXAS is often progressive and transfusion-dependence is associated with a poorer prognosis. A macrocytic anaemia is a prominent feature of the condition. • The absence or low proportion of vacuoles in bone marrow should not prevent us from testing for VEXAS if clinically indicated. • No established protocols for VEXAS yet exist. Haematological management is supportive. Anti-inflammatory treatment is based on collective observed evidence, including the use of tocilizumbab (anti-IL6) and janus kinase (JAK) inhibitors. Allogenic haemopoietic stem cell transplantation has been successful in few patients and may represent an option for patients with severe disease.

Analysis of Molecular Testing for Suspected Myeloproliferative Neoplasm at a Hybrid Community-Academic Health System

Testing for somatic mutations in JAK2, MPL, and CALR genes is a crucial element in the diagnosis of myeloproliferative neoplasms (MPN). This may have inadvertently led to increased requests for testing to “rule out MPN,” including clinical situations with low pre-test probability. This paper examines JAK2, MPL, and CALR testing by next generation sequencing (NGS) with the goal of formulating practical guidelines to make test utilization more efficient and effective. NGS results from 1482 patients tested between 2015 and March 2022 were retrieved, along with corresponding bone marrow biopsies and CBC results performed within 90 days prior to NGS and 245 (16.5%) cases were positive for pathogenic variants in JAK2, MPL, or CALR genes.The findings showed an increase in the proportion of positive cases with patient age, and a statistically significant difference in RBC counts and platelet counts among patients with positive versus negative results. Utilizing these factors, simple algorithms were constructed to predict positive results with a maximum sensitivity of 91%, while potentially eliminating 28% of negative tests. However, these models still failed to identify ∼9% of patients with MPNs. Among these “missed” patients, many had either primary myelofibrosis or MDS/MPN. Considering a simple triage model to help guide MPN testing could represent a more cost effective approach, particularly if “missed” patients could be further reduced.

Deciphering the toxicity of polyhexamethylene guanidine phosphate in lung carcinogenesis: Mutational profiles and molecular mechanisms

Polyhexamethylene guanidine (PHMG) is widely utilized in personal hygiene products due to its bactericidal, non-volatile, and hydrophilic properties. However, the long-term toxic effects and underlying mechanisms associated with respiratory exposure to the commonly used form, PHMG phosphate (PHMG-p), are still insufficiently understood. This study aims to elucidate the types of pulmonary lesions and the incidence of lung cancer associated with varying concentrations of PHMG-p and observation periods, along with the molecular mechanisms underlying this relationship. To assess these effects, CT scans and pathological analyses were conducted for up to 54 weeks following initial exposure to PHMG-p. Furthermore, to investigate the underlying causes of pulmonary toxicity, TGF-beta-activated kinase 1 was identified as a PHMG-p-binding protein, and its associated signaling pathways, including necroptosis, apoptosis, and MKK7, were explored. Somatic mutational signature, and gene ontology (GO) analyses were performed to investigate the genetic characteristics of PHMG-p-induced lung carcinogenesis. PHMG-p exposure led to somatic mutations in lung cancer-related genes, including TP53, SOS1, KMT2D, MDM2, ERBB2, SETD2, MET, ARID1A, RBM10, and CDKN2A as well as in genes such as RAB31, WASHC1, DDX11, ECD, STAB2, MUC2, and MUC5AC. The mutated genes were primarily associated with impaired DNA repair mechanisms. GO analysis highlighted the activation of pathways related to cell cycle checkpoints, necroptosis, MAPK, and idiopathic pulmonary fibrosis, while also revealing the suppression of signaling pathways associated with natural killer cells, GADD45, LXR/RXR activation, and IL-15 production. Gain-of-function experiments confirmed the oncogenic roles of PLAU and HMGA2, as well as the tumor-suppressive functions of TBX4 and GPX3. These findings suggest that PHMG-p activates necroptosis and MAPK signaling, increases the frequency of somatic mutations, and inhibits apoptosis, thus fostering an environment conducive to carcinogenesis. This underscores the importance of understanding the potential health risks associated with PHMG-p exposure and provides insights for future research and regulatory considerations regarding the safety of personal hygiene products.

Investigation of the Relationship Between DNA Mismatch Repair Genes and Microsatellite Instability in Solid Tumors

Aim: In this study, we aimed to detect the MSI status and somatic mutations in MMR genes (MSH2, MSH6, MLH1, PMS2) of a total of 55 solid tumors diagnosed with colorectal, endometrium and ovarian cancer by NGS method and to reveal the relationship between them. Material and Method: DNA isolation was performed by taking 10-micron sections from paraffin-embedded tissue samples of 55 patients diagnosed with kolorektal, endometrium ve ovarian solid tümörlerinin and Kapa NGS DNA extraction kit was used for sequence analysis. The purity and concentration of the DNA obtained was measured by Qubit fluoremeter, and NadPrep DNA Universal Library Preparation Kit was used for high quality library preparation. Bioinformatics analyses were performed on the Genomize Seq platform. The MSI value was analysed by Roche Navify Mutation Caller software and percentage MSI values were determined using the MSIsensor2 pipeline for secondary analysis of NGS. Results: All ovarian tumors were in the MSI-Stable category. The average MSI value was 2.01. One sample had an MSI of zero. In addition, no mutations in the MSH2 and MLH1 genes were detected in any of the ovarian tumors. 3 of 4 endometrial tumors were in the MSI-Stable category, and 1 tumor was in the MSI-low (MSI value: 13.2) category. No variants were detected in the MSH2 and PMS2 genes in endometrial tumors. 2 of the 41 colorectal tumors (Case4, Case16) were in the MSI-High category. No variants were detected in the MMR genes in 19 tumors. Conclusion: Although frame-shift and stop-gain mutations were detected in 23 tumors that would cause protein deficiency in MMR genes, MSI-H was not detected, as expected, except for two colorectal tumors. Therefore, the results of our study emphasise the need to define new predictive biomarkers clinically within the framework of algorithms to predict response to immunotherapy and determine prognosis.

VEXAS syndrome: an adult-onset autoinflammatory disorder with underlying somatic mutation.

VEXAS syndrome (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) was first described in 2020, where in a cohort of adults with unexplained fever or inflammation, systematic genetic testing was performed and 25 men with a median age of 64 years and somatic mutations in the UBA1 gene were identified. In the current review, we aim to discuss the relevant literature from January 2023 until July 2024 to give new insights into the pathophysiology, epidemiology, diagnosis and treatment of VEXAS. VEXAS affects 1 : 4269 in men over the age of 50. Janus-Kinase-inhibitors (JAKi) and IL-6-inhibitors are more effective immunosuppressants against hyperinflammation. Ruxolitinib is more effective than other JAKi. Azacitidine induces remission in many patients, but only few MDS-associated patients were treated. Allogeneic stem cell transplantation is feasible for selected cases. Infections are the major cause of death. Prognosis is still poor with a 5-year mortality rate of 18-40%. In the current review, we discuss the novelties for VEXAS, including pathogenic pathways, epidemiological data, diagnostic criteria and algorithms, treatment options and complications. We hope that this review may improve rheumatologists understanding of VEXAS. We strongly recommend enrolling VEXAS patients in registries and clinical trials, to improve prognosis of VEXAS in the future.

Laser-Based Microdissection of Single Cells from Tissue Sections and PCR Analysis of Rearranged Immunoglobulin Genes from Isolated Normal and Malignant Human B Cells.

Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the DNA of the isolated cells, rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of IGV gene subgroup-specific primers recognizing nearly all IGV genes together with primers for the J genes. By sequence analysis of IGV region genes from distinct cells, the clonal relationship of the B-lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D and J gene usage. Moreover, the presence and pattern of somatic IGV gene mutations give valuable insight into the differentiation stage of the B cells.

Pheochromocytoma-Paraganglioma Syndrome: A Multiform Disease with Different Genotype and Phenotype Features.

Pheochromocytoma and paraganglioma (PPGL) are rare tumors derived from the adrenal medulla and extra-adrenal chromaffin cells. Diagnosis is often challenging due to the great variability in clinical presentation; the complexity of management due to the dangerous effects of catecholamine excess and the potentially malignant behavior require in-depth knowledge of the pathology and multidisciplinary management. Nowadays, diagnostic ability has certainly improved and guidelines and consensus documents for treatment and follow-up are available. A major impulse to the development of this knowledge has come from the new findings on the genetic and molecular characteristics of PPGLs. Germline mutation in susceptibility genes is detected in 40% of subjects, with a mutation frequency of 10-12% also in patients with sporadic presentation and genetic testing should be incorporated within clinical care. PPGL susceptibility genes include "old genes" associated with Neurofibromatosis type 1 (NF1 gene), Von Hippel Lindau syndrome (VHL gene) and Multiple Endocrine Neoplasia type 2 syndrome (RET gene), the family of SDHx genes (SDHA, SDHB, SDHC, SDHD, SDHAF2), and genes less frequently involved such as TMEM, MAX, and FH. Each gene has a different risk of relapse, malignancy, and other organ involvement; for mutation carriers, affected or asymptomatic, it is possible to define a tailored long-life surveillance program according to the gene involved. In addition, molecular characterization of the tumor has allowed the identification of somatic mutations in other driver genes, bringing to 70% the PPGLs for which we know the mechanisms of tumorigenesis. This has expanded the catalog of tumor driver genes, which are identifiable in up to 70% of patients Integrated genomic and transcriptomic data over the last 10 years have revealed three distinct major molecular signatures, triggered by pathogenic variants in susceptibility genes and characterized by the activation of a specific oncogenic signaling: the pseudo hypoxic, the kinase, and the Wnt signaling pathways. These molecular clusters show a different biochemical phenotype and clinical behavior; they may also represent the prerequisite for implementing customized therapy and follow-up.

Two-hit model for the development of aldosterone-producing adenoma: supporting from two new cases.

Recently, a two-hit model for the development of aldosterone-producing adenoma (APA) was proposed but until now, only two cases supporting the model have been reported. Here, we present two new cases of primary aldosteronism (PA), both of which had large functional adenomas with somatic mutations in aldosterone-driving genes. Furthermore, the first patient, who had a history of colorectal cancer, was found to have a germline and an additional somatic mutation in APC, and APC inactivation was confirmed by immunohistochemistry. The other patient had pathogenic somatic mutation inCTNNB1. These pro-proliferation mutations resulted in abnormal activation of the Wnt/β-catenin pathway. Two consecutive events apparent in these patients, namely, the first event leading to cell proliferation and the second driving hormonal hypersecretion, supported the two-hit model of APA development. The two-hit model usually occurs in the larger adenomas, and the driving factors of the first hit that promote cell proliferation still require further research and exploration.

Somatic mutations in autoinflammatory and autoimmune disease.

Somatic mutations (also known as acquired mutations) are emerging as common, age-related processes that occur in all cells throughout the body. Somatic mutations are canonically linked to malignant processes but over the past decade have been increasingly causally connected to benign diseases including rheumatic conditions. Here we outline the contribution of somatic mutations to complex and monogenic immunological diseases with a detailed review of unique aspects associated with such causes. Somatic mutations can cause early- or late-onset rheumatic monogenic diseases but also contribute to the pathogenesis of complex inflammatory and immune-mediated diseases, affect disease progression and define new clinical subtypes. Although even variants with a low variant allele fraction can be pathogenic, clonal dynamics could lead to changes over time in the proportion of mutant cells, with possible phenotypic consequences for the individual. Thus, somatic mutagenesis and clonal expansion have relevant implications in genetic testing and counselling. On the basis of both increased recognition of somatic diseases in clinical practice and improved technical and bioinformatic processes, we hypothesize that there will be an ever-expanding list of somatic mutations in various genes leading to inflammatory conditions, particularly in late-onset disease.

Low-level mosaic GCK mutations in children with diazoxide-unresponsive congenital hyperinsulinism.

Some children with diazoxide-unresponsive congenital hyperinsulinism (HI) lack any detectable disease-causing mutation in peripheral blood DNA. To examine whether somatic post-zygotic mutations of known HI genes are responsible for disease in children with diazoxide-unresponsive HI requiring surgery with histology not classified as focal or Localized Islet Nuclear Enlargement (LINE), and without detectable mutations by standard genetic testing of peripheral blood DNA. Next-generation sequencing (NGS) was performed on specimens of pancreas from 10 children with diazoxide-unresponsive HI. Four unique GCK mutations were identified at low levels of mosaicism ranging from 4.4-10.1% in pancreatic DNA from five of these 10 children. The GCK mutations were not detectable in peripheral blood DNA by NGS in three cases from which peripheral blood DNA was available for testing. All four GCK mutations have been previously published as activating HI mutations. The histology was consistent with diffuse-HI in four of the five cases with mosaic GCK mutations. In one of these, hypomethylation of IC2 on chromosome 11p was identified in pancreatic and peripheral blood DNA. Histology of the fifth case revealed minor islet abnormalities suggestive of Beckwith Wiedemann Spectrum (BWSp) although molecular analysis for 11pUPD was negative in pancreas. These results indicate that post-zygotic somatic GCK mutations are responsible for some cases of non-focal diazoxide-unresponsive hyperinsulinism.