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BackgroundConstitutive 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 β-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 β-catenin paradox.MethodsWe analysed the expression patterns of SMAD4, p53 and β-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.ResultsEighty-four percent of CRCs with high nuclear β-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.ConclusionsThe 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.

During the maturation of pre-B cells, the recombination activating gene 1 and 2 (RAG1/2) endonuclease complex plays a crucial role in coordinating V(D)J recombination by introducing DNA breaks in immunoglobulin (Ig) loci. Dysregulation of RAG1/2 has been linked to the onset of B cell malignancies, yet the mechanisms controlling RAG1/2 in pre-B cells exposed to excessive DNA damage are not fully understood. In this study, we show that DNA damage-induced activation of p53 initiates a negative-feedback loop which rapidly downregulates RAG1 levels. This feedback loop involves ataxia telangiectasia mutated activation, subsequent stabilization of p53, and modulation of microRNA-34a (miR-34a) levels, which is one of the p53 targets. Notably, this loop incorporates transcription factor forkhead box P1 as a downstream effector. The absence of p53 resulted in an increased proportion of IgM + cells prompted to upregulate RAG1/2 and to undergo Ig light chain recombination. Similar results were obtained in primary pre-B cells with depleted levels of miR-34a. We propose that in pre-B cells undergoing Ig gene recombination, the DNA breaks activate a p53/miR-34a/forkhead box P1-mediated negative-feedback loop that contributes to the rapid downregulation of RAG. This regulation limits the RAG-dependent DNA damage, thereby protecting the stability of the genome during V(D)J rearrangement in developing B cells., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In developing B cells, V(D)J gene recombination is initiated by the RAG1/2 endonuclease complex, introducing double-stranded DNA breaks (DSBs) in V, D, and J genes and resulting in the formation of the hypervariable parts of immunoglobulins (Ig). Persistent or aberrant RAG1/2 targeting is a potential threat to genome integrity. While RAG1 and RAG2 have been shown to bind various regions genome-wide, the in vivo off-target DNA damage instigated by RAG1/2 endonuclease remains less well understood. In the current study, we identified regions containing RAG1/2-induced DNA breaks in mouse pre-B cells on a genome-wide scale using a global DNA DSB detection strategy. We detected 1489 putative RAG1/2-dependent DSBs, most of which were located outside the Ig loci. DNA sequence motif analysis showed a specific enrichment of RAG1/2-induced DNA DSBs at GA- and CA-repeats and GC-rich motifs. These findings provide further insights into RAG1/2 off-target activity. The ability of RAG1/2 to introduce DSBs on the non-Ig loci during the endogenous V(D)J recombination emphasizes its genotoxic potential in developing lymphocytes., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

Juvenile xanthogranuloma (JXG) is a histiocytic neoplasm that usually presents in the skin. Rarely, extracutaneous localizations occur; the genetic drivers of this clinical variant of JXG remain incompletely characterized. We present detailed clinicopathologic and molecular data of 16 children with extracutaneous JXG and 5 adults with xanthogranulomas confined to the central nervous system (CNS) or soft tissue. Tissue samples were obtained through the Dutch Nationwide Pathology Databank and analyzed with an innovative sequencing technique capable of detecting both small genomic variants and gene rearrangements. Targetable kinase alterations were detected in 16/16 children and 1/5 adults. Alterations included CLTC::SYK fusions in 6 children and CSF1R mutations in 7 others - all below 2 years old with soft tissue tumors. One child had a CSF1R mutation and MRC1::PDGFRB fusion. Most were treated surgically, although spontaneous regression occurred in 1/6 with CLTC::SYK and 2/7 with CSF1R mutations - underscoring that treatment is not always necessary. Tumors with CLTC::SYK fusions generally lacked Touton giant cells, but exhibited many other histologic features of JXG and concordant methylation profiles. Using multispectral immunofluorescence, phosphorylated-SYK expression was localized to CD163+ histiocytes; tumors with CLTC::SYK fusions also demonstrated mTOR activation, Cyclin D1 expression, and variable phosphorylated-ERK expression. BRAFV600E was detected in 1 child and 1 adult with CNS xanthogranulomas; both responded to BRAF inhibition. Finally, a TPM3::NTRK1 fusion or MAP2K1 deletion were detected in 2 children with systemic JXG who experienced spontaneous disease regression. This study advances the molecular understanding of histiocytic neoplasms and may guide diagnostics and clinical management., (Copyright © 2024 American Society of Hematology.)

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., (© 2023 The Authors. Journal of the European Academy of Dermatology and Venereology published by John Wiley & Sons Ltd on behalf of European Academy of Dermatology and Venereology.)

Capillary malformations (CM) (port-wine stains) are congenital skin lesions that are characterized by dilated capillaries and postcapillary venules. CMs are caused by altered functioning of the vascular endothelium. Somatic genetic mutations have predominantly been identified in the endothelial cells of CMs, providing an opportunity for the development of targeted therapies. However, there is currently limited in-depth mechanistic insight into the pathophysiology and a lack of preclinical research approaches. In a monocenter exploratory study of 17 adult patients with CMs, we found somatic sequence variants in the GNAQ (p.R183Q, p.R183G, or p.Q209R) or GNA11 (p.R183C) genes. We applied an endothelial-selective cell isolation protocol to culture primary endothelial cells from skin biopsies from these patients. We successfully expanded patient-derived cells in culture in 3 of the 17 cases while maintaining endothelial specificity as demonstrated by vascular endothelial-cadherin immunostainings. In addition, we tested the angiogenic capacity of endothelial cells from a patient with a GNAQ (p.R183G) sequence substitution. These proof-of-principle results reveal that primary cells isolated from CMs may represent a functional research model to investigate the role of endothelial somatic mutations in the etiology of CMs, but improved isolation and culture methodologies are urgently needed to advance the field., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands)., Methods: Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18-21, and KRAS exon 2-3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance., Results: A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of >0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752_I759del) (69%), EGFR p.(N771_H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately., Conclusions: Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine., (© Association for Diagnostics & Laboratory Medicine 2024.)

Aims: The discovery of somatic genetic alterations established many histiocytic disorders as haematologic neoplasms. We aimed to investigate the demographic characteristics and additional haematologic cancers of patients diagnosed with histiocytic disorders in The Netherlands., Methods and Results: We retrieved data on histiocytosis patients from the Dutch Nationwide Pathology Databank (Palga). During 1993 to 2022, more than 4000 patients with a pathologist-assigned diagnosis of a histiocytic disorder were registered in Palga. Xanthogranulomas were the most common subtype, challenging the prevailing assumption that Langerhans cell histiocytosis (LCH) is the most common histiocytic disorder. LCH and juvenile xanthogranuloma (JXG) had a peak incidence in the first years of life; males were overrepresented among all histiocytosis subgroups. 118 patients had a histiocytic disorder and an additional haematologic malignancy, including 107 (91%) adults at the time of histiocytosis diagnosis. In 16/118 patients, both entities had been analysed for the same genetic alteration(s). In 11 of these 16 patients, identical genetic alterations had been detected in both haematologic neoplasms. This included two patients with PAX5 p.P80R mutated B cell acute lymphoblastic leukaemia and secondary histiocytic sarcoma, further supporting that PAX5 alterations may predispose (precursor) B cells to differentiate into the myeloid lineage. All 4/11 patients with myeloid neoplasms as their additional haematologic malignancy had shared N/KRAS mutations., Conclusions: This population-based study highlights the frequency of xanthogranulomas. Furthermore, our data add to the growing evidence supporting clonal relationships between histiocytic/dendritic cell neoplasms and additional myeloid or lymphoid malignancies. Particularly adult histiocytosis patients should be carefully evaluated for the development of these associated haematologic cancers., (© 2024 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Background: Distinguishing congenital pulmonary airway malformations (CPAMs) from pleuropulmonary blastoma (PPB) can be challenging. Previously diagnosed patients with CPAM may have been misdiagnosed and we may have missed DICER1-associated PPBs, a diagnosis with important clinical implications for patients and their families. To gain insight in potential misdiagnoses, we systematically assessed somatic DICER1 gene mutation status in an unselected, retrospective cohort of patients with a CPAM diagnosis., Methods: In the Amsterdam University Medical Center (the Netherlands), it has been standard policy to resect CPAM lesions. We included all consecutive cases of children (age 0-18 years) with a diagnosis of CPAM between 2007 and 2017 at this center. Clinical and radiographic features were reviewed, and DICER1 gene sequencing was performed on DNA retrieved from CPAM tissue samples., Results: Twenty-eight patients with a surgically removed CPAM were included. CPAM type 1 and type 2 were the most common subtypes (n = 12 and n = 13). For 21 patients a chest CT scan was available for reassessment by two pediatric radiologists. In 9 patients (9/21, 43%) the CPAM subtype scored by the radiologists did not correspond with the subtype given at pathology assessment. No pathogenic mutations and no copy number variations of the DICER1 gene were found in the DNA extracted from CPAM tissue (0/28)., Conclusions: Our findings suggest that the initial CPAM diagnoses were correct. These findings should be validated through larger studies to draw conclusions regarding whether systematic DICER1 genetic testing is required in children with a pathological confirmed diagnosis of CPAM or not., Level of Evidence: Level IV., Competing Interests: Conflicts of interest None., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In this retrospective study, BRAF mutation status did not correlate with disease extent or (event-free) survival in 156 adults with Langerhans cell histiocytosis. BRAFV600E was associated with an increased incidence of second malignancies, often comprising hematological cancers, which may be clonally related., (© 2023 by The American Society of Hematology.)

Purpose: Anal cancer is increasing in HIV+ men who have sex with men (MSM). Treatment options for its precursor, high-grade anal intraepithelial neoplasia (HGAIN), are suboptimal. In this phase I to II dose-finding study, we assessed the safety and efficacy of the human papillomavirus type 16 (HPV16) synthetic long peptide vaccine (SLP-HPV-01) in HIV+ MSM with HPV16-positive HGAIN., Patients and Methods: Four dosage schedules (1-5-10; 5-10-20; 10-20-40; and 40-40-40-40 μg) of SLP-HPV-01 were administered intradermally with a 3-week interval in 10 patients per dose level (DL). In each dose group, 5 patients also received 1 μg/kg pegylated IFNα-2b subcutaneously. Primary endpoints were safety and regression of HGAIN at 3, 6, and 12 months., Results: Eighty-one of 134 screened patients (60%) had HPV16-negative HGAIN lesions, leaving 53 eligible patients. Thirteen patients were excluded, leaving 40 men. The vaccine was well tolerated. One patient developed a generalized rash. The highest dosage level induced the strongest immune responses. There was no indication for stronger reactivity in the IFNα groups. Up to 18 months of follow-up, 8/38 intention-to-treat patients had a complete clinical and histologic response and one had a partial response (in total 9/38, 23.7%). At the highest dosage level, the clinical response was 4/10 (40%). Stronger immune responses were detected among clinical responders., Conclusions: The highest DL is safe, immunogenic, and associated with clinical responses to HPV16-induced lesions. However, as the majority of HGAIN is caused by the other HPV types, further studies should aim at pan-HPV vaccination to prevent or treat HGAIN., (©2023 American Association for Cancer Research.)

Introduction: Patients with serrated polyposis syndrome (SPS) have an increased risk to develop colorectal cancer (CRC). Due to an abundance of serrated polyps, these CRCs are assumed to arise mainly through the serrated neoplasia pathway rather than through the classical adenoma-carcinoma pathway. We aimed to evaluate the pathogenetic routes of CRCs in patients with SPS., Methods: We collected endoscopy and pathology data on CRCs and polyps of patients with SPS under treatment in our center. Our primary end point was the proportion of BRAFV600E mutated CRCs, indicating serrated pathway CRCs (sCRCs). CRCs lacking BRAFV600E most likely inferred a classical adenoma-carcinoma origin (aCRCs). We assessed patient, polyp, and CRC characteristics and stratified for BRAFV600E mutation status., Results: Thirty-five patients with SPS harbored a total of 43 CRCs. Twenty-one CRCs (48.8%) carried a BRAFV600E mutation, 10 of which lacked MLH1 staining and 17 (81%) were located in the proximal colon. Twenty-two CRCs (51.1%) did not carry a BRAFV600E mutation and were MLH1 proficient. Of these 22 putatively aCRCs, 17 (77.3%) were located distally and one-third (36.4%) harbored a pathogenic KRAS or NRAS mutation. In patients with BRAFwt -CRCs, a higher ratio of the median number of conventional adenomas versus serrated polyps was found (4 vs 13) than patients with BRAFV600E -CRCs (1 vs 14)., Discussion: Our study indicates that in patients with SPS, the ratio of sCRCs:aCRCs on average is 50:50. This elevated sCRC:aCRC ratio in patients with SPS, when compared with non-SPS patients, correlates well with the differences in the ratios of the numbers of sessile serrated lesions and conventional adenomas in patients with SPS and non-SPS patients, respectively., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Background: Serrated polyposis syndrome (SPS) is the most prevalent colonic polyposis syndrome and is associated with an increased colorectal cancer risk. A recent study in resected appendices of SPS patients reported that 6/23 (26.1 %) of identified serrated polyps had histological dysplasia. We evaluated the prevalence and clinical relevance of appendiceal lesions in a large SPS cohort., Methods: Prospective data from 2007 to 2020 for a cohort of 199 SPS patients were analyzed. Data were retrieved from endoscopy and pathology reports. Patients who underwent (pre)clearance colonoscopies, surveillance colonoscopies, or colorectal surgery including the appendix were separately evaluated for the presence of appendiceal lesions. The primary outcome was the prevalence of adenocarcinomas and serrated polyps/adenomas with advanced histology in the surgery group., Results: 171 patients were included, of whom 110 received endoscopic surveillance and 34 underwent surgery. Appendiceal lesion prevalence in the surgery group was 14 /34 (41.2 %, 95 %CI 24.7 %-59.3 %); none were advanced on histology. Detection rates in the (pre)clearance group were 1 /171 (0.6 %, 95 %CI 0.01 %-3.2 %) for advanced and 3 /171 (1.8 %, 95 %CI 0.04 %-5.0 %) for nonadvanced appendiceal lesions, all of which were sessile serrated lesions. During 522 patient-years of surveillance, no advanced appendiceal lesions were detected at endoscopy, and in 1 /110 patients (0.9 %, 95 %CI 0.02 %-5.0 %) was a nonadvanced lesion detected., Conclusion: Appendiceal lesions are common in SPS patients. The discrepancy between the endoscopic detection rate of appendiceal lesions and the reported prevalence in surgically resected appendices suggests a substantial miss-rate of appendiceal lesions during colonoscopy. Advanced appendiceal lesions are however rare and no appendiceal adenocarcinomas occurred, implying limited clinical relevance of these lesions., Competing Interests: B.A.J. Bastiaansen has received speakers’ fees from Olympus, Tillotts Pharma AG, and Ovesco Endoscopy AG. E. Dekker has received a research grant from Fujifilm, consultancy fees from Tillots, Olympus, Fujifilm, GI Supply, CPP-FAP, PAION, and Ambu, and speakers' fees from Olympus, GI Supply, Norgine, IPSEN, PAION, and Fujifilm.D.E.F.W.M. van Toledo, J.E.G. IJspeert, A.G.C. Bleijenberg, and C.J.M. van Noesel declare that have no conflicts of interest., (Thieme. All rights reserved.)

Metabolic alterations are important cancer-associated features that allow cancer cell transformation and survival under stress conditions. Multiple myeloma (MM) plasma cells show increased glycolysis and oxidative phosphorylation (OXPHOS), which are characteristics associated with recurrent genetic aberrations that drive the proliferation and survival of MM cells. The protein kinase B/AKT acts as a central node in cellular metabolism and is constitutively active in MM cells. Despite the known role of AKT in modulating cellular metabolism, little is known about the downstream factors of AKT that control the metabolic adaptability of MM cells. Here, we demonstrate that negative regulation of the forkhead box O (FOXO) transcription factors (TFs) by AKT is crucial to prevent the metabolic shutdown in MM cells, thus contributing to their metabolic adaptability. Our results demonstrate that the expression of several key metabolic genes involved in glycolysis, the tricarboxylic acid (TCA) cycle, and OXPHOS are repressed by FOXO TFs. Moreover, the FOXO-dependent repression of glycolysis- and TCA-associated genes correlates with a favorable prognosis in a large cohort of patients with MM. Our data suggest that repression of FOXO by AKT is essential to sustain glycolysis and the TCA cycle activity in MM cells and, as such, predicts patient survival., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is a protease and scaffold protein essential in propagating B-cell receptor (BCR) signaling to NF-κB. The deubiquitinating enzyme cylindromatosis (CYLD) is a recently discovered MALT1 target that can negatively regulate NF-κB activation. Here, we show that low expression of CYLD is associated with inferior prognosis of diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) patients, and that chronic BCR signaling propagates MALT1-mediated cleavage and, consequently, inactivation and rapid proteasomal degradation of CYLD. Ectopic overexpression of WT CYLD or a MALT1-cleavage resistant mutant of CYLD reduced phosphorylation of IκBα, repressed transcription of canonical NF-κB target genes and impaired growth of BCR-dependent lymphoma cell lines. Furthermore, silencing of CYLD expression rendered BCR-dependent lymphoma cell lines less sensitive to inhibition of NF-κΒ signaling and cell proliferation by BCR pathway inhibitors, e.g., the BTK inhibitor ibrutinib, indicating that these effects are partially mediated by CYLD. Taken together, our findings identify an important role for MALT1-mediated CYLD cleavage in BCR signaling, NF-κB activation and cell proliferation, which provides novel insights into the underlying molecular mechanisms and clinical potential of inhibitors of MALT1 and ubiquitination enzymes as promising therapeutics for DLBCL, MCL and potentially other B-cell malignancies., (© 2023. The Author(s).)

Background: Here we present updated survival of the CAIRO2 trial and assessed whether the addition of anti-EGFR to anti-VEGF therapy could still be an effective treatment option for patients with extended RAS/BRAF wildtype and left-sided metastatic colorectal cancer (mCRC)., Materials and Methods: Retrospective updated survival and extended RAS and BRAF V600E mutational analysis were performed in the CAIRO2 trial, a multicenter, randomized phase III trial on the effect of adding cetuximab to a combination of capecitabine, oxaliplatin (CAPOX), and bevacizumab in mCRC., Results: Updated survival analysis confirmed that the addition of cetuximab did not provide a benefit on either progression free (PFS) or overall survival (OS) in the intention-to-treat population. With the extended mutational analyses 31 KRAS, 31 NRAS and 12 BRAF V600E additional mutations were found. No benefit of the addition of cetuximab was observed within the extended wildtype group, even when selecting only left-sided tumors (PFS HR 0.96, p = 0.7775). However, compared to the original trial an increase of 6.5 months was seen for patients with both extended wildtype and left-sided tumors (median OS 28.6 months)., Conclusion: Adding cetuximab to CAPOX and bevacizumab does not provide clinical benefit in patients with mCRC, even in the extended wildtype group with left-sided tumors. However, in the extended wildtype group we did observe clinically relevant higher survival compared to the initial trial report, indicating that it is important to analyze a broader panel of RAS and BRAF variants using more recent sequencing techniques when assessing survival benefit after anti-EGFR therapy., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Langerhans cell histiocytosis (LCH) is a rare neoplastic disorder caused by somatic genetic alterations in hematopoietic precursor cells differentiating into CD1a+/CD207+ histiocytes. LCH clinical manifestation is highly heterogeneous. BRAF and MAP2K1 mutations account for ∼80% of genetic driver alterations in neoplastic LCH cells. However, their clinical associations remain incompletely understood. Here, we present an international clinicogenomic study of childhood LCH, investigating 377 patients genotyped for at least BRAFV600E. MAPK pathway gene alterations were detected in 300 (79.6%) patients, including 191 (50.7%) with BRAFV600E, 54 with MAP2K1 mutations, 39 with BRAF exon 12 mutations, 13 with rare BRAF alterations, and 3 with ARAF or KRAS mutations. Our results confirm that BRAFV600E associates with lower age at diagnosis and higher prevalence of multisystem LCH, high-risk disease, and skin involvement. Furthermore, BRAFV600E appeared to correlate with a higher prevalence of central nervous system (CNS)-risk bone lesions. In contrast, MAP2K1 mutations associated with a higher prevalence of single-system (SS)-bone LCH, and BRAF exon 12 deletions seemed to correlate with more lung involvement. Although BRAFV600E correlated with reduced event-free survival in the overall cohort, neither BRAF nor MAP2K1 mutations associated with event-free survival when patients were stratified by disease extent. Thus, the correlation of BRAFV600E with inferior clinical outcome is (primarily) driven by its association with disease extents known for high rates of progression or relapse, including multisystem LCH. These findings advance our understanding of factors underlying the remarkable clinical heterogeneity of LCH but also question the independent prognostic value of lesional BRAFV600E status., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Biallelic MSH3 germline variants are a rare cause of adenomatous polyposis as yet reported in two small families only. We describe the phenotype of a third family, the largest thus far, with adenomatous polyposis related to compound heterozygous MSH3 pathogenic variants. The index patient was a 55-years old male diagnosed with rectal cancer and adenomatous polyposis (cumulatively 52 polyps), with a family history of colorectal polyposis with unknown cause. Next-generation sequencing and copy number variation analysis of a panel of genes associated with colorectal cancer and polyposis revealed compound heterozygous germline pathogenic variants in the MSH3 gene. Nine out of 11 siblings were genotyped. Three siblings carried the same compound heterozygous MSH3 variants. Colonoscopy screening showed predominantly right-sided adenomatous polyposis in all compound heterozygous siblings, with a cumulative number of adenomas ranging from 18 to 54 in an average of four colonoscopies, and age at first adenoma detection ranging from 46 to 59. Microsatellite analysis demonstrated alterations at selected tetranucleotide repeats (EMAST) in DNA retrieved from the rectal adenocarcinoma, colorectal adenomas as well as of normal colonic mucosa. Gastro-duodenoscopy did not reveal adenomas in any of the four patients. Extra-intestinal findings included a ductal adenocarcinoma in ectopic breast tissue in one female sibling at the age of 46, and liver cysts in three affected siblings. None of the three heterozygous or wild type siblings who previously underwent colonoscopy had adenomatous polyposis. We conclude that biallelic variants in MSH3 are a rare cause of attenuated adenomatous polyposis with an onset in middle age., (© 2022. The Author(s).)

Background: Circulating tumor DNA (ctDNA) has predictive and prognostic value in localized and metastatic cancer. This study analyzed the prognostic value of baseline and on-treatment ctDNA in metastatic gastroesophageal cancer (mGEC) using a region-specific next generation sequencing (NGS) panel., Methods: Cell free DNA was isolated from plasma of patients before start of first-line palliative systemic treatment and after 9 and 18 weeks. Two NGS panels were designed comprising the most frequently mutated genes and targetable mutations in GEC. Tumor-derived mutations in matched metastatic biopsies were used to validate that the sequencing panels assessed true tumor-derived variants. Tumor volumes were calculated from baseline CT scans and correlated to variant allele frequency (VAF). Survival analyses were performed using univariable and multivariable Cox-regression analyses., Results: ctDNA was detected in pretreatment plasma in 75% of 72 patients and correlated well with mutations in metastatic biopsies (86% accordance). The VAF correlated with baseline tumor volume (Pearson's R 0.53, p < 0.0001). Detection of multiple gene mutations at baseline in plasma was associated with worse overall survival (OS, HR 2.16, 95% CI 1.10-4.28; p = 0.027) and progression free survival (PFS, HR 2.71, 95% CI 1.28-5.73; p = 0.009). OS and PFS were inferior in patients with residual detectable ctDNA after 9 weeks of treatment (OS: HR 4.95, 95% CI 1.53-16.04; p = 0.008; PFS: HR 4.08, 95% CI 1.31-12.75; p = 0.016)., Conclusion: Based on our NGS panel, the number of ctDNA mutations before start of first-line chemotherapy has prognostic value. Moreover, residual ctDNA after three cycles of systemic treatment is associated with inferior survival., (© 2022. The Author(s).)

A significant proportion of colorectal cancer (CRC) patients develop peritoneal metastases (PM) in the course of their disease. PMs are associated with a poor quality of life, significant morbidity and dismal disease outcome. To improve care for this patient group, a better understanding of the molecular characteristics of CRC-PM is required. Here we present a comprehensive molecular characterization of a cohort of 52 patients. This reveals that CRC-PM represent a distinct CRC molecular subtype, CMS4, but can be further divided in three separate categories, each presenting with unique features. We uncover that the CMS4-associated structural protein Moesin plays a key role in peritoneal dissemination. Finally, we define specific evolutionary features of CRC-PM which indicate that polyclonal metastatic seeding underlies these lesions. Together our results suggest that CRC-PM should be perceived as a distinct disease entity., (© 2022. The Author(s).)

Introduction: Anal cancer precursors, or high-grade anal intraepithelial neoplasia (HGAIN), are highly prevalent in HIV-seropositive (HIV+) men who have sex with men (MSM). Around 30% of lesions regress within 1 year, but current histopathological assessment is unable to distinguish between HGAIN likely to regress and HGAIN likely to persist or progress to cancer. We aim to assess if host cell DNA methylation markers can predict regression of HGAIN, thus determining the need for immediate treatment or active surveillance. This could reduce overtreatment and the associated anal and psycho-sexual morbidity., Methods and Analysis: This is an active surveillance cohort study in three centres located in Amsterdam, the Netherlands, in 200 HIV+ MSM diagnosed with HGAIN. Participants will not be treated, but closely monitored during 24 months of follow-up with 6 monthly visits including cytology, and high-resolution anoscopy with biopsies. The primary study endpoint is histopathological regression of each baseline HGAIN lesion at the end of the study. Regression is defined as ≤low grade anal intraepithelial neoplasia in the exit biopsy at 24 months. Regression proportions in lesions with low versus high methylation levels ( ASCL1 , ZNF582 ), other biomarkers (HPV genotype, HPV-E4, p16 INK4A , Ki-67) and immunological markers at baseline will be compared. Main secondary endpoints are the histological and clinical outcome (ie, the number of octants affected by HGAIN) of each baseline HGAIN lesion and overall HGAIN disease (i.e., all lesions combined) after each visit. The health-related quality of life of the study group will be compared with that of a control group of 50 HIV+ MSM receiving regular HGAIN treatment., Ethics and Dissemination: Ethics approval was obtained from the Institutional Review Board of the Academic Medical Center (Amsterdam, The Netherlands; reference no. 2021&#95;099). Participants are required to provide written informed consent. Findings will be disseminated through publication in peer-reviewed scientific journals and presentations at international scientific conferences; dissemination to policy makers and the target patient group will be achieved through our (inter-)national network, professional associations and collaboration with a patient representative organisation., Trial Registration Number: NL9664., Competing Interests: Competing interests: RDMS is minority stockholder of Self-screen, a spin-off company of VUmc, which owns patents on methylation markers and HPV detection. HJCdV received financial compensation or goods for research from Medigene, Gilead and MSD; financial compensation for presentations from Abbott and Janssen and financial compensation for advice to Medigene and Novartis. MFSvdL served on advisory boards of Merck. All other authors reported no potential conflicts., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples., Methods: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity., Results: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed., Conclusions: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies., (© American Association for Clinical Chemistry 2022.)

T-cell redirecting bispecific antibodies hold high promise for treatment of B-cell malignancies. B-cell maturation antigen (BCMA) exhibits high expression on normal and malignant mature B cells including plasma cells, which can be enhanced by inhibition of γ-secretase. BCMA is considered a validated target in multiple myeloma but whether mature B-cell lymphomas can be targeted by the BCMAxCD3 T-cell redirector teclistamab is currently unknown. BCMA expression on B-cell non-Hodgkin lymphoma and primary chronic lymphocytic leukemia (CLL) cells was assessed by flow cytometry and/or IHC. To assess teclistamab efficacy, cells were treated with teclistamab in presence of effector cells with/without γ-secretase inhibition. BCMA could be detected on all tested mature B-cell malignancy cell lines, while expression levels varied per tumor type. γ-secretase inhibition universally increased BCMA surface expression. These data were corroborated in primary samples from patients with Waldenstrom's macroglobulinemia, CLL, and diffuse large B-cell lymphoma. Functional studies with the B-cell lymphoma cell lines revealed teclistamab-mediated T-cell activation, proliferation, and cytotoxicity. This was independent of the level of BCMA expression, but generally lower in mature B-cell malignancies compared with multiple myeloma. Despite low BCMA levels, healthy donor T cells and CLL-derived T cells induced lysis of (autologous) CLL cells upon addition of teclistamab. These data show that BCMA is expressed on various B-cell malignancies and that lymphoma cell lines and primary CLL can be targeted using teclistamab. Further studies to understand the determinants of response to teclistamab are required to identify which other diseases might be suitable for teclistamab targeting., Significance: Besides reported BCMA expression on multiple myeloma, we demonstrate BCMA can be detected and enhanced using γ-secretase inhibition on cell lines and primary material of various B-cell malignancies. Furthermore, using CLL we demonstrate that low BCMA-expressing tumors can be targeted efficiently using the BCMAxCD3 DuoBody teclistamab., Competing Interests: N.W.C.J. van de Donk reports grants and other from Janssen Pharmaceuticals during the conduct of the study; grants and other from Amgen, Celgene/BMS, Novartis, Cellectis; other from Takeda, Roche, Bayer, Adaptive, and Servier outside the submitted work. H.C. Adams reports other from Janssen R&D outside the submitted work; in addition, H.C. Adams has a patent to PRD4087 issued. E. Eldering reports other from Janssen during the conduct of the study; grants from Janssen outside the submitted work. R. Verona reports other from Janssen R&D during the conduct of the study; other from Janssen R&D outside the submitted work; in addition, R. Verona has a patent to 63/194,470 pending. A.P. Kater reports grants from Janssen during the conduct of the study; grants and other from Abbvie, Genentech, AstraZeneca, BMS, and other from LAVA outside the submitted work; in addition, A.P. Kater has a patent to BCMA-bispecific for lymphoma pending. No other disclosures were reported., (© 2022 The Authors; Published by the American Association for Cancer Research.)

Gene-expression profiling (GEP) is used to study the molecular biology of lymphomas. Here, advancing insights from GEP studies in diffuse large B-cell lymphoma (DLBCL) lymphomagenesis are discussed. GEP studies elucidated subtypes based on cell-of-origin principles and profoundly changed the biological understanding of DLBCL with clinical relevance. Studies integrating GEP and next-generation DNA sequencing defined different molecular subtypes of DLBCL entities originating at specific anatomical localizations. With the emergence of high-throughput technologies, the tumor microenvironment (TME) has been recognized as a critical component in DLBCL pathogenesis. TME studies have characterized so-called "lymphoma microenvironments" and "ecotypes". Despite gained insights, unexplained chemo-refractoriness in DLBCL remains. To further elucidate the complex biology of DLBCL, we propose a novel targeted GEP consortium panel, called BLYM-777. This knowledge-based biology-driven panel includes probes for 777 genes, covering many aspects regarding B-cell lymphomagenesis (f.e., MYC signature, TME, immune surveillance and resistance to CAR T-cell therapy). Regarding lymphomagenesis, upcoming DLBCL studies need to incorporate genomic and transcriptomic approaches with proteomic methods and correlate these multi-omics data with patient characteristics of well-defined and homogeneous cohorts. This multilayered methodology potentially enhances diagnostic classification of DLBCL subtypes, prognostication, and the development of novel targeted therapeutic strategies.

Cancer therapy frequently fails due to the emergence of resistance. Many tumors include phenotypically immature tumor cells, which have been implicated in therapy resistance. Neuroblastoma cells can adopt a lineage-committed adrenergic (ADRN) or an immature mesenchymal (MES) state. They differ in epigenetic landscape and transcription factors, and MES cells are more resistant to chemotherapy. Here we analyzed the response of MES cells to targeted drugs. Activating anaplastic lymphoma kinase ( ALK ) mutations are frequently found in neuroblastoma and ALK inhibitors (ALKi) are in clinical trials. ALKi treatment of ADRN neuroblastoma cells with a tumor-driving ALK mutation induced cell death. Conversely, MES cells did not express either mutant or wild-type ALK and were resistant to ALKi, and MES cells formed tumors that progressed under ALKi therapy. In assessing the role of MES cells in relapse development, TRAIL was identified to specifically induce apoptosis in MES cells and to suppress MES tumor growth. Addition of TRAIL to ALKi treatment of neuroblastoma xenografts delayed relapses in a subset of the animals, suggesting a role for MES cells in relapse formation. While ADRN cells resembled normal embryonal neuroblasts, MES cells resembled immature precursor cells, which also lacked ALK expression. Resistance to targeted drugs can therefore be an intrinsic property of immature cancer cells based on their resemblance to developmental precursors. SIGNIFICANCE: In neuroblastoma, mesenchymal tumor cells lack expression of the tumor-driving ALK oncogene and are resistant to ALKi, but dual treatment with ALKi and mesenchymal cell-targeting TRAIL delays tumor relapse., (©2021 American Association for Cancer Research.)

ALK-positive histiocytosis is a rare subtype of histiocytic neoplasm first described in 2008 in 3 infants with multisystemic disease involving the liver and hematopoietic system. This entity has subsequently been documented in case reports and series to occupy a wider clinicopathologic spectrum with recurrent KIF5B-ALK fusions. The full clinicopathologic and molecular spectra of ALK-positive histiocytosis remain, however, poorly characterized. Here, we describe the largest study of ALK-positive histiocytosis to date, with detailed clinicopathologic data of 39 cases, including 37 cases with confirmed ALK rearrangements. The clinical spectrum comprised distinct clinical phenotypic groups: infants with multisystemic disease with liver and hematopoietic involvement, as originally described (Group 1A: 6/39), other patients with multisystemic disease (Group 1B: 10/39), and patients with single-system disease (Group 2: 23/39). Nineteen patients of the entire cohort (49%) had neurologic involvement (7 and 12 from Groups 1B and 2, respectively). Histology included classic xanthogranuloma features in almost one-third of cases, whereas the majority displayed a more densely cellular, monomorphic appearance without lipidized histiocytes but sometimes more spindled or epithelioid morphology. Neoplastic histiocytes were positive for macrophage markers and often conferred strong expression of phosphorylated extracellular signal-regulated kinase, confirming MAPK pathway activation. KIF5B-ALK fusions were detected in 27 patients, whereas CLTC-ALK, TPM3-ALK, TFG-ALK, EML4-ALK, and DCTN1-ALK fusions were identified in single cases. Robust and durable responses were observed in 11/11 patients treated with ALK inhibition, 10 with neurologic involvement. This study presents the existing clinicopathologic and molecular landscape of ALK-positive histiocytosis and provides guidance for the clinical management of this emerging histiocytic entity., (© 2022 by The American Society of Hematology.)

Anal cancer has increasing incidence and is preceded by high-grade anal intraepithelial neoplasia (HGAIN; AIN2-3). Previously, we identified and validated several methylation markers for accurate detection of anal cancer and HGAIN with cancer risk in HIV-positive (HIV+) men who have sex with men (MSM). This study aimed to evaluate these markers in HIV-negative risk groups. A cross-sectional series of 176 tissue samples of anal cancer, AIN3, AIN2, AIN1 and control biopsies obtained in HIV-negative women and men was tested for six methylation markers (ASCL1, LHX8, SST, WDR17, ZIC1 and ZNF582). Accuracy for detection of AIN3 and cancer (AIN3+) was determined by univariable and multivariable mixed-effect ordinal logistic regression. Methylation levels of all markers increased with increasing severity of disease (P < 0.0001) and were comparable to results in HIV+ MSM. All markers showed high accuracy for AIN3+ detection [area under the curve (AUC): 0.83-0.86]. The optimal marker panel (ASCL1 and ZIC1; AUC = 0.85 for AIN3+) detected 98% of cancers at 79% specificity. In conclusion, DNA methylation markers show a high diagnostic performance for AIN3+ detection in HIV+ and HIV-negative risk groups, justifying broad application of methylation analysis for anal cancer prevention programmes., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Objective: Anal cancer precursor lesions high-grade anal intraepithelial neoplasia (HGAIN) are highly prevalent among HIV+ MSM. Treatment of HGAIN is frustrated by high recurrence rates. We investigated the efficacy of the quadrivalent human papillomavirus (qHPV) vaccine as posttreatment adjuvant in preventing HGAIN recurrence in HIV+ MSM., Design: Randomized, double-blind, placebo-controlled, multicentre trial., Setting: Three HIV outpatient clinics in Amsterdam, the Netherlands., Subjects: HIV+ MSM with CD4+ cell count more than 350 cells/μl, biopsy-proven intra-anal HGAIN successfully treated in the past year, and lesions still in remission at enrolment, as assessed by high-resolution anoscopy (HRA)., Intervention: Participants were randomized to three doses of qHPV (Gardasil-4, MSD) or placebo with vaccinations at 0, 2, and 6 months. HRA was repeated at 6, 12, and 18 months., Main Outcome Measure: The primary outcome was cumulative, biopsy-proven HGAIN recurrence rate at 18 months, evaluated in an intention-to-treat (ITT) (received all vaccinations) and per-protocol analysis (all vaccinations and complete follow-up)., Results: We randomized 126 participants of which 64 (50.8%) received qHPV and 62 (49.2%) placebo. All participants received three vaccinations, and in both groups for two participants follow-up was incomplete. We found no difference (P = 0.38) in cumulative HGAIN recurrence rates between the qHPV (44/64, 68.8%) and placebo group (38/62, 61.3%) in the ITT analysis [absolute risk reduction -7.5 (95% confidence interval (CI) -24.1 to 9.2)]. This was similar in the per-protocol analysis., Conclusion: Despite adequate serological responses to qHPV vaccination, short-term recurrence of HGAIN was not prevented. These findings do not support qHPV vaccination as a treatment adjuvant to prevent HGAIN recurrence in HIV+ MSM., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: High-grade anal intraepithelial neoplasia (HGAIN; AIN2-3) is highly prevalent in HIV+ men, but only a minority of these lesions progress towards cancer. Currently, cancer progression risk cannot be established; therefore, no consensus exists on whether HGAIN should be treated. This study aimed to validate previously identified host cell DNA methylation markers for detection and cancer risk stratification of HGAIN., Methods: A large independent cross-sectional series of 345 anal cancer, AIN3, AIN2, AIN1, and normal control biopsies of HIV+ men was tested for DNA methylation of 6 genes using quantitative methylation-specific PCR. We determined accuracy for detection of AIN3 and cancer (AIN3+) by univariable and multivariable logistic regression analysis, followed by leave-one-out cross-validation. Methylation levels were assessed in a series of 10 anal cancer cases with preceding HGAIN at similar anatomic locations, and compared with the cross-sectional series., Results: Methylation levels of all genes increased with increasing severity of disease (P < .05). HGAIN revealed a heterogeneous methylation pattern, with a subset resembling cancer. ZNF582 showed highest accuracy (AUC = 0.88) for AIN3+ detection, slightly improved by addition of ASCL1 and SST (AUC = 0.89), forming a marker panel. In the longitudinal series, HGAIN preceding cancer displayed high methylation levels similar to cancers., Conclusions: We validated the accuracy of 5 methylation markers for the detection of anal (pre-) cancer. High methylation levels in HGAIN were associated with progression to cancer. These markers provide a promising tool to identify HGAIN in need of treatment, preventing overtreatment of HGAIN with a low cancer progression risk., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets multiple organs and causes severe coagulopathy. Histopathological organ changes might not only be attributable to a direct virus-induced effect, but also the immune response. The aims of this study were to assess the duration of viral presence, identify the extent of inflammatory response, and investigate the underlying cause of coagulopathy., Methods: This prospective autopsy cohort study was done at Amsterdam University Medical Centers (UMC), the Netherlands. With informed consent from relatives, full body autopsy was done on 21 patients with COVID-19 for whom autopsy was requested between March 9 and May 18, 2020. In addition to histopathological evaluation of organ damage, the presence of SARS-CoV-2 nucleocapsid protein and the composition of the immune infiltrate and thrombi were assessed, and all were linked to disease course., Findings: Our cohort (n=21) included 16 (76%) men, and median age was 68 years (range 41-78). Median disease course (time from onset of symptoms to death) was 22 days (range 5-44 days). In 11 patients tested for SARS-CoV-2 tropism, SARS-CoV-2 infected cells were present in multiple organs, most abundantly in the lungs, but presence in the lungs became sporadic with increased disease course. Other SARS-CoV-2-positive organs included the upper respiratory tract, heart, kidneys, and gastrointestinal tract. In histological analyses of organs (sampled from nine to 21 patients per organ), an extensive inflammatory response was present in the lungs, heart, liver, kidneys, and brain. In the brain, extensive inflammation was seen in the olfactory bulbs and medulla oblongata. Thrombi and neutrophilic plugs were present in the lungs, heart, kidneys, liver, spleen, and brain and were most frequently observed late in the disease course (15 patients with thrombi, median disease course 22 days [5-44]; ten patients with neutrophilic plugs, 21 days [5-44]). Neutrophilic plugs were observed in two forms: solely composed of neutrophils with neutrophil extracellular traps (NETs), or as aggregates of NETs and platelets.., Interpretation: In patients with lethal COVID-19, an extensive systemic inflammatory response was present, with a continued presence of neutrophils and NETs. However, SARS-CoV-2-infected cells were only sporadically present at late stages of COVID-19. This suggests a maladaptive immune response and substantiates the evidence for immunomodulation as a target in the treatment of severe COVID-19., Funding: Amsterdam UMC Corona Research Fund., (© 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license.)

The phosphatidylinositide-3 kinases and the downstream mediator AKT drive survival and proliferation of multiple myeloma (MM) cells. AKT signaling is active in MM and has pleiotropic effects; however, the key molecular aspects of AKT dependency in MM are not fully clear. Among the various downstream AKT targets are the Forkhead box O (FOXO) transcription factors (TFs) and glycogen synthase kinase 3 (GSK3), which are negatively regulated by AKT signaling. Here we show that abrogation of AKT signaling in MM cells provokes cell death and cell cycle arrest, which crucially depends on both FOXO TFs and GSK3. Based on gene expression profiling, we defined a FOXO-repressed gene set that has prognostic significance in a large cohort of patients with MM, indicating that AKT-mediated gene activation is associated with inferior overall survival. We further show that AKT signaling stabilizes the antiapoptotic myeloid cell leukemia 1 (MCL1) protein by inhibiting FOXO- and GSK3-mediated MCL1 turnover. In concordance, abrogation of AKT signaling greatly sensitized MM cells for an MCL1-targeting BH3-mimetic, which is currently in clinical development. Taken together, our results indicate that AKT activity is required to restrain the tumor-suppressive functions of FOXO and GSK3, thereby stabilizing the antiapoptotic protein MCL1 in MM. These novel insights into the role of AKT in MM pathogenesis and MCL1 regulation provide opportunities to improve targeted therapy for patients with MM., (© 2020 by The American Society of Hematology.)

Objective: Patients with Sjӧgren's syndrome (SS) have an increased risk of developing malignant B cell lymphomas, particularly mucosa-associated lymphoid tissue (MALT)-type lymphomas. We have previously shown that a predominant proportion of patients with SS-associated salivary gland MALT lymphoma express somatically hypermutated IgM with strong amino acid sequence homology with stereotypic rheumatoid factors (RFs). The present study was undertaken in a larger cohort of patients with SS-associated MALT lymphoma to more firmly assess the frequency of RF reactivity and the significance of somatic IGV-region mutations for RF reactivity., Methods: B cell antigen receptors (BCRs) of 16 patients with SS-associated salivary gland MALT lymphoma were analyzed. Soluble recombinant IgM was produced of 12 MALT lymphoma samples, including 1 MALT lymphoma sample that expressed an IgM antibody fitting in a novel IGHV3-30-encoded stereotypic IGHV subset. For 4 of the 12 IgM antibodies from MALT lymphoma samples, the somatically mutated IGHV and IGKV gene sequences were reverted to germline configurations. Their RF activity and binding affinity were determined by enzyme-linked immunosorbent assay and surface plasmon resonance, respectively., Results: Nine (75%) of the 12 IgM antibodies identified in patients with SS-associated salivary gland MALT lymphoma displayed strong monoreactive RF activity. Reversion of the IGHV and IGKV mutations to germline configuration resulted in RF affinities for IgG that were significantly lower for 3 of the 4 somatically mutated IgM antibodies. In stereotypic IGHV3-7/IGKV3-15-encoded RFs, a recurrent replacement mutation in the IGKV3-15-third complementarity-determining region was found to play a pivotal role in the affinity for IgG-Fc., Conclusion: A majority of patients with SS-associated salivary gland MALT lymphoma express somatically mutated BCRs that are selected for monoreactive, high-affinity binding of IgG-Fc. These data underscore the notion that soluble IgG, most likely in immune complexes in inflamed tissues, is the principal autoantigen in the pathogenesis of a variety of B cell lymphomas, particularly SS-associated MALT lymphomas., (© 2020 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

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.

Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder of hematopoietic origin characterized by inflammatory lesions containing clonal histiocytes (LCH-cells) intermixed with various immune cells, including T cells. In 50-60% of LCH-patients, the somatic BRAF V600E driver mutation, which is common in many cancers, is detected in these LCH-cells in an otherwise quiet genomic landscape. Non-synonymous mutations like BRAF V600E can be a source of neoantigens capable of eliciting effective antitumor CD8 + T cell responses. This requires neopeptides to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8 + T cells at tumor sites. Here, we demonstrate substantial heterogeneity in CD8 + T cell density in n = 101 LCH-lesions, with BRAF V600E mutated lesions displaying significantly lower CD8 + T cell:CD1a + LCH-cell ratios ( p = 0.01) than BRAF wildtype lesions. Because LCH-lesional CD8 + T cell density had no significant impact on event-free survival, we investigated whether the intracellularly expressed BRAF V600E protein is degraded into neopeptides that are naturally processed and presented by cell surface HLA class I molecules. Epitope prediction tools revealed a single HLA class I binding BRAF V600E derived neopeptide (KIGDFGLAT E K), which indeed displayed strong to intermediate binding capacity to HLA-A * 03:01 and HLA-A * 11:01 in an in vitro peptide-HLA binding assay. Mass spectrometry-based targeted peptidomics was used to investigate the presence of this neopeptide in HLA class I presented peptides isolated from several BRAF V600E expressing cell lines with various HLA genotypes. While the HLA-A * 02:01 binding BRAF wildtype peptide KIGDFGLATV was traced in peptides isolated from all five cell lines expressing this HLA subtype, KIGDFGLAT E K was not detected in the HLA class I peptidomes of two distinct BRAF V600E transduced cell lines with confirmed expression of HLA-A * 03:01 or HLA-A * 11:01. These data indicate that the in silico predicted HLA class I binding and proteasome-generated neopeptides derived from the BRAF V600E protein are not presented by HLA class I molecules. Given that the BRAF V600E mutation is highly prevalent in chemotherapy refractory LCH-patients who may qualify for immunotherapy, this study therefore questions the efficacy of immune checkpoint inhibitor therapy in LCH., (Copyright © 2020 Kemps, Zondag, Steenwijk, Andriessen, Borst, Vloemans, Roelen, Voortman, Verdijk, van Noesel, Cleven, Hawkins, Lang, de Ru, Janssen, Haasnoot, Franken, van Eijk, Solleveld-Westerink, van Wezel, Egeler, Beishuizen, van Laar, Abla, van den Bos, van Veelen and van Halteren.)

Background: Signal transducer and activator of transcription 3 hyper-IgE syndrome (STAT3-HIES) is caused by heterozygous mutations in the STAT3 gene and is associated with eczema, elevated serum IgE, and recurrent infections resembling severe atopic dermatitis, while clinically relevant specific IgE is almost absent., Methods: To investigate the impact of STAT3 signaling on B-cell responses, we assessed lymph node and bone marrow, blood B and plasma cell subsets, somatic hypermutations in Ig genes, and in vitro proliferation and antibody production in STAT3-HIES patients and healthy controls., Results: Lymph nodes of STAT3-HIES patients showed normal germinal center architecture and CD138 + plasma cells residing in the paracortex, which expressed IgE, IgG, and IgM but not IgA. IgE + plasma cells were abundantly present in STAT3-HIES bone marrow. Proliferation of naive B cells upon stimulation with CD40L and IL-4 was similar in patients and controls, while patient cells showed reduced responses to IL-21. IgE, IgG1, IgG3 and IgA1 transcripts showed reduced somatic hypermutations. Peripheral blood IgE + memory B-cell frequencies were increased in STAT3-HIES, while other memory B-cell frequencies except for IgG4 + cells were decreased., Conclusions: Despite impaired STAT3 signaling, STAT3-HIES patients can mount in vivo T-cell-dependent B-cell responses, while circulating memory B cells, except for those expressing IgG4 and IgE, were reduced. Reduced molecular maturation demonstrated the critical need of STAT3 signaling for optimal affinity maturation and B-cell differentiation, supporting the need for immunoglobulin substitution therapy and explaining the high IgE serum level in the majority with absent allergic symptoms., (© 2019 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)