Background
Standard care for patients with high-risk myelodysplastic syndrome (MDS) is hypomethylating agents such as azacitidine (AZA), which can induce expression of methylated tumor-associated ...antigens and therefore potentiate immunotherapeutic targeting.
Method
In this phase 1 trial, we combined AZA with a therapeutic peptide vaccine targeting antigens encoded from
NY-ESO-1, MAGE-A3, PRAME,
and
WT-1
, which have previously been demonstrated to be upregulated by AZA treatment.
Result
Five patients who had responded to AZA monotherapy were included in the study and treated with the vaccine. The combination therapy showed only few adverse events during the study period, whereof none classified as serious. However, no specific immune responses could be detected using intracellular cytokine staining or ELISpot assays. Minor changes in the phenotypic composition of immune cells and their expression of stimulatory and inhibitory markers were detected. All patients progressed to AML with a mean time to progression from inclusion (TTP) of 5.2 months (range 2.8 to 7.6). Mean survival was 18.1 months (range 10.9 to 30.6) from MDS diagnosis and 11.3 months (range 4.3 to 22.2) from inclusion. Sequencing of bone marrow showed clonal expansion of malignant cells, as well as appearance of novel mutations.
Conclusion
The patients progressed to AML with an average time of only five months after initiating the combination therapy. This may be unrelated to the experimental treatment, but the trial was terminated early as there was no sign of clinical benefit or immunological response.
Why the manuscript is especially interesting
This study is the first to exploit the potential synergistic effects of combining a multi-peptide cancer vaccine with epigenetic therapy in MDS. Although our results are negative, they emphasize challenges to induce immune reactivity in patients with high-risk MDS.
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EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OILJ, PNG, SAZU, SBCE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Mycosis fungoides is the most frequent subtype of primary cutaneous T-cell lymphomas. The diagnosis is based on a thorough clinic-pathologic correlation, which can, especially in early-stage disease, ...be challenging due to similarities with several benign skin disorders such as psoriasis and atopic dermatitis. Here, we present a case of an 81-year-old man with a 20-year-long medical history of skin problems treated as psoriasis with limited effect. Since December 2021, the patient experienced worsening of his skin symptoms with rapidly growing tumors and widespread patches and plaques. Positron emission tomography/computed tomography evaluation revealed markedly metabolic activity related to the skin tumors and increased FDG uptake in several retroperitoneal lymph nodes. Histological assessment of skin biopsies demonstrated a highly proliferative T-cell lymphoma with a γ/δ+ and CD8+ cytotoxic phenotype. The morphology of the tumor cells appeared blastic with an abnormal immunephenotype CD3+, CD2−, CD5
, CD4−, CD8+, CD56−, and CD30−. Next-generation sequencing detected a likely pathogenic
mutation with an allele frequency of 72% as well as a
variant of unknown significance. This case highlights the diagnostic complexity of an indolent skin lymphoma evolving into an aggressive cytotoxic lymphoma.
Lynch syndrome-associated cancer develops due to germline pathogenic variants in one of the mismatch repair (MMR) genes,
,
,
or
. Somatic second hits in tumors cause MMR deficiency, testing for which ...is used to screen for Lynch syndrome in colorectal cancer and to guide selection for immunotherapy. Both MMR protein immunohistochemistry and microsatellite instability (MSI) analysis can be used. However, concordance between methods can vary for different tumor types. Therefore, we aimed to compare methods of MMR deficiency testing in Lynch syndrome-associated urothelial cancers.
Ninety-seven urothelial (61 upper tract and 28 bladder) tumors diagnosed from 1980 to 2017 in carriers of Lynch syndrome-associated pathogenic MMR variants and their first-degree relatives (FDR) were analyzed by MMR protein immunohistochemistry, the MSI Analysis System v1.2 (Promega), and an amplicon sequencing-based MSI assay. Two sets of MSI markers were used in sequencing-based MSI analysis: a panel of 24 and 54 markers developed for colorectal cancer and blood MSI analysis, respectively.
Among the 97 urothelial tumors, 86 (88.7%) showed immunohistochemical MMR loss and 68 were successfully analyzed by the Promega MSI assay, of which 48 (70.6%) were MSI-high and 20 (29.4%) were MSI-low/microsatellite stable. Seventy-two samples had sufficient DNA for the sequencing-based MSI assay, of which 55 (76.4%) and 61 (84.7%) scored as MSI-high using the 24-marker and 54-marker panels, respectively. The concordance between the MSI assays and immunohistochemistry was 70.6% (p = 0.003), 87.5% (p = 0.039), and 90.3% (p = 1.00) for the Promega assay, the 24-marker assay, and the 54-marker assay, respectively. Of the 11 tumors with retained MMR protein expression, four were MSI-low/MSI-high or MSI-high by the Promega assay or one of the sequencing-based assays.
Our results show that Lynch syndrome-associated urothelial cancers frequently had loss of MMR protein expression. The Promega MSI assay was significantly less sensitive, but the 54-marker sequencing-based MSI analysis showed no significant difference compared to immunohistochemistry. Data from this study alongside previous studies, suggest that universal MMR deficiency testing of newly diagnosed urothelial cancers, using immunohistochemistry and/or sequencing-based MSI analysis of sensitive markers, offer a potentially useful approach to identification of Lynch syndrome cases.
Complement component C5 is crucial for experimental animal inflammatory tissue damage; however, its involvement in human inflammation is incompletely understood. The responses to Gram-negative ...bacteria were here studied taking advantage of human genetic complement-deficiencies--nature's own knockouts--including a previously undescribed C5 defect. Such deficiencies provide a unique tool for investigating the biological role of proteins. The experimental conditions allowed cross-talk between the different inflammatory pathways using a whole blood model based on the anticoagulant lepirudin, which does not interfere with the complement system. Expression of tissue factor, cell adhesion molecules, and oxidative burst depended highly on C5, mediated through the activation product C5a, whereas granulocyte enzyme release relied mainly on C3 and was C5a-independent. Release of cytokines and chemokines was mediated to varying degrees by complement and CD14; for example, interleukin (IL)-1β and IL-8 were more dependent on complement than IFN-γ and IL-6, which were highly dependent on CD14. IL-1 receptor antagonist (IL-1ra) and IFN-γ inducible protein 10 (IP-10) were fully dependent on CD14 and inversely regulated by complement, that is, complement deficiency and complement inhibition enhanced their release. Granulocyte responses were mainly complement-dependent, whereas monocyte responses were more dependent on CD14. Notably, all responses were abolished by combined neutralization of complement and CD14. The present study provides important insight into the comprehensive role of complement in human inflammatory responses to Gram-negative bacteria.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Optimized and reliable DNA/RNA extraction protocols are a vital tool in clinical practice in the context of molecular testing. Here, we present our successful attempt to enhance the quantity of RNA ...isolated from clinical specimens, which we originally found challenging (breast and testis). We compared several purification methods with special focus on two AllPrep system-based protocols (QIAGEN). Our data suggest that addition of proteinase K may markedly increase RNA and, in some cases, also DNA yield. The extraction kit used, AllPrep DNA/RNA/miRNA universal kit, provides RNA amounts comparable with the phenol-chloroform extraction method; however, part of the final yield consisted of small RNAs, visible as a thick band in the bioanalyzer gel-like image (5S peak). The 5S peak, albeit in some cases dominating the bioanalyzer image, plays only a small role in RT-qPCR analysis, and Qubit or NanoDrop measurements can still be used as a reliable estimate of starting amounts of mRNA for downstream analyses. In conclusion, we showed that implementing a protocol containing a step of proteinase K digestion markedly increases RNA yield. The AllPrep DNA/RNA/miRNA Universal Kit can be successfully used for simultaneous extraction of DNA and total RNA, irrespective of the tissue of origin, and does not present inconveniences related to phenol-chloroform extraction.
Inactivating mutations in Bruton's tyrosine kinase (BTK) in patients with follicular lymphoma (FL) have recently been reported. These mutations were found in BTK inhibitor‐treatment naïve patients. ...Here, we report the BTK mutation status in a real‐world cohort of patients with non‐Hodgkin lymphoma. We found primary BTK mutations in 7.7% of patients with large B‐cell lymphoma (LBCL) and in 14.1% of patients with FL. All patients with BTK‐mutated LBCL were BCL2 translocation positive, and the correlation between BCL2 translocation and BTK mutation persisted even when patients with known transformation from FL were excluded.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
We report a male infant who presented at 8 months of age with atypical hemolytic uremic syndrome (aHUS) responsive to plasma therapy. Investigation showed him to have complement factor H (CFH) ...deficiency associated with a homozygous CFH mutation (c.2880delT p.Phe960fs). Mutation screening of the child's parents revealed that the father was heterozygous for this change but that it was not present in his mother. Chromosome 1 uniparental isodisomy of paternal origin was confirmed by genotyping chromosome 1 SNPs. CD46 SNP genotyping was undertaken in this individual and another patient with CFH deficiency associated with chromosome 1 uniparental isodisomy. This showed a homozygous aHUS risk haplotype ( CD46 GGAAC ) in the patient with aHUS and a homozygous glomerulonephritis risk haplotype ( CD46 AAGGT ) in the patient with endocapillary glomerulonephritis. We also showed that FHL-1 (factor H–like protein 1) was present in the patient with aHUS and absent in the patient with glomerulonephritis. This study emphasizes that modifiers such as CD46 and FHL-1 may determine the kidney phenotype of patients who present with homozygous CFH deficiency.
Abstract C1q deficiency is a rare condition associated with a systemic lupus erythematosus (SLE)-like syndrome and recurrent infections. Here we present the molecular basis behind C1q deficiency in ...three sisters of Inuit origin. Initial examination for complement deficiency showed no function of the classical complement activation pathway in the patients; the lectin and alternative pathways were intact. No C1q or low molecular weight C1q was detected in sera and no anti-C1q autoantibodies were found. Sequencing of the C1q genes revealed a novel missense mutation (Gly-Arg) in codon 217 of the B chain. All sisters were homozygous for the mutation: both parents were heterozygous. None of 100 healthy controls carried the mutation. Our findings define a third class of molecular mechanisms behind C1q deficiency, where missense mutations cause a lack of detectable C1q-antigen in serum.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Abstract
Introduction
Integration of molecular characterization of lymphomas in clinical diagnostics may improve subclassification and risk‐stratification, and we implemented a next generation ...sequencing (NGS) analysis as part of routine diagnostic work‐up of all mature B‐cell non‐Hodgkin's lymphoma (B‐NHL). Here, we present data of mutational profiles with potential complementary diagnostic, prognostic, and predictive value detected in our consecutive non‐selected cohort of B‐NHL patients.
Methods
NGS results from 298 patients with both newly diagnosed and relapsed/refractory disease were included as a single center study. NGS was performed as routine analysis together with standard diagnostic work‐up using a custom‐made amplicon PCR‐based multiplex NGS panel covering all coding exons and consensus splice sites in 59 genes.
Results
Mutations were detected in 94% of the 298 samples. Most lymphomas could be classified definitively, but 24 cases were classified as small B‐cell lymphomas without defining characteristics. Of these, 50% (12/24 cases) could retrospectively be assigned a likely diagnostic subtype according to mutational findings.
Conclusion
Implementation of a 59 gene exome sequencing panel added diagnostic value to 50% of unclassified cases and provided in 94% of the cases possible biomarkers for disease monitoring as well as potential diagnostic, prognostic, and predictive markers for future studies.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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