Cell-of-origin classification using the Hans and Lymph2Cx algorithms in primary cutaneous large B-cell lymphomas

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT) and primary cutaneous follicle center lymphoma with a diffuse population of large cells (PCFCL-LC) are both primary cutaneous B-cell lymphomas with large-cell morphology (CLBCL) but with different clinical characteristics and behavior. In systemic diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS), gene-expression profiling (GEP) revealed two molecular subgroups based on their cell-of-origin (COO) with prognostic significance: the germinal center B-cell-like (GCB) subtype and the activated B-cell-like (ABC) subtype. This study investigated whether COO classification is a useful tool for classification of CLBCL. For this retrospective study, 51 patients with PCDLBCL-LT and 15 patients with PCFCL-LC were analyzed for their COO according to the immunohistochemistry-based Hans algorithm and the NanoString GEP-based Lymph2Cx algorithm. In PCFCL-LC, all cases (100%) classified as GCB by both Hans and Lymph2Cx. In contrast, COO classification in PCDLBCL-LT was heterogeneous. Using Hans, 75% of the PCDLBCL-LT patients classified as non-GCB and 25% as GCB, while Lymph2Cx classified only 18% as ABC, 43% as unclassified/intermediate, and 39% as GCB. These COO subgroups did not differ in the expression of BCL2 and IgM, mutations in MYD88 and/or CD79B, loss of CDKN2A, or survival. In conclusion, PCFCL-LC uniformly classified as GCB, while PCDLBCL-LT classified along the COO spectrum of DLBCL-NOS using the Hans and Lymph2Cx algorithms. In contrast to DLBCL-NOS, the clinical relevance of COO classification in CLBCL using these algorithms has limitations and cannot be used as an alternative for the current multiparameter approach in differentiation of PCDLBCL-LT and PCFCL-LC

ATBF1 and NQO1 as candidate targets for allelic loss at chromosome arm 16q in breast cancer: Absence of somatic ATBF1 mutations and no role for the C609T NQO1 polymorphism

<p>Abstract</p> <p>Background</p> <p>Loss of heterozygosity (LOH) at chromosome arm 16q is frequently observed in human breast cancer, suggesting that one or more target tumor suppressor genes (TSGs) are located there. However, detailed mapping of the smallest region of LOH has not yet resulted in the identification of a TSG at 16q. Therefore, the present study attempted to identify TSGs using an approach based on mRNA expression.</p> <p>Methods</p> <p>A cDNA microarray for the 16q region was constructed and analyzed using RNA samples from 39 breast tumors with known LOH status at 16q.</p> <p>Results</p> <p>Five genes were identified to show lower expression in tumors with LOH at 16q compared to tumors without LOH. The genes for NAD(P)H dehydrogenase quinone (<it>NQO1</it>) and AT-binding transcription factor 1 (<it>ATBF1</it>) were further investigated given their functions as potential TSGs. <it>NQO1 </it>has been implicated in carcinogenesis due to its role in quinone detoxification and in stabilization of p53. One inactive polymorphic variant of <it>NQO1 </it>encodes a product showing reduced enzymatic activity. However, we did not find preferential targeting of the active <it>NQO1 </it>allele in tumors with LOH at 16q. Immunohistochemical analysis of 354 invasive breast tumors revealed that NQO1 protein expression in a subset of breast tumors is higher than in normal epithelium, which contradicts its proposed role as a tumor suppressor gene.</p> <p><it>ATBF1 </it>has been suggested as a target for LOH at 16q in prostate cancer. We analyzed the entire coding sequence in 48 breast tumors, but did not identify somatic sequence changes. We did find several in-frame insertions and deletions, two variants of which were reported to be somatic pathogenic mutations in prostate cancer. Here, we show that these variants are also present in the germline in 2.5% of 550 breast cancer patients and 2.9% of 175 healthy controls. This indicates that the frequency of these variants is not increased in breast cancer patients. Moreover, there is no preferential LOH of the wildtype allele in breast tumors.</p> <p>Conclusion</p> <p>Two likely candidate TSGs at 16q in breast cancer, <it>NQO1 </it>and <it>ATBF1</it>, were identified here as showing reduced expression in tumors with 16q LOH, but further analysis indicated that they are not target genes of LOH. Furthermore, our results call into question the validity of the previously reported pathogenic variants of the <it>ATBF1 </it>gene.</p

Single nucleotide polymorphism (SNP) rs3751143 in P2RX7 is associated with therapy failure in chronic Q fever while rs7125062 in MMP1 is associated with fewer complications

OBJECTIVES: Chronic Q fever is a persistent infection with the intracellular bacterium Coxiella burnetii. Development of chronic Q fever is associated with single nucleotide polymorphisms (SNPs) in genes encoding for pattern recognition receptors, for phagolysosomal pathway components and for matrix metalloproteinases (MMPs). We evaluated the association of SNPs in these innate-immunity and MMP genes with clinical outcomes. METHODS: SNPs were selected from previous association studies and analysed in a cohort of patients with chronic Q fever. The primary outcome was all-cause mortality; secondary outcomes were therapy failure and chronic Q fever-related complications. Subdistribution hazard ratios (SHR) were calculated. RESULTS: Nineteen SNPs were analysed in 134 patients with proven and 29 with probable chronic Q fever. In multivariable analysis, none of the selected SNPs was associated with all-cause mortality. However, SNP rs3751143 located in P2RX7 appeared to be associated with therapy failure (SHR 2.42; 95% confidence interval, 1.16-5.05; p 0.02), which is in line with other reports, showing that a loss of function of the P2X7 receptor leads to inefficient killing of intracellular organisms. In addition, SNP rs7125062 located in MMP1, involved in the cleavage of extracellular matrix, was associated with fewer chronic Q fever-related complications such as acute aneurysms (SHR 0.49; 95% confidence interval, 0.29-0.83; p 0.008). CONCLUSIONS: A polymorphism in P2RX7, known to lead to loss of function of the receptor and inefficient killing of intracellular organisms, and a polymorphism in MMP1 were respectively associated with more therapy failures and fewer complications such as acute aneurysms in patients with chronic Q fever

Genetic Stability of Driver Alterations in Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type and Their Relapses:A Rationale for the Use of Molecular-Based Methods for More Effective Disease Monitoring

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT) is a rare, aggressive cutaneous lymphoma with a 5-year disease-specific survival of only ~55%. Despite high response rates to initial immune-polychemotherapy, most patients experience a disease relapse. The genetic evolution of primary and relapsed/refractory disease has only scarcely been studied in PCDLBCL-LT patients. Therefore, in this retrospective cohort study, 73 primary/pre-treatment and relapsed/refractory biopsies of 57 patients with PCDLBCL-LT were molecularly characterized with triple FISH and targeted next-generation sequencing for 52 B-cell-lymphoma-relevant genes, including paired analysis in 16 patients. In this cohort, 95% of patients harboured at least one of the three main driver alterations (mutations in MYD88/CD79B and/or CDKN2A-loss). In relapsed/refractory PCDLBCL-LT, these oncogenic aberrations were persistently present, demonstrating genetic stability over time. Novel alterations in relapsed disease affected mostly CDKN2A, MYC, and PIM1. Regarding survival, only MYC rearrangements and HIST1H1E mutations were statistically significantly associated with an inferior outcome. The stable presence of one or more of the three main driver alterations (mutated MYD88/CD79B and/or CDKN2A-loss) is promising for targeted therapies addressing these alterations and serves as a rationale for molecular-based disease monitoring, improving response evaluation and early identification and intervention of disease relapses in these poor-prognostic PCDLBCL-LT patients

Endovascular treatment in anterior circulation stroke beyond 6.5 hours after onset or time last seen well:results from the MR CLEAN Registry

BACKGROUND: Randomised controlled trials with perfusion selection have shown benefit of endovascular treatment (EVT) for ischaemic stroke between 6 and 24 hours after symptom onset or time last seen well. However, outcomes after EVT in these late window patients without perfusion imaging are largely unknown. We assessed their characteristics and outcomes in routine clinical practice. METHODS: The Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands Registry, a prospective, multicentre study in the Netherlands, included patients with an anterior circulation occlusion who underwent EVT between 2014 and 2017. CT perfusion was no standard imaging modality. We used adjusted ordinal logistic regression analysis to compare patients treated within versus beyond 6.5 hours after propensity score matching on age, prestroke modified Rankin Scale (mRS), National Institutes of Health Stroke Scale, Alberta Stroke Programme Early CT Score (ASPECTS), collateral status, location of occlusion and treatment with intravenous thrombolysis. Outcomes included 3-month mRS score, functional independence (defined as mRS 0–2), and death. RESULTS: Of 3264 patients who underwent EVT, 106 (3.2%) were treated beyond 6.5 hours (median 8.5, IQR 6.9–10.6), of whom 93 (87.7%) had unknown time of stroke onset. CT perfusion was not performed in 87/106 (80.2%) late window patients. Late window patients were younger (mean 67 vs 70 years, p<0.04) and had slightly lower ASPECTS (median 8 vs 9, p<0.01), but better collateral status (collateral score 2–3: 68.3% vs 57.7%, p=0.03). No differences were observed in proportions of functional independence (43.3% vs 40.5%, p=0.57) or death (24.0% vs 28.9%, p=0.28). After matching, outcomes remained similar (adjusted common OR for 1 point improvement in mRS 1.04, 95% CI 0.56 to 1.93). CONCLUSIONS: Without the use of CT perfusion selection criteria, EVT in the 6.5–24-hour time window was not associated with poorer outcome in selected patients with favourable clinical and CT/CT angiography characteristics. randomised controlled trials with lenient inclusion criteria are needed to identify more patients who can benefit from EVT in the late window

Whole Gene Capture Analysis of 15 CRC Susceptibility Genes in Suspected Lynch Syndrome Patients

Background and Aims Lynch Syndrome (LS) is caused by pathogenic germline variants in one of the mismatch repair (MMR) genes. However, up to 60% of MMR-deficient colorectal cancer cases are categorized as suspected Lynch Syndrome (sLS) because no pathogenic MMR germline variant can be identified, which leads to difficulties in clinical management. We therefore analyzed the genomic regions of 15 CRC susceptibility genes in leukocyte DNA of 34 unrelated sLS patients and 11 patients with MLH1 hypermethylated tumors with a clear family history. Methods Using targeted next-generation sequencing, we analyzed the entire non-repetitive genomic sequence, including intronic and regulatory sequences, of 15 CRC susceptibility genes. In addition, tumor DNA from 28 sLS patients was analyzed for somatic MMR variants. Results Of 1979 germline variants found in the leukocyte DNA of 34 sLS patients, one was a pathogenic variant (MLH1 c.1667+1delG). Leukocyte DNA of 11 patients with MLH1 hypermethylated tumors was negative for pathogenic germline variants in the tested CRC susceptibility genes and for germline MLH1 hypermethylation. Somatic DNA analysis of 28 sLS tumors identified eight (29%) cases with two pathogenic somatic variants, one with a VUS predicted to pathogenic and LOH, and nine cases (32%) with one pathogenic somatic variant (n = 8) or one VUS predicted to be pathogenic (n = 1). Conclusions This is the first study in sLS patients to include the entire genomic sequence of CRC susceptibility genes. An underlying somatic or germline MMR gene defect was identified in ten of 34 sLS patients (29%). In the remaining sLS patients, the underlying genetic defect explaining the MMRdeficiency in their tumors might be found outside the genomic regions harboring the MMR and other known CRC susceptibility genes

No preclinical rationale for IGF1R directed therapy in chondrosarcoma of bone

Background: Chondrosarcoma is a malignant cartilage forming bone tumour for which no effective systemic treatment is available. Previous studies illustrate the need for a better understanding of the role of the IGF pathway in chondrosarcoma to determine if it can be a target for therapy, which was therefore explored in this study. Methods: Expression of mediators of IGF1R signalling and phosphorylation status of IRS1 was determined in chondrosarcoma cell lines by qRT-PCR and western blot. The effect of activation and inhibition of IGF1R signalling on downstream targets was assessed by western blot. Ten chondrosarcoma cell lines were treated with OSI-906 (IGF1R and IR dual inhibitor) after which cell proliferation and migration were determined by a viability assay and the xCELLigence system, respectively. In addition, four chondrosarcoma cell lines were treated with a combination of doxorubicin and OSI-906. By immunohistochemistry, IGF1R expression levels were determined in tissue microarrays of 187 cartilage tumours and ten paraffin embedded cell lines. Results: Mediators of IGF1R signalling are heterogeneously expressed and phosphorylated IRS1 was detected in 67 % of the tested chondrosarcoma cell lines, suggesting that IGF1R signalling is active in a subset of chondrosarcoma cell lines. In the cell lines with phosphorylated IRS1, inhibition of IGF1R signalling decreased phosphorylated Akt levels and increased IGF1R expression, but it did not influence MAPK or S6 activity. In line with these findings, treatment with IGF1R/IR inhibitors did not impact proliferation or migration in any of the chondrosarcoma cell lines, even upon stimulation with IGF1. Although synergistic effects of IGF1R/IR inhibition with doxorubicin are described for other cancers, our results demonstrate that this was not the case for chondrosarcoma. In addition, we found minimal IGF1R expression in primary tumours in contrast to the high expression detected in chondrosarcoma cell lines, even if both were derived from the same tumour, suggesting that in vitro culturing upregulates IGF1R expression. Conclusions: The results from this study indicate that the IGF pathway is not essential for chondrosarcoma growth, migration or chemoresistance. Furthermore, IGF1R is only minimally expressed in chondrosarcoma primary tumours. Therefore, the IGF pathway is not expected to be an effective therapeutic target for chondrosarcoma of bone. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2522-8) contains supplementary material, which is available to authorized users

Comparative Performance Analysis of IdyllaTM and ArcherTM in the Detection of Gene Fusions in Spitzoid Melanocytic Tumors

Melanocytic neoplasms with spitzoid histomorphology are often difficult to classify without identifying genetic drivers such as kinase fusions. Traditional diagnostic methods, such as immunohistochemistry, can yield inconclusive results, and advanced techniques such as the Archer fusion assay are often inaccessible and costly. The Idylla GeneFusion Assay might offer a rapid and cost-effective alternative. This study compared Idylla and Archer in identifying ALK, pan-NTRK, RET, and ROS1 gene fusions. Of the 147 samples where next-generation sequencing did not detect genetic drivers, 89 (60.5%) meeting the tissue requirements were further analyzed using Idylla (Cohort A). Idylla demonstrated a sensitivity of 75% and a specificity of 100% in detecting these fusions. Additionally, among 27 randomly selected cases (Cohort B) that failed to meet the inclusion criteria, Idylla maintained the same levels of sensitivity and specificity. Our findings also show that Idylla can be effectively conducted with isolated RNA, broadening its applicability beyond tissue samples. Although the Idylla assay may not replace more comprehensive molecular assays such as Archer, it could serve as a valuable initial screening tool in diagnosing spitzoid melanocytic tumors

Association studies of up to 1.2 million individuals yield new insights into the genetic etiology of tobacco and alcohol use.

Tobacco and alcohol use are leading causes of mortality that influence risk for many complex diseases and disorders1. They are heritable2,3 and etiologically related4,5 behaviors that have been resistant to gene discovery efforts6-11. In sample sizes up to 1.2 million individuals, we discovered 566 genetic variants in 406 loci associated with multiple stages of tobacco use (initiation, cessation, and heaviness) as well as alcohol use, with 150 loci evidencing pleiotropic association. Smoking phenotypes were positively genetically correlated with many health conditions, whereas alcohol use was negatively correlated with these conditions, such that increased genetic risk for alcohol use is associated with lower disease risk. We report evidence for the involvement of many systems in tobacco and alcohol use, including genes involved in nicotinic, dopaminergic, and glutamatergic neurotransmission. The results provide a solid starting point to evaluate the effects of these loci in model organisms and more precise substance use measures