CANTRK: A Canadian Ring Study to Optimize Detection of NTRK Gene Fusions by Next-Generation RNA Sequencing.

The Canadian NTRK (CANTRK) study is an interlaboratory comparison ring study to optimize testing for neurotrophic receptor tyrosine kinase (NTRK) fusions in Canadian laboratories. Sixteen diagnostic laboratories used next-generation sequencing (NGS) for NTRK1, NTRK2, or NTRK3 fusions. Each laboratory received 12 formalin-fixed, paraffin-embedded tumor samples with unique NTRK fusions and two control non-NTRK fusion samples (one ALK and one ROS1). Laboratories used validated protocols for NGS fusion detection. Panels included Oncomine Comprehensive Assay v3, Oncomine Focus Assay, Oncomine Precision Assay, AmpliSeq for Illumina Focus, TruSight RNA Pan-Cancer Panel, FusionPlex Lung, and QIAseq Multimodal Lung. One sample was withdrawn from analysis because of sample quality issues. Of the remaining 13 samples, 6 of 11 NTRK fusions and both control fusions were detected by all laboratories. Two fusions, WNK2::NTRK2 and STRN3::NTRK2, were not detected by 10 laboratories using the Oncomine Comprehensive or Focus panels, due to absence of WNK2 and STRN3 in panel designs. Two fusions, TPM3::NTRK1 and LMNA::NTRK1, were challenging to detect on the AmpliSeq for Illumina Focus panel because of bioinformatics issues. One ETV6::NTRK3 fusion at low levels was not detected by two laboratories using the TruSight Pan-Cancer Panel. Panels detecting all fusions included FusionPlex Lung, Oncomine Precision, and QIAseq Multimodal Lung. The CANTRK study showed competency in detection of NTRK fusions by NGS across different panels in 16 Canadian laboratories and identified key test issues as targets for improvements.

A Pan-Canadian Validation Study for the Detection of EGFR T790M Mutation Using Circulating Tumor DNA From Peripheral Blood.

INTRODUCTION: Genotyping circulating tumor DNA (ctDNA) is a promising noninvasive clinical tool to identify the EGFR T790M resistance mutation in patients with advanced NSCLC with resistance to EGFR inhibitors. To facilitate standardization and clinical adoption of ctDNA testing across Canada, we developed a 2-phase multicenter study to standardize T790M mutation detection using plasma ctDNA testing. METHODS: In phase 1, commercial reference standards were distributed to participating clinical laboratories, to use their existing platforms for mutation detection. Baseline performance characteristics were established using known and blinded engineered plasma samples spiked with predetermined concentrations of T790M, L858R, and exon 19 deletion variants. In phase II, peripheral blood collected from local patients with known EGFR activating mutations and progressing on treatment were assayed for the presence of EGFR variants and concordance with a clinically validated test at the reference laboratory. RESULTS: All laboratories in phase 1 detected the variants at 0.5 % and 5.0 % allele frequencies, with no false positives. In phase 2, the concordance with the reference laboratory for detection of both the primary and resistance mutation was high, with next-generation sequencing and droplet digital polymerase chain reaction exhibiting the best overall concordance. Data also suggested that the ability to detect mutations at clinically relevant limits of detection is generally not platform-specific, but rather impacted by laboratory-specific practices. CONCLUSIONS: Discrepancies among sending laboratories using the same assay suggest that laboratory-specific practices may impact performance. In addition, a negative or inconclusive ctDNA test should be followed by tumor testing when possible.

Profiling non-small cell lung cancer reveals that PD-L1 is associated with wild type EGFR and vascular invasion, and immunohistochemistry quantification of PD-L1 correlates weakly with RT-qPCR.

Most lung cancer patients are diagnosed at an advanced stage, limiting their treatment options with very low response rate. Lung cancer is the most common cause of cancer death worldwide. Therapies that target driver gene mutations (e.g. EGFR, ALK, ROS1) and checkpoint inhibitors such anti-PD-1 and PD-L1 immunotherapies are being used to treat lung cancer patients. Identification of correlations between driver mutations and PD-L1 expression will allow for the best management of patient treatment. 851 cases of non-small cell lung cancer cases were profiled for the presence of biomarkers EGFR, KRAS, BRAF, and PIK3CA mutations by SNaPshot/sizing genotyping. Immunohistochemistry was used to identify the protein expression of ALK and PD-L1. Total PD-L1 mRNA expression (from unsorted tumor samples) was quantified by RT-qPCR in a sub-group of the cohort to assess its correlation with PD-L1 protein level in tumor cells. Statistical analysis revealed correlations between the presence of the mutations, PD-L1 expression, and the pathological data. Specifically, increased PD-L1 expression was associated with wildtype EGFR and vascular invasion, and total PD-L1 mRNA levels correlated weakly with protein expression on tumor cells. These data provide insights into driver gene mutations and immune checkpoint status in relation to lung cancer subtypes and suggest that RT-qPCR is useful for assessing PD-L1 levels.

Molecular profiling of non-small cell lung cancer.

Lung cancer is generally treated with conventional therapies, including chemotherapy and radiation. These methods, however, are not specific to cancer cells and instead attack every cell present, including normal cells. Personalized therapies provide more efficient treatment options as they target the individual's genetic makeup. The goal of this study was to identify the frequency of causal genetic mutations across a variety of lung cancer subtypes in the earlier stages. 833 samples of non-small cell lung cancer from 799 patients who received resection of their lung cancer, were selected for molecular analysis of six known mutations, including EGFR, KRAS, BRAF, PIK3CA, HER2 and ALK. A SNaPshot assay was used for point mutations and fragment analysis searched for insertions and deletions. ALK was evaluated by IHC +/- FISH. Statistical analysis was performed to determine correlations between molecular and clinical/pathological patient data. None of the tested variants were identified in most (66.15%) of cases. The observed frequencies among the total samples vs. only the adenocarcinoma cases were notable different, with the highest frequency being the KRAS mutation (24.49% vs. 35.55%), followed by EGFR (6.96% vs. 10.23%), PIK3CA (1.20% vs. 0.9%), BRAF (1.08% vs. 1.62%), ALK (0.12% vs. 0.18%), while the lowest was the HER2 mutation (0% for both). The statistical analysis yielded correlations between presence of a mutation with gender, cancer type, vascular invasion and smoking history. The outcome of this study will provide data that helps stratify patient prognosis and supports development of more precise treatments, resulting in improved outcomes for future lung cancer patients.

Low-depth sequencing for copy number abnormalities in multiple myeloma supersedes fluorescent in situ hybridization in scope and resolution.

Multiple myeloma (MM) is an incurable hematological malignancy that relies on cytogenetic determination of copy number abnormalities (CNAs) for prognosis and management. Low-depth whole genome sequencing (LD-WGS) is a cost-effective alternative to targeted genomics for CNA detection, but its value has yet to be explored in MM. DNA from CD138+ cells from MM patients were sequenced using an Illumina NextSeq at <1x depth (ultralow-depth). Subsampling analysis and window size adjustment were performed for determining sensitivity limits and results compared to fluorescent in-Situ hybridization (FISH). CNA calls made down to 5 million (M) reads were comparable to those at 20 M reads at a window size of 100 kb had a sensitivity and specificity of 93%, 92% and an area under the curve of 0.94. All CNAs detected by FISH on the MM samples were also detected by LD-WGS; the latter detected a further 36 focal CNAs not detected by FISH. Cost per sample of LD-WGS was significantly lower for our organization than FISH testing. LD-WGS for MM is significantly more sensitive than targeted technologies such as FISH in CNA detection and resolution, provides a more cost-effective option for clinical purposes and potential for exploring prognostically relevant and drug discovery targets.

Genetic profiles of different subsets of Merkel cell carcinoma show links between combined and pure MCPyV-negative tumors.

Tumorigenesis in Merkel cell carcinoma (MCC) is driven by (1) clonal integration of the Merkel cell polyomavirus (MCPyV) in neoplastic cells and/or (2) genetic damage by ultraviolet (UV) light. A higher mutational burden, a UV-mutational signature, and many mutations in the TP53 and RB1 genes characterize the virus-negative subset. MCPyV-negative MCCs include combined (often squamous and neuroendocrine) and pure (neuroendocrine) tumors. Because a combined morphology could elude detection microscopically, we sought a genetic link between combined and pure virus-negative tumors. From a global cohort of 46 cases, 9 pure MCPyV-positive, 9 pure MCPyV-negative, and 10 combined MCPyV-negative MCCs were studied by genome-wide microarray in search of copy number aberrations. The entire cohort (n=46) was evaluated by next-generation sequencing for mutations in selected tumor suppressor genes and oncogenes. More copy number aberrations and a greater fraction of the genome were changed in combined and pure MCPyV-negative tumors relative to MCPyV-positive cases (P<.01 for all comparisons). No difference in these parameters was found between the 2 MCPyV-negative groups. Copy number loss of RB1 or an inactivating RB1 mutation (either or both) was common in combined (8/10, 80%) and pure (7/9, 78%) MCPyV-negative tumors but not MCPyV-positive cases (1/9, 11%). A similar trend was seen for TP53 (combined [2/10, 20%] and pure virus-negative tumors [5/9, 56%] showed gene copy number loss or mutations contrasted with pure virus-positive cases [0/9, 0%]). The shared genetic profiles of combined and pure MCPyV-negative tumors link these subsets and separate them from MCPyV-positive tumors.

ALK+ lung adenocarcinoma in never smokers and long-term ex-smokers: prevalence and detection by immunohistochemistry and fluorescence in situ hybridization.

ALK gene rearrangements are identified in 2-5 % of all non-small cell lung cancer and are more common in lifetime non-smokers with adenocarcinoma, but the prevalence of ALK rearrangements is not as well characterized in long-term ex-smokers (quit >10 years prior to diagnosis). Accurate and timely diagnosis of ALK-rearranged tumors is of clinical importance given the remarkable response to targeted inhibitors. ALK gene rearrangement may be detected by fluorescence in situ hybridization (FISH), and abnormal expression of ALK protein may be detected by immunohistochemistry (IHC), the latter of which is faster and less expensive. The aim of this study is to evaluate the prevalence of ALK rearrangement in non-smokers and long-term ex-smokers with lung adenocarcinoma and to assess the performance of IHC for the detection of ALK+ tumors when compared to FISH. Two hundred fifty-one cases of resected lung adenocarcinoma were retrospectively reviewed, including non-smokers (n = 79) or long-term ex-smokers (n = 172). ALK IHC and ALK FISH were performed on each case. Four cases demonstrated ALK rearrangement by FISH (4/251; 1.6 %). All cases were non-smokers (4/79; 5.1 %), and all were positive for ALK by IHC. No additional cases were considered positive by IHC, and only 26 (10.4 %) cases were considered equivocal using a conservative approach to interpretation, resulting in a sensitivity of 100 % and specificity of 89.5 %. ALK rearrangement was not observed in lung adenocarcinoma arising in long-term ex-smokers, whereas it is seen in up to 5.1 % of lifetime non-smokers. ALK IHC using the 5A4 antibody demonstrates high sensitivity, supporting its use as a screening test.

Cutaneous manifestations of angioimmunoblastic T-cell lymphoma: clinical and pathological characteristics.

Angioimmunoblastic T-cell lymphoma (AITL), an uncommon variant of peripheral T-cell lymphoma, affects the skin in approximately 50% of cases. Its protean clinical and histopathological cutaneous manifestations pose a challenge in diagnosis, particularly when these precede the diagnosis of AITL on a lymph node biopsy. In this retrospective study, we compared 11 cases of AITL with cutaneous manifestations (mean age 67 years; male:female ratio 1:0.8; 24 skin biopsies) with 20 control cases of inflammatory and non-AITL lymphomatous diseases (mean age 52 years; male:female ratio 1:1.5; 26 skin biopsies). Clinical, histopathological, immunohistochemical, and molecular data were documented. New insights into the clinical evolution of cutaneous involvement by AITL (C-AITL), from early macular, through papular to nodular stages, were observed. Microscopically, a parallel increment in the density of the dermal infiltrate and in the detection of lymphocyte cytological atypia was noted over time. Identification and quantification of follicular T-helper cells (Tfh), the neoplastic lineage, by immunohistochemistry helped to separate cases of C-AITL from inflammatory controls, offering promise as a useful diagnostic adjunct. The presence of T-cell clonality did not have discriminatory value between the 2 groups. Our work suggests that the early maculopapular phase of C-AITL eludes identification on pathological grounds alone and that features such as cytological atypia and high endothelial venules lack diagnostic specificity. In the context of (1) a rash that simulates a drug/viral exanthem or an acute manifestation of a connective tissue disorder, but proves recalcitrant, (2) constitutional abnormalities and/or lymphadenopathy that persist, and (3) a Tfh cell-rich perivascular dermatitis, the diagnosis of early C-AITL can be suspected, but not confirmed, without the benefit of a lymph node biopsy. The later nodular phase of C-AITL occurring in a similar constitutional background, can usually be discerned as lymphomatous, clinically and pathologically. Here a Tfh cell-rich infiltrate is a clue to the specific diagnosis, but confirmation by a nodal evaluation remains mandatory. Despite the difficulty in establishing a diagnosis of C-AITL in its early stages, and speculation that the initial eruptions might be reactive in nature, our sequential data support the concept that these are lymphomatous ab initio. To address the diagnostic challenge presented by this disease, meaningful integration of clinical and pathological data is imperative.

Germline mutations in MAP3K6 are associated with familial gastric cancer.

Gastric cancer is among the leading causes of cancer-related deaths worldwide. While heritable forms of gastric cancer are relatively rare, identifying the genes responsible for such cases can inform diagnosis and treatment for both hereditary and sporadic cases of gastric cancer. Mutations in the E-cadherin gene, CDH1, account for 40% of the most common form of familial gastric cancer (FGC), hereditary diffuse gastric cancer (HDGC). The genes responsible for the remaining forms of FGC are currently unknown. Here we examined a large family from Maritime Canada with FGC without CDH1 mutations, and identified a germline coding variant (p.P946L) in mitogen-activated protein kinase kinase kinase 6 (MAP3K6). Based on conservation, predicted pathogenicity and a known role of the gene in cancer predisposition, MAP3K6 was considered a strong candidate and was investigated further. Screening of an additional 115 unrelated individuals with non-CDH1 FGC identified the p.P946L MAP3K6 variant, as well as four additional coding variants in MAP3K6 (p.F849Sfs*142, p.P958T, p.D200Y and p.V207G). A somatic second-hit variant (p.H506Y) was present in DNA obtained from one of the tumor specimens, and evidence of DNA hypermethylation within the MAP3K6 gene was observed in DNA from the tumor of another affected individual. These findings, together with previous evidence from mouse models that MAP3K6 acts as a tumor suppressor, and studies showing the presence of somatic mutations in MAP3K6 in non-hereditary gastric cancers and gastric cancer cell lines, point towards MAP3K6 variants as a predisposing factor for FGC.

Canadian anaplastic lymphoma kinase study: a model for multicenter standardization and optimization of ALK testing in lung cancer.

INTRODUCTION: Fluorescence in situ hybridization (FISH) is currently the standard for diagnosing anaplastic lymphoma kinase (ALK)-rearranged (ALK+) lung cancers for ALK inhibitor therapies. ALK immunohistochemistry (IHC) may serve as a screening and alternative diagnostic method. The Canadian ALK (CALK) study was initiated to implement a multicenter optimization and standardization of laboratory developed ALK IHC and FISH tests across 14 hospitals. METHODS: Twenty-eight lung adenocarcinomas with known ALK status were used as blinded study samples. Thirteen laboratories performed IHC using locally developed staining protocols for 5A4, ALK1, or D5F3 antibodies; results were assessed by H-score. Twelve centers conducted FISH using protocols based on Vysis' ALK break-apart FISH kit. Initial IHC results were used to optimize local IHC protocols, followed by a repeat IHC study to assess the results of standardization. Three laboratories conducted a prospective parallel IHC and FISH analysis on 411 consecutive clinical samples using post-validation optimized assays. RESULTS: Among study samples, FISH demonstrated 22 consensus ALK+ and six ALK wild type tumors. Preoptimization IHC scores from 12 centers with 5A4 and the percent abnormal cells by FISH from 12 centers showed intraclass correlation coefficients of 0.83 and 0.68, respectively. IHC optimization improved the intraclass correlation coefficients to 0.94. Factors affecting FISH scoring and outliers were identified. Post-optimization concurrent IHC/FISH testing in 373 informative cases revealed 100% sensitivity and specificity for IHC versus FISH. CONCLUSIONS: Multicenter standardization study may accelerate the implementation of ALK testing protocols across a country/region. Our data support the use of an appropriately validated IHC assay to screen for ALK+ lung cancers.

Novel splice-site mutation in ATP8B1 results in atypical progressive familial intrahepatic cholestasis type 1.

BACKGROUND AND AIM: Our objective was to identify the molecular genetic basis of an Alagille-like condition not linked to JAG1 or NOTCH2 in two related sibships. METHODS: Because of common ancestry, and an autosomal recessive mode of inheritance, it was hypothesized that all affected and no unaffected individuals would be homozygous for the same haplotype in the region of the causative gene. Single nucleotide polymorphism arrays were therefore used to genotype 3 affected individuals from two sibships, their mothers and four unaffected siblings, to identify regions of homozygosity. Genes within the largest regions were prioritized and sequenced for mutations. Mutant RNA transcripts were also sequenced. RESULTS: A novel splice acceptor site mutation in the ATP8B1 gene was identified (a G-C preceding exon 16 resulting in a 4 bp deletion and frameshift from the 5' end of exon 16). This result was unexpected because ATP8B1 mutations are associated with progressive familial intrahepatic cholestasis type 1 (PFIC1). Intrahepatic bile duct paucity, cardiac anomalies, renal tubular acidosis and hypothyroidism led to an initial diagnosis of Alagille syndrome. However, in retrospect, abnormal sweat chloride, normal gamma-glutamyl transferase, normal to low cholesterol, and an autosomal recessive mode of inheritance were consistent with PFIC1. Renal tubular acidosis, hypothyroidism and cardiac anomalies have not previously been associated with PFIC1. CONCLUSION: This work expands the phenotypic spectrum of PFIC1, and highlights the overlap in clinical phenotype between Alagille syndrome and PFIC1. Knowledge of the causative mutation allows for carrier testing and prenatal diagnosis in this community.

Multiplex ligation-dependent probe amplification versus multiprobe fluorescence in situ hybridization to detect genomic aberrations in chronic lymphocytic leukemia: a tertiary center experience.

Cytogenetic abnormalities play a major role in the prognosis of patients with chronic lymphocytic leukemia (CLL). Several methods have emerged to try to best identify these abnormalities. We used fluorescence in situ hybridization (FISH) to determine the frequency of cytogenetic changes in our CLL patient population. We also evaluated the effectiveness of multiplex ligation-dependent probe amplification (MLPA) in detecting these abnormalities. Sixty-two B-CLL patients and 20 healthy controls were enrolled, and FISH and MLPA analyses were performed on peripheral blood samples. Using FISH, genomic aberrations were found in 73% of patients and presented as follows: single 13q14.3 deletion (60%), trisomy 12 (7%), ATM deletion (6%), 17p13.1 deletion (2%). MLPA analyses done on 61/62 patients showed sensitivity and specificity values of 90% and 100% respectively. MLPA revealed several additional copy number changes, the most common being 19p13 (LDLR and CDKN2D). Moreover, the cost for MLPA analysis, including technical time and reagents, is 86% less than FISH. In conclusion, cytogenetic abnormalities are a common finding in CLL patients, and MLPA is a reliable approach that is more cost effective and faster than FISH. Despite MLPA limitations of sensitivity, it can be used as a first-line screen and complementary test to FISH analysis.

JAK2 V617F positive polycythemia Vera in a child with neurofibromatosis type I.

We report a child with polycythemia vera (PV). This patient demonstrates the acquired somatic JAK2 V617F mutation and also has neurofibromatosis type I (NF1). NF1, while not previously associated with PV, is associated with another childhood MPD, juvenile myelomonocytic leukemia (JMML). Thus we examined a number of genetic abnormalities identified in JMML patients, but found no association in this case. Neurofibromin sequencing failed to identify a causative mutation. An unknown genetic abnormality resulting in NF1 may have predisposed this young child to acquiring the common JAK2 mutation.

An autosomal recessive form of Alagille-like syndrome that is not linked to JAG1.

PURPOSE: Alagille syndrome is an autosomal dominant condition characterized by a paucity of interlobular bile ducts and chronic cholestasis, cardiac disease, skeletal abnormalities, ocular abnormalities, and characteristic facies. Most cases harbor a mutation in JAG1. We describe a large consanguineous family with five individuals affected with an Alagille-like syndrome that appears to be autosomal recessive. Our objective was to characterize the disorder clinically and determine whether affected individuals had inherited a mutation in JAG1. METHODS: Clinical data were obtained through questioning and patient chart review. Linkage analysis using microsatellite markers was used to assess the possibility of a JAG1 mutation. RESULTS: The clinical phenotype of patients was not entirely consistent with classic Alagille syndrome. All affected individuals had neonatal cholestasis with intrahepatic bile duct paucity, with three having pulmonary stenosis, but the presentation was unusually uniform and severe in childhood. There was no evidence of posterior embryotoxon or vertebral anomalies. Cardiac abnormalities were inconsistent between patients. Most significantly, the pedigree suggested an autosomal recessive form of inheritance. Linkage analysis excluded a mutation in JAG1. CONCLUSIONS: We have identified a kindred with an Alagille-like syndrome with an autosomal recessive form of inheritance not caused by a mutation in JAG1.

Acute promyelocytic leukemia: a novel PML/RARalpha fusion that generates a frameshift in the RARalpha transcript and ATRA resistance.

Acute promyelocytic leukemia (APL) is characterized by increased promyelocytes in the marrow that harbor a t(15;17) and promyelocyte leukemia (PML)/RARalpha fusion gene. The oncogenic gene product is believed to act through disruption of the transcription-modulating function of RARalpha. Differentiation of promyelocytes and remission is achieved with all transretinoic acid (ATRA) therapy usually in combination with chemotherapy. This report describes a patient with the t(15;17) who did not respond typically to ATRA and IDAC induction chemotherapy, although achieved and remains in complete remission five years following induction and one consolidation with high dose cytarabine (HIDAC). RT-PCR and sequencing revealed a novel fusion of RARalpha exon 3 to PML exon 5 that creates a frameshift and premature stop codon in the RARalpha portion of the transcript. Since none of the RARalpha functional domains are maintained, this case highlights the possibility that PML/RARalpha may directly affect promyelocyte differentiation through disruption of PML function.

Detection of tyrosinase mRNA in the sentinel lymph nodes of melanoma patients is not a predictor of short-term disease recurrence.

Sentinel lymph node evaluation has enabled identification of patients with cutaneous melanoma who might benefit from elective regional lymph node dissection. Sentinel nodes are currently assessed by histologic and reverse transcription polymerase chain reaction (RT-PCR) evaluation for melanocyte-specific markers. The clinical significance of positive findings by RT-PCR in the absence of histologic evidence of metastasis (HIS(NEG)/PCR(POS)) remains unclear. Examination of 264 lymph nodes from 139 patients revealed histopathologic positivity in 34 patients (24.5%), in which 26 also demonstrated simultaneous RT-PCR positivity (HIS(POS)/PCR(POS)). Of 35 HIS(NEG)/PCR(POS) patients (25.2%), five also had nodal capsular nevi. In total, capsular nevi were detected in 13 patients (9.4%). A total of 70 patients (50.4%) had negative sentinel nodes by both histopathology and RT-PCR (HIS(NEG)/PCR(NEG)). Over a median follow-up of 25 months, local and/or systemic recurrence developed in 31 patients (22.3%). Recurrence rates were similar among patients with histopathologic evidence of sentinel lymph node metastasis, irrespective of RT-PCR status (HIS(POS)/PCR(POS) 62%; HIS(POS)/PCR(NEG) 75%). In contrast, only 10% of HIS(NEG)/PCR(NEG) patients developed recurrence, significantly less than those in either HIS(POS) group (P<0.0001). Recurrence in the HIS(NEG)/PCR(POS)/CN(NEG) group (7.7%) was comparable to that in HIS(NEG)/PCR(NEG) patients and significantly lower than that in either HIS(POS) group (P<0.0001). The only independent prognostic factors identified by multivariate analysis were the Breslow thickness of the primary tumour and histopathologic positivity of sentinel nodes. Our findings support previous observations that histopathologic evidence of metastatic melanoma in sentinel lymph nodes is an independent predictor of disease recurrence. In contrast, detection of tyrosinase mRNA by RT-PCR alone does not appear to increase the likelihood of short-term disease recurrence.

Rheumatoid arthritis association with the FCRL3 -169C polymorphism is restricted to PTPN22 1858T-homozygous individuals in a Canadian population.

OBJECTIVE: Variants in genes encoding the Fc receptor-like 3 (FcRL-3) and the class II major histocompatibility complex (MHC) transactivator proteins have been associated with an increased risk of rheumatoid arthritis (RA) in Japanese and Nordic populations, respectively. The aim of this study was to investigate these associations in a Canadian Caucasian cohort of RA cases and healthy controls. METHODS: A total of 1,187 RA patients and 462 healthy controls were genotyped for FCRL3 and MHC2TA gene variants associated with RA. Epistasis between the FCRL3 -169C and the PTPN22 1858T variants was also examined. RESULTS: An association was detected between RA and both the FCRL3 -169C allele (OR 1.19, P = 0.023) and the homozygous genotype (OR 1.41, P = 0.027), but association of the MHC2TA promoter region variant (-168G) with RA was not replicated. Stratification of the RA cohort by PTPN22 genotypes revealed the FCRL3 risk variant and RA association was stronger in the patient subgroup lacking PTPN22 1858T variants (P = 0.004) and was not detectable in the subgroup with PTPN22 1858T variants (P = 0.52). The PTPN22 association with RA was greater in the absence than in the presence of the FCRL3 -169C allele (P = 0.0008 versus P = 0.001). The PTPN22 1858T variant also increased the risk of autoimmune thyroid disease (AITD) in the RA patients, whereas the FCRL3 risk variant was protective against AITD. CONCLUSION: Our findings support an association of RA with an FCRL3 functional polymorphism and reveal that this association is stronger in the absence of PTPN22 risk genotypes. These findings support a genetic heterogeneity across RA populations, suggesting that both the FCRL3 and PTPN22 genes play roles in RA susceptibility, but in different individuals.

Association of the lymphoid tyrosine phosphatase R620W variant with rheumatoid arthritis, but not Crohn's disease, in Canadian populations.

OBJECTIVE: A single-nucleotide polymorphism in the PTPN22 gene encoding the lymphoid protein tyrosine phosphatase (Lyp) has recently been identified as a functional variant associated with susceptibility to rheumatoid arthritis (RA), type 1 diabetes, and systemic lupus erythematosus. To determine whether association of this variant (PTPN22 1858T) with RA is reproducible and is also observed in another autoimmune condition, Crohn's disease, we investigated the association between the PTPN22 1858T allele and RA and Crohn's disease in a Canadian population. METHODS: Two RA case-control cohorts representing a total of 1,234 patients and 791 healthy controls as well as a cohort of 455 patients with Crohn's disease and 190 controls were genotyped for the PTPN22 C1858T polymorphism, and genotype frequencies were compared between patients and controls. RESULTS: Significant association of the PTPN22 1858T allele with RA was detected in both the Toronto-based RA cohort (P = 1.6 x 10(-6), odds ratio [OR] 1.8) and the Halifax-based RA cohort (P = 9.4 x 10(-4), OR 1.94). Association of the risk allele with RA was not affected by sex, age at disease onset, or the presence of either rheumatoid factor or rheumatoid nodules. No association between the PTPN22 risk allele and Crohn's disease was detected. CONCLUSION: These observations confirm the association of RA susceptibility with the PTPN22 1858T allele. However, the data also reveal a lack of association between this variant and Crohn's disease, suggesting that the PTPN22 1858T allele is a risk allele for multiple, but not all, autoimmune diseases.

Intravascular T-cell lymphoma with bowel involvement: case report and literature review.

Intravascular lymphoma (IVL) is a rare form of non-Hodgkin lymphoma characterized by massive proliferation of large, neoplastic cells in small- and medium-sized blood vessels. Most cases of IVL are of B-cell immunophenotype; fewer than 15 cases of T-cell IVL have been reported. A 23-year-old male presented with acute abdominal pain, fever, and tender lower abdomen. Pathology at laparotomy revealed infiltration of colonic vessels with large lymphoid cells compatible with IVL. We reviewed all cases of IVL diagnosed at the Queen Elizabeth II Health Sciences Centre in Halifax, Nova Scotia, from August 1992 to August 2002. A literature review was also performed. Five additional cases of IVL were identified at this institution during a 10-year period. Three patients presented with neurological symptoms, and two with abdominal pain. In 4 of 5 cases, patients died of lymphoma within 3 months of presentation; one patient experienced a 10-month remission. While visceral involvement with IVL is common at autopsy, IVL presenting as an acute abdomen in an immunocompetent patient has not previously been described. Among the 15 cases of T-cell IVL reported in the literature, only two occurred in people under age 30. Given the rarity of T-cell IVL, it is remarkable that three cases of T-cell IVL have been diagnosed at our institution during a 10-year period.

Partial regression of primary cutaneous melanoma: is there an association with sub-clinical sentinel lymph node metastasis?

Whether partial regression of a primary melanoma has an adverse impact on prognosis is controversial. As an indirect mechanism of addressing this question we drew a correlation between the histopathological characteristics of 107 cutaneous melanomas and the presence of sub-clinical metastasis in corresponding sentinel lymph nodes. Partial regression of the primary tumor, defined as focal replacement of the lesion by a scar, unrelated to a previous biopsy, was observed in 20 (19%) cases in the group as a whole. Excluding cases in which an accurate Breslow thickness of the primary melanoma could not be established and/or the presence of a capsular nevus was detected in the sentinel node, a total of 97 remained. Seventeen cases (Breslow thickness 0.63-9.7; mean 2.4 mm) showed partial regression and 80 (Breslow thickness 0.25-7.00; mean 1.8 mm) were devoid of regression. Of the 17 cases with regression 5 (29%) had nodal metastasis (by histopathology and/or molecular analysis) and of the 80 cases without regression 23 (29%) had nodal metastasis (by one or both evaluations). Our data reveals no association between partial regression of the primary melanoma and sentinel node involvement by the disease. The Breslow thickness proved to be the only significant independent variable related to nodal metastasis. Of interest, ulceration of the primary lesion was significantly associated with nodal disease on univariate, but not on multivariate, analysis. While acknowledging that the cohort size may lack the statistical power to demonstrate subtle associations, our data supports the known relevance of tumor thickness and ulceration to regional lymph node metastasis and thereby, to outcome of melanoma in its early stages, but fails to support a similar role for partial regression.