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.

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.

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.

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.

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.

Familial skewed X-chromosome inactivation linked to a component of the cohesin complex, SA2.

The gene dosage inequality between females with two X-chromosomes and males with one is compensated for by X-chromosome inactivation (XCI), which ensures the silencing of one X in every somatic cell of female mammals. XCI in humans results in a mosaic of two cell populations: those expressing the maternal X-chromosome and those expressing the paternal X-chromosome. We have previously shown that the degree of mosaicism (the X-inactivation pattern) in a Canadian family is directly related to disease severity in female carriers of the X-linked recessive bleeding disorder, haemophilia A. The distribution of X-inactivation patterns in this family was consistent with a genetic trait having a co-dominant mode of inheritance, suggesting that XCI choice may not be completely random. To identify genetic elements that could be responsible for biased XCI choice, a linkage analysis was undertaken using an approach tailored to accommodate the continuous nature of the X-inactivation pattern phenotype in the Canadian family. Several X-linked regions were identified, one of which overlaps with a region previously found to be linked to familial skewed XCI. SA2, a component of the cohesin complex is identified as a candidate gene that could participate in XCI through its association with CTCF.

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.

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.

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.

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.

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.

Heritable skewed X-chromosome inactivation leads to haemophilia A expression in heterozygous females.

Factor VIII gene, F8, mutations cause haemophilia A (HA), an X-linked recessive disorder. Expression in heterozygous females has been ascribed to skewed X-chromosome inactivation (XCI). To investigate the cause of HA in three heterozygous females within an Atlantic Canadian kindred, the proband (severely affected girl, FVIII activity: 2%) and 17 relatives across three generations were studied. F8 genotype, FVIII activity, XCI ratio (XCIR) (paternal active X: maternal active X), karyotype, submegabase resolution tiling set array competitive genome hybridization (competitive genomic hybridization (SMRT)), and microsatellite analyses were utilized. A positive linear relationship between FVIII activity and percentage-activated normal X-chromosome was found in HA heterozygous females (R(2)=0.87). All affected, but no unaffected females, had an XCIR skewed toward activation of the mutant X-chromosome (proband 92:8, SD 2). Unexpectedly, high numbers of females have dramatically skewed XCIRs (>80:20 or <20:80) (P<0.05). The distribution of XCIR frequencies within this family was significantly different than predicted by normal population data or models of random XCI (P<0.025), with more females having higher degrees of skewing. Known causes of skewing, such as chromosomal abnormalities, selection against deleterious alleles, and X-inactive-specific transcript mutations, are not consistent with our results. This study shows that FVIII activity in HA heterozygous females can be directly related to XCI skewing, and that low FVIII activity in females in this family is due to unfavourable XCI skewing. Further, the findings suggest that these XCI ratios are genetically influenced, consistent with a novel heritable human X controlling element (XCE) functioning similarly to the mouse Xce.

Polymorphisms of multidrug resistance gene (MDR1) and cyclosporine absorption in de novo renal transplant patients.

BACKGROUND: Several single nucleotide polymorphisms (SNPs) in the multidrug resistance (MDR1) gene may play a role in the interindividual variation of cyclosporine A (CsA) absorption in renal transplant patients. METHODS: An analysis of CsA absorption measured by the dose- and weight-adjusted 4 hr area under the time-concentration curve, AUC(0-4)/mg doseCsA/kg, was conducted on day 3 after transplantation, in 69 de novo renal transplant patients who were genotyped for MDR1 SNPs. Follow-up pharmacogenomic analysis at 1 month posttransplant was performed utilizing dose- and weight-adjusted 2-hour postdose CsA concentration (C2). RESULTS: AUC(0-4)/mg doseCsA/kg was significantly higher (P=0.024) in (C/C)3435 individuals than in a grouped population of (C/T)3435 and (T/T)3435 patients on postoperative day 3. G2677T variants were not significantly correlated with CsA absorption (P=0.084). The number of C3435-G2677 haplotypes was the best predictor of CsA exposure. At 1 month posttransplant, no correlation was seen between MDR1 SNPs and CsA exposure. The frequency of wild-type variants for C3435T and G2677T were 61% and 77.6%, respectively. SNPs at G2677T and C3435T loci were found to be in linkage disequilibrium. CONCLUSIONS: MDR1 polymorphisms are associated with differences in CsA exposure only in the first posttransplant week.

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.

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.

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.

Human X-chromosome inactivation pattern distributions fit a model of genetically influenced choice better than models of completely random choice.

In eutherian mammals, one X-chromosome in every XX somatic cell is transcriptionally silenced through the process of X-chromosome inactivation (XCI). Females are thus functional mosaics, where some cells express genes from the paternal X, and the others from the maternal X. The relative abundance of the two cell populations (X-inactivation pattern, XIP) can have significant medical implications for some females. In mice, the 'choice' of which X to inactivate, maternal or paternal, in each cell of the early embryo is genetically influenced. In humans, the timing of XCI choice and whether choice occurs completely randomly or under a genetic influence is debated. Here, we explore these questions by analysing the distribution of XIPs in large populations of normal females. Models were generated to predict XIP distributions resulting from completely random or genetically influenced choice. Each model describes the discrete primary distribution at the onset of XCI, and the continuous secondary distribution accounting for changes to the XIP as a result of development and ageing. Statistical methods are used to compare models with empirical data from Danish and Utah populations. A rigorous data treatment strategy maximises information content and allows for unbiased use of unphased XIP data. The Anderson-Darling goodness-of-fit statistics and likelihood ratio tests indicate that a model of genetically influenced XCI choice better fits the empirical data than models of completely random choice.

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.

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.