Debra Saxe

Incidence and significance of chromosome mosaicism involving an autosomal structural abnormality diagnosed prenatally through amniocentesis: A collaborative study

Among 179 663 prenatal diagnosis cases collected from ten institutions and two publications, 555 (0·3 per cent) were diagnosed as having chromosome mosaicism. Of these, 57 (10·3 per cent) were mosaic for an autosomal structural abnormality, 28 (5 per cent) for a sex chromosome structural abnormality, and 85 (15·3 per cent) were mosaic for a marker chromosome. Ninety‐five cases of prenatally diagnosed mosaicism with a structural abnormality in an autosome and a normal cell line, and with a known phenotypic outcome, were collected for karyotype–phenotype correlations through our collaboration (40 cases), a prior survey (26 cases), and published reports (29 cases). They included 13 balanced reciprocal translocations, one unbalanced reciprocal translocation, four balanced Robertsonian translocations, four unbalanced Robertsonian translocations, four inversions, 17 deletions, three ring chromosomes, 19 i(20q), seven +i(12p), six other isochromosomes, and 17 partial trisomies resulting from a duplication or other rearrangement. All cases mosaic for a balanced structural rearrangement resulted in a normal phenotype. All cases of 46/46,i(20q) resulted in normal liveborns. Five of seven cases with 46/47,+i(12p) had an abnormal phenotype compatible with Killian–Pallister syndrome. The overall risk for an abnormal outcome for a mosaic case with an unbalanced structural abnormality, excluding 46/46,i(20q) and 46/47,+i(12p), is 40·4 per cent. In the same category, the study also suggested a correlation between the percentage of abnormal cells and an abnormal phenotype. For mosaicism involving a terminal deletion, the possibility of a familial fragile site should be considered.

Neural-tube defects are associated with low concentrations of cobalamin (vitamin B12) in amniotic fluid.

While folate supplementation reduces the risk of recurrent neural‐tube defects (NTD), both folate and cobalamin deficiencies may be independent risk‐factors for neural‐tube defects. Folate‐dependence and impaired remethylation of homocysteine are implicated as mechanisms for NTD. There are few references reported for folate, cobalamin, homocysteine and methionine in the fetal compartment. This case‐controlled pilot study of amniotic fluid (AF) samples derived from 16 NTD pregnancies and 64 age‐matched controls quantitates total homocysteine (tHcy), total cysteine (tCys), folate, cobalamin (B12), and methionine. Only decreased AF B12 concentrations were found (150 pg/ml versus 540 pg/ml, P<0·02). Since cobalamin, folate and homocysteine participate in the remethylation of homocysteine, via methyl transfer from 5‐methyltetrahydrofolate to B12, to methionine, we compared ratios of these methionine synthase (EC 2.1.1.13) ‐related intermediates. The ratio of B12/folate for NTD versus controls was 48 (34–90) versus 126 (123–182), P<0·001. The ratio of methionine/(folate×tHcy) was 1·4 (1·2–2·2) versus 2·7 (2·4–3·3), P<0·001. We conclude that AF from pregnancies with NTD have lower B12 concentrations, and that ratios of product to substrate(s) of homocysteine remethylation suggest impaired methionine synthase in the fetal compartment through the early second trimester.

NUT Midline Carcinoma in a Newborn with Multiorgan Disseminated Tumor and a 2-Year-Old with a Pancreatic/Hepatic Primary

NUT midline carcinoma (NMC) is a rare and aggressive malignant epithelial tumor defined by rearrangement of the NUT gene on chromosome 15. In two thirds of cases, NUT is involved in a balanced translocation with BDR4 on chromosome 19, while in the remaining cases, NUT is rearranged with variant fusion partners such as BRD3. These undifferentiated tumors primarily affect midline structures, usually in the upper aerodigestive tract and mediastinum. Most reported cases have followed a rapidly lethal clinical course. We report the clinical and pathological findings of NMC in the youngest patients identified so far. The 1st case involves a newborn who presented with a supraorbital mass and extensive multiorgan involvement, including the spine, lungs, liver, pancreas, adrenal glands, and subcutaneous tissue. The 2nd patient was a 2-year-old male with an abdominal mass involving the liver and pancreas with pulmonary metastasis. Histopathological analysis of both tumors showed undifferentiated malignant neoplasms, and immunohistochemistry showed positivity for epithelial markers. Both tumors demonstrated t(15;19), and immunohistochemistry with NUT monoclonal antibodies and fluorescent in situ hybridization confirmed NUT rearrangement. The patients died from disease at 1 and 2 months postpresentation. Thus far, 25 cases have been reported, including our 2 current cases. Presentation ages range from 0 to 78 years (mean, 23 years). Herein, we report the 2 youngest reported cases of NMC, including the 1st congenital case and the 1st case arising within the liver/pancreas. Increased awareness and further molecular studies are required for a better understanding of NMC pathobiology and improved therapeutic outcomes.

Analysis of graft survival in a trial of stem cell transplant in ALS

The first US Food and Drug Administration–approved clinical trial to treat amyotrophic lateral sclerosis (ALS) with neural stem cell–based therapy is in progress. The goal of the current study was to identify and assess the survival of human spinal cord–derived neural stem cells (HSSCs) transplanted into the spinal cord in patients with ALS.

Peripheral blood monitoring of chronic myeloid leukemia during treatment with imatinib, second-line agents, and beyond

The current study was conducted to compare simultaneously obtained bone marrow (BM) cytogenetics (CTG), peripheral blood (PB) and BM fluorescence in situ hybridization (FISH), and quantitative real‐time polymerase chain reaction (Q‐PCR) for BCR‐ABL1 in monitoring response to treatment with tyrosine kinase inhibitors and homoharringtonine (HHT) in patients with chronic myeloid leukemia (CML).

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies

We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated ≥500× coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at ≥5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n = 27), deletions (n = 5), insertions (n = 3), and multinucleotide variants (n = 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines.

The role of CD11c expression in the diagnosis of mantle cell lymphoma.

Flow cytometric immunophenotyping (FCI) aids in the differentiation of chronic lymphocytic leukemia (CLL) from mantle cell lymphoma (MCL); however, overlapping phenotypes may occur. CD11c expression has been reported in up to 90% of CLL cases but has rarely been reported in MCL. Whether CD11c can be used to exclude MCL has not been directly addressed. FCI reports were reviewed for 90 MCL cases (44 patients) and 355 CLL/small lymphocytic lymphoma (SLL) cases (158 patients). MCL cases were confirmed by cyclin D1 immunoreactivity and/or t(11;14) detection by karyotyping or fluorescence in situ hybridization. Cases with typical MCL immunophenotypes did not express CD11c. The 2 MCL cases displaying dim CD11c positivity (2 of 44 patients) expressed other markers not typical of MCL. CD11c was detected in 96 (27.0%) of 355 cases of CLL/SLL representing 53 of 158 patients. CD11c expression is rare in MCL and may aid in differentiation of CD5+ B-cell neoplasms, particularly when small samples limit further ancillary testing.

A Novel Flow Cytometric Antibody Panel for Distinguishing Burkitt Lymphoma From CD10+ Diffuse Large B-Cell Lymphoma

Rapid and accurate differential diagnosis between Burkitt lymphoma (BL) and CD10+ diffuse large B-cell lymphoma (DLBCL) is imperative because their treatment differs. Recent studies have characterized several antigens differentially expressed in these 2 types of lymphoma. Our goal was to determine whether use of these markers would aid in the differential diagnosis of BL vs CD10+ DLBCL by flow cytometric immunophenotyping (FCI). Twenty-three cases of CD10+ B-cell lymphomas with available cryopreserved samples were identified (13 BL and 10 CD10+ DLBCL). Multiparameter FCI was performed using the following antibodies: CD18, CD20, CD43, CD44, and CD54 and isotype controls. Expression of CD44 and CD54 was detected at a significantly lower level in BL compared with CD10+ DLBCL (P = .001 and P = .01, respectively). There was not a significant difference in expression of CD18 and CD43. Our data show that expression of CD44 and CD54 differs significantly between BL and CD10+ DLBCL.

The role of immunohistochemical analysis in the evaluation of EML4-ALK gene rearrangement in lung cancer

Background:Among the mutations described in non–small cell lung carcinoma is a rearrangement resulting from an inversion within chromosome 2p leading to the formation of a fusion gene, echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK). Fluorescence in situ hybridization (FISH) is the gold standard for the detection of ALK gene rearrangements. However, molecular methods are not readily available in all pathology laboratories. Immunohistochemistry (IHC) using an antibody directed against the EML4-ALK fusion protein provides a widely available alternative method of detection. We assessed whether IHC is a comparable and cost-effective alternative to FISH analysis for the detection of ALK gene rearrangements. Design:A total of 110 non–small cell lung carcinoma cases (63 surgical/biopsy and 47 cytology specimens), previously tested for ALK gene rearrangements by FISH [7 (6.4%) positive for the rearrangement], were probed for the EML4-ALK fusion protein using a monoclonal EML4-ALK antibody, clone 5A4. Cells were considered to stain positive for ALK if >5% of cells showed cytoplasmic staining of at least grade 1 intensity (scale: 0 to 3). A cost analysis was performed using ALK IHC as a screening test. Results:The sensitivity and specificity of the EML4-ALK IHC stain compared with ALK FISH analysis were 100% and 96%, respectively. All 7 FISH-positive cases stained positive by IHC, whereas 4 FISH-negative cases demonstrated positive staining. One of the 4 FISH-negative, IHC-positive cases harbored an EML4-ALK rearrangement by RT-PCR yielding 3 false-positive results overall. The &kgr; agreement between IHC and FISH methods is 0.76 (substantial/excellent). The potential savings of implementing the ALK IHC as a screening method would be

Characteristics and outcomes of diffuse large B-cell lymphoma presenting in leukaemic phase

10,418.21. Conclusions:Sensitivity of the EML4-ALK IHC stain is excellent (100%) but due to its suboptimal specificity, IHC cannot reliably supplant FISH analysis for the detection of ALK gene rearrangements. IHC shows promise as a screening tool to prevent unnecessary costly FISH analysis.