JoAnn C. Kelly

Plasmablastic lymphoma with MYC translocation: evidence for a common pathway in the generation of plasmablastic features

Plasmablastic lymphoma, which is considered a subtype of diffuse large B-cell lymphoma, shares many similar morphological and immunophenotypic features with plasmablastic transformation of plasma cell myeloma. In the setting of human immunodeficiency virus (HIV) infection, both types of neoplasms can be associated with Epstein–Barr virus (EBV), thus making their distinction challenging. Moreover, the biological relationship between these entities remains unclear. We report four unique cases of plasmablastic lymphoma occurring in the setting of HIV infection that had overlapping clinical and genetic features with plasma cell myeloma. We reviewed the clinical, morphological, and cytogenetic findings and performed immunohistochemistry, in situ hybridization for EBV, chromosome analysis, and fluorescent in situ hybridization (FISH) using the MYC break-apart rearrangement probe. All patients were males with a median age of 45 years. In addition to extra-nodal disease, plasmablastic morphology, and phenotype typical of plasmablastic lymphoma, three of the four cases also showed clinical findings overlapping with plasma cell myeloma, that is, monoclonal serum immunoglobulin and lytic bone lesions. Furthermore, these cases showed complex cytogenetic changes that are more commonly observed in plasma cell myeloma. A unique feature was the presence of MYC (8q24.1) rearrangement confirmed by FISH in all four cases. MYC translocation has been associated with tumor progression in multiple myeloma but has only rarely been previously reported in plasmablastic lymphoma. These cases show a clinical and biological relationship between plasmablastic lymphoma and the plasmablastic variant of plasma cell myeloma. Dysregulation of MYC may be a common genetic mechanism that imparts plasmablastic morphology and aggressive clinical course to B-cell neoplasms at a later stage of differentiation.

Exon scanning by reverse transcriptase–polymerase chain reaction for detection of known and novel EML4–ALK fusion variants in non–small cell lung cancer

Chromosomal inversions within chromosome 2p, resulting in fusions between the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes, are a recent focus of treatment options for non-small cell lung cancer. Thirteen EML4-ALK fusion variants have been identified, affecting eight EML4 exons. We have developed an exon scanning approach using multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify known and potential variants involving the first 22 EML4 exons. A total of 55 formalin-fixed, paraffin-embedded lung cancer tumors were screened, of which 5 (9%) were positive for EML4-ALK fusions. Four positive cases harbored known fusion variants: variant 3a, 3b, or both in three cases and variant 1 in one case. The fifth positive specimen harbored two novel variants, designated 8a and 8b, involving exon 17 of EML4. Fluorescence in situ hybridization confirmed the presence of EML4-ALK fusions in three of the four RT-PCR-positive specimens with sufficient tissue for examination, and also confirmed absence of fusions in all 19 RT-PCR-negative specimens tested. Immunohistochemistry analysis confirmed ALK protein expression in the sample containing the novel 8a and 8b variants. This RT-PCR-based exon scanning approach avoids the limitations of screening only for previously identified EML4-ALK fusions and provides a simple molecular assay for fusion detection in a clinical diagnostics setting.

Deletions of the PRKAR1A Locus at 17q24.2-q24.3 in Carney Complex: Genotype-Phenotype Correlations and Implications for Genetic Testing

BACKGROUND Carney complex (CNC) is a multiple neoplasia syndrome caused by PRKAR1A-inactivating mutations. One-third of the patients, however, have no detectable PRKAR1A coding sequence defects. Small deletions of the gene were previously reported in few patients, but large deletions of the chromosomal PRKAR1A locus have not been studied systematically in a large cohort of patients with CNC. SETTING A tertiary care referral center was the setting for analysis of an international cohort of patients with CNC. METHODS Methods included genome-wide array analysis followed by fluorescent in situ hybridization, mRNA, and other studies as well as a retrospective analysis of clinical information and phenotype-genotype correlation. RESULTS We detected 17q24.2-q24.3 deletions of varying size that included the PRKAR1A gene in 11 CNC patients (of 51 tested). Quantitative PCR showed that these patients had significantly lower PRKAR1A mRNA levels. Phenotype varied but was generally severe and included manifestations that are not commonly associated with CNC, presumably due to haploinsufficiency of other genes in addition to PRKAR1A. CONCLUSIONS A significant number (21.6%) of patients with CNC that are negative in currently available testing may have PRKAR1A haploinsufficiency due to genomic defects that are not detected by Sanger sequencing. Array-based studies are necessary for diagnostic confirmation of these defects and should be done in patients with unusual and severe phenotypes who are PRKAR1A mutation-negative.

Incidence and patterns of ALK FISH abnormalities seen in a large unselected series of lung carcinomas

BackgroundAnaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements have been reported in 2-13% of patients with non-small cell lung cancer (NSCLC). Patients with ALK rearrangements do not respond to EGFR-specific tyrosine kinase inhibitors (TKIs); however, they do benefit from small molecule inhibitors targeting ALK.ResultsIn this study, fluorescence in situ hybridization (FISH) using a break-apart probe for the ALK gene was performed on formalin fixed paraffin-embedded tissue to determine the incidence of ALK rearrangements and hybridization patterns in a large unselected cohort of 1387 patients with a referred diagnosis of non-small cell lung cancer (1011 of these patients had a histologic diagnosis of adenocarcinoma). The abnormal FISH signal patterns varied from a single split signal to complex patterns. Among 49 abnormal samples (49/1387, 3.5%), 32 had 1 to 3 split signals. Fifteen samples had deletions of the green 5′ end of the ALK signal, and 1 of these 15 samples showed amplification of the orange 3′ end of the ALK signal. Two patients showed a deletion of the 3′ALK signal. Thirty eight of these 49 samples (38/1011, 3.7%) were among the 1011 patients with confirmed adenocarcinoma. Five of 8 patients with ALK rearrangements detected by FISH were confirmed to have EML4-ALK fusions by multiplex RT-PCR. Among the 45 ALK-rearranged samples tested, only 1 EGFR mutation (T790M) was detected. Two KRAS mutations were detected among 24 ALK-rearranged samples tested.ConclusionsIn a large unselected series, the frequency of ALK gene rearrangement detected by FISH was approximately 3.5% of lung carcinoma, and 3.7% of patients with lung adenocarcinoma, with variant signal patterns frequently detected. Rare cases with coexisting KRAS and EGFR mutations were seen.

Long-term persistence of nonpathogenic clonal chromosome abnormalities in donor hematopoietic cells after allogeneic stem cell transplantation.

We describe the cases of two unrelated patients who exhibited multiple chromosomal abnormalities in donor cells after allogeneic peripheral blood stem cell transplantation (PBSCT). The patients were diagnosed with chronic myeloid leukemia and chronic lymphocytic leukemia, respectively, and both underwent nonmyeloablative conditioning with cyclophosphamide and fludarabine followed by PBSCT from their HLA-matched opposite-sex siblings. Post-transplant bone marrow cytogenetics showed full engraftment, and the early post-transplant studies demonstrated only normal donor metaphases. Subsequent studies of both patients, however, revealed a population of metaphase cells with abnormal, but apparently balanced, donor karyotypes. Chromosome studies performed on peripheral blood cells collected from both donors after transplantation were normal. Both patients remained in clinical remission during follow-up of approximately 8 years in one case, and 6 years in the other case, despite the persistence of the abnormal clones. Chromosomal abnormalities in residual recipient cells after bone marrow or PBSCT are not unusual. In contrast, only rare reports of chromosome abnormalities in donor cells exist, all of which have been associated with post-bone marrow transplant myelodysplastic syndrome or acute leukemias. The present cases demonstrate the rare phenomenon of persistent clonal nonpathogenic chromosome aberrations in cells of donor origin.

Isochromosome 22 in trisomy 22 mosaic with five cell lines

This report describes a full‐term male infant with trisomy 22 due to an isochromosome 22. Prenatal diagnosis with amniotic fluid showed two cell lines, one with an isochromosome 22 and the other with a deleted isochromosome 22. Subsequent cytogenetic analyses of cord blood, umbilical cord tissue, and placenta revealed additional cell lines. A normal cell line was found in umbilical cord tissue and two of three placental sites. The newborn had numerous dysmorphic features and died within 48 hrs of birth.

Abstract 4675: Detection of ALK, ROS1, and RET translocations in non-small cell lung cancer (NSCLC) patients by intragenic differential expression analysis

BACKGROUND: ALK, ROS1, and RET translocations are frequently detected in NSCLC patients. Crizotinib, a tyrosine kinase inhibitor (TKI), was approved by the FDA in 2011 to treat NSCLC in patients harboring ALK translocations as detected by an FDA-approved assay. However, the FDA-approved ALK FISH assay is technically challenging, with failures due to pre-analytic variables. Another approach, intragenic differential expression (IDE), detects translocations by comparing expression levels of the 5′ end with the 3′ end of target gene transcripts. In this study we developed and evaluated a rapid IDE assay to screen for ALK, ROS1, and RET translocations, independent of the fusion partner. METHODS: A total of 419 samples (408 randomly-selected NSCLC clinical samples, ALK positive and ROS1 positive cell lines (2 each), and 7 previously-tested RET-positive clinical samples) were used to develop and evaluate performance characteristics of the IDE assays. To determine IDE scores, levels of ALK, ROS1, and RET expression were first determined by quantitative RT-PCR measurement of the 5′- and 3′- ends of the respective transcripts. The differences in expression levels were calculated as ΔCt (Ct5′ - Ct3′). High ΔCt values indicate presumptive presence of gene translocations. 212/408 NSCLC samples were analyzed by ALK FISH and EML4-ALK RT-PCR, and 196/408 samples were analyzed by EML4-ALK RT-PCR. RESULTS: Thirty-one of the 408 (7.6%) clinical samples tested positive for ALK rearrangements by IDE. Among them, 20 were confirmed by FISH and/or EML4-ALK (true positive, 64.5%), while 11 were negative by FISH and/or EML4-ALK (false positive, 35.5%). One of 10 ALK FISH positive samples tested negative by both ALK IDE and EML4-ALK RT-PCR analysis (false negative), while one of 202 FISH-negative sample tested positive by both EML4-ALK and ALK IDE. ALK IDE exhibited 94.5% (189/200) concordance with ALK FISH and 96.0% (356/371) concordance with the EML4-ALK assay. For ROS1, both ROS1-positive cell lines and 4/408 (1.0%) NSCLC samples tested positive for ROS1 by IDE. Among the 4 IDE-positive NSCLC samples, 1 was confirmed by ROS1 FISH. For RET, all 7 known positives and 10/408 (2.5%) NSCLC samples tested positive by IDE. Three of six RET IDE positive NSCLC samples were confirmed by RET FISH. Overall, ALK, ROS1, and RET translocations were mutually exclusive in NSCLC patients. The lung IDE assay had a failure rate of 3.7%. CONCLUSION: These findings demonstrate the feasibility of using IDE to detect ALK, ROS1, and RET gene translocations. These assays may have potential as a screening tool to select patients for further confirmation by FISH for TKI-targeted therapy. The IDE concept can be applied to a wide range of somatic translocations. Citation Format: Shih-Min Cheng, Cindy Barlan, Feras Hantash, Heather R. Sanders, Patricia H. Chan, Vladimira Sulcova, Marc A. Sanidad, Kevin Qu, Joann C. Kelly, Fatih Z. Boyar, Anthony D. Sferruzza, Frederic M. Waldman. Detection of ALK, ROS1, and RET translocations in non-small cell lung cancer (NSCLC) patients by intragenic differential expression analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4675. doi:10.1158/1538-7445.AM2014-4675

Abstract 3745: Detection of various ALK translocations using intragenic differential expression (IDE) in patients with non-small cell lung cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Efforts to target EML4-ALK fusions with ALK inhibitors in non-small cell lung cancer (NSCLC) have shown promising results in patients harboring a paracentric inversion on chromosome 2, inv(2)(p21p23). In effect, the clinical utility of these drugs is dependent on the presence of ALK gene activation. Reliable testing for ALK activation by translocation is important for selecting patients for this therapy. Although 11 variants for translocation are known, new variants have recently been reported. The aim of this study was to develop a reliable molecular assay to detect translocation of the ALK gene irrespective of the breakpoint on the EML4 gene or the partner gene. To achieve this, we used intragenic differential expression (IDE) of ALK gene comparing expression levels of the 5′ end with the 3′ end. Methods: ALK IDE was determined by measuring the levels of both 5′ and 3′ transcript regions by quantitative RT-PCR. IDE scores were obtained by calculating the endogenous control (ABL1) normalized differences in 5′ and 3′ ALK levels (IDE = 5′ALK/ABL1 - 3′ALK/ABL1). High IDE score, relative to normal, indicates the presence of ALK rearrangement. Relative expression of ALK (independent of rearrangement) was also established. A subset of samples were also analyzed by fluorescence in situ hybridization (FISH). All study samples were analyzed for direct detection of EML4-ALK by RT-PCR performed in a parallel study. Results: The ALK IDE value of an EML4-ALK fusion-positive cell line (NCI-H2228) was 0.7 whereas it was 0.0 for 2 EML4-ALK-negative NSCLC cell lines (NCI H838 and NCI H1299). The positive control value (0.7) was set as the cutoff to distinguish positive vs. negative ALK rearrangement. Eleven percent (6/56) of the lung cancer tissue samples were IDE positive. Eighty-three percent (5/6) of these positives were confirmed by direct detection of EML4-ALK fusion transcript by RT-PCR, including one specimen harboring the previously undescribed variants 8a and 8b. Five IDE-positive samples and 5 with slightly-to-moderately elevated levels of ALK transcript (3′ ALK > 0.1) were further analyzed by FISH. Of these 10 samples, 80% (8/10) showed ALK rearrangement and/or gene amplification. All samples interpreted as having ALK rearrangements by FISH were also positive by IDE (3/3). Two IDE positive (1 confirmed, 1 unconfirmed by RT-PCR) were interpreted as rearrangement negative by FISH. Conclusions: ALK IDE accurately categorized all FISH-confirmed rearrangements as positive and detected rearrangements in at least 1 other confirmed case not identified by FISH. This method is useful for detection of EML4-ALK rearrangements and may function as a universal molecular assay for determining ALK rearrangements in multiple tumor types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3745.