Multiplexed automated digital quantification of fusion transcripts: comparative study with fluorescent in-situ hybridization (FISH) technique in acute leukemia patients.

BACKGROUND: The World Health Organization (WHO) classification system defines recurrent chromosomal translocations as the sole diagnostic and prognostic criteria for acute leukemia (AL). These fusion transcripts are pivotal in the pathogenesis of AL. Clinical laboratories universally employ conventional karyotype/FISH to detect these chromosomal translocations, which is complex, labour intensive and lacks multiplexing capacity. Hence, it is imperative to explore and evaluate some newer automated, cost-efficient multiplexed technologies to accommodate the expanding genetic landscape in AL. METHODS: "nCounter® Leukemia fusion gene expression assay" by NanoString was employed to detect various fusion transcripts in a large set samples (n = 94) utilizing RNA from formalin fixed paraffin embedded (FFPE) diagnostic bone marrow biopsy specimens. This series included AL patients with various recurrent translocations (n = 49), normal karyotype (n = 19), or complex karyotype (n = 21), as well as normal bone marrow samples (n = 5). Fusion gene expression data were compared with results obtained by conventional karyotype and FISH technology to determine sensitivity/specificity, as well as positive /negative predictive values. RESULTS: Junction probes for PML/RARA; RUNX1-RUNX1T1; BCR/ABL1 showed 100 % sensitivity/specificity. A high degree of correlation was noted for MLL/AF4 (85 sensitivity/100 specificity) and TCF3-PBX1 (75 % sensitivity/100 % specificity) probes. CBFB-MYH11 fusion probes showed moderate sensitivity (57 %) but high specificity (100 %). ETV6/RUNX1 displayed discordance between fusion transcript assay and FISH results as well as rare non-specific binding in AL samples with normal or complex cytogenetics. CONCLUSIONS: Our study presents preliminary data with high correlation between fusion transcript detection by a throughput automated multiplexed platform, compared to conventional karyotype/FISH technique for detection of chromosomal translocations in AL patients. Our preliminary observations, mandates further vast validation studies to explore automated molecular platforms in diagnostic pathology.

Differential expression of Toll-like receptor (TLR) and B cell receptor (BCR) signaling molecules in primary diffuse large B-cell lymphoma of the central nervous system.

Primary diffuse large B-cell lymphoma of the central nervous system (CNS DLBCL) is a distinct and aggressive lymphoma that is confined to CNS. Since, central nervous system is barrier-protected and immunologically silent; role of TLR/BCR signaling in pathogenesis and biology of CNS DLBCL is intriguing. Genomic mutations in key regulators of TLR/BCR signaling pathway (MYD88/CD79B/CARD11) have recently been reported in this disease. These observations raised possible implications in novel targeted therapies; however, expression pattern of molecules related to TLR/BCR pathways in this lymphoma remains unknown. We have analyzed the expression of 19 genes encoding TLR/BCR pathways and targets in CNS DLBCLs (n = 20) by Nanostring nCounter™ analysis and compared it with expression patterns in purified reactive B-lymphocytes and systemic diffuse large B cell lymphoma (DLBCL) (n = 20). Relative expression of TLR4, TLR5, TLR9, CD79B and BLNK was higher in CNS DLBCLs than in control B-lymphocytes; where as TLR7, MALT1, BCL10, CD79A and LYN was lower in CNS DLBCLs (P < 0.0001). When compared with systemic DLBCL samples, higher expression of TLR9, CD79B, CARD11, LYN and BLNK was noted in CNS DLBCL (>1.5 fold change; P < 0.01). The B cell receptor molecules like BLNK and CD79B were also associated with higher expression of MYD88 dependent TLRs (TLR4/5/9). In conclusion, we have shown over expression of TLR/BCR related genes or their targets, where genomic mutations have commonly been identified in CNS DLBCL. We have also demonstrated that TLR over expression closely relate with up regulation of genes associated with BCR pathway like CD79B/BLNK and CARD11, which play an important role in NF-kB pathway activation. Our results provide an important insight into the possibility of TLR and/or B-cell receptor signaling molecules as possible therapeutic targets in CNS DLBCL.

High dose salvage therapy with dose intensive cyclophosphamide, etoposide and cisplatin may increase transplant rates for relapsed/refractory aggressive non-Hodgkin lymphoma.

Only one-quarter to one-third of patients with relapsed/refractory aggressive non-Hodgkin lymphoma (r/r-aNHL) treated with common salvage chemotherapy regimens and autologous stem cell transplant (ASCT) achieve 5-year progression-free survival (PFS). Worse outcomes have been reported after failure of prior rituximab-containing induction, initial time to progression (TTP) < 1 year or age-adjusted International Prognostic Index (aaIPI) = 2-3 at relapse. In Calgary, we have treated patients with r/r-aNHL with dose-intensive cyclophosphamide 5.25 g/m(2), etoposide 1.05 g/m(2) and cisplatin 105 mg/m(2) (DICEP) for both re-induction therapy and autologous blood stem cell mobilization. In this study we retrospectively analyzed 113 consecutive transplant-eligible patients with r/r-aNHL who received one cycle of DICEP (n = 93) or R-DICEP (n = 20) from 1995 to 2009. Patient characteristics included: median age = 49 years (22-69); TTP < 1 year = 85; elevated lactate dehydrogenase (LDH) = 60; Eastern Cooperative Oncology Group performance status (ECOG) 2-4 = 42; aaIPI 2-3 = 59; bulk > 10 cm = 26, prior rituximab = 27. The median number of CD34 + cells collected was 19 × 10(6)/kg (0.3-142), 83.5% responded and 90% (102) proceeded to ASCT. The 5-year PFS rate was 42% for all patients, 32% for those with relapse aaIPI = 2-3, 35% for initial TTP < 1 year and 56% for those who failed initial rituximab induction. In conclusion, (R)DICEP is an effective re-induction regimen for r/r-aNHL, leading to excellent stem cell mobilization, a high chance of proceeding to ASCT and encouraging long-term PFS rates.