DIAGNOSTIC%20TOOLS%20IN%20ONCO-HEMATOLOGY

CENTRO DI RIFERIMENTO ONCOLOGICO

Technology Transfer Opportunities Contact: Ermes Mestroni emestroni@cro.it

DIAGNOSTIC TOOLS IN ONCO-HEMATOLOGY Technology Overview The Clinical and Experimental Oncohematology Division of CRO Aviano constantly identifies novel molecular and immunophenotypic diagnostic / prognostic profiles of hematological malignancies.

Likewise, research efforts are aimed at clarifying various aspects of the physiopathology of B cell chronic lymphocytic leukemia (B-CLL).

Lab operation uses progressive state-of-the-art technologies and procedures to evaluate chromosomal translocations and other gene abnormalities. 7 thermal cyclers (MJ Research PCT) for qualitative Polymerase Chain Reaction detection, ABI Prism® 7700 Real Time PCR machine. For DNA sequencing of rearranged products and others, a multi-color fluorescence-based 4 parallel-capillary ABI PRISM® 3130 Genetic Analyzer offers high-quality data and efficient sample processing. Core facility and Agilent G2565AA scanner for GEP analysis.

Advantages √ All the PCR protocols are characterized by the same cycling conditions and ready-to-use PCR tubes for each specific translocation. √ Up-to-date laboratories equipped with core facility for “gene expression profiling” (GEP), instruments, reagents, study designed and data analysis. √ Fresh and frozen primary CLL cells available, vials from at least 400 CLL cases well characterized for IGHV gene status and expression of prognosticators (CD38, CD49d, ZAP-70 etc.), viable frozen samples of a wide panel of human leukemia/lymphoma B cell lines. √ Quickness of execution. √ Expertise and extensive international network of contacts.

Development Stage Several PCR protocols have been developed to detect the most important genetic alterations involved in the onco-hematological diseases. Study and identification of novel molecular diagnostic and prognostic markers to be introduced in routine of clinical activities. Main genetic alterations detected (since 1996): Leukemias: -

BCR/ABL [t(9;22) M-BCR] BCR/ABL [t(9;22) m-BCR] BCR/ABL [t(9;22) µ-BCR] AML1/ETO [t(8;21)] PML/RAR [t(15;17)] CBFß/MYH11 [inv(16)] MLL/AF4 [t(4;11)] E2A/PBX1 [t(1;19)] TEL/AML1 [t(12;21)] DEK-CAN [t(6;9)]

Lymphomas: -

ALK/NPM [t(2;5)] BCL2/IgH [t(14;18) MBR] BCL2/IgH [t(14;18) mcr] BCL1/IgH [t(11;14) MTC] API2/MLT [t(11;18)] B cell Clonality: (FR1-JH/CDR3-JH) T cell Clonality: TCR gamma

Myeloproliferative disorders: -

JAK-2 detection by Amplification Refractory Mutation System (ARMS-PCR)

CENTRO DI RIFERIMENTO ONCOLOGICO

Technology Transfer Opportunities Contact: Ermes Mestroni emestroni@cro.it

Chronic Lymphocytic Leukemia (CLL) Patients with CLL from diagnosis to prognosis to therapeutic monitoring are investigated by use of stateof-the-art technologies. A definitive diagnosis of CLL is obtained with morphology and flow-cytometry, then, physicians can stratify risk with use of FISH and molecular testing.

IgVH Mutation Analysis in CLL The Immunoglobulin Variable Region Heavy chain gene encodes antibodies that function in the immune response. ~50-70% of CLL patients have evidence of IgVH somatic mutations whose status identifies 2 CLL subtypes with differing clinical course: indolent and aggressive. * Patients with hypermutated IgVH have a better prognosis (median survival 293 months); * Patients without hypermutated IgVH, poorer prognosis (117 months).

RNA is reverse transcribed into cDNA and amplified by the PCR using primer sets for the IgVH gene.

PCR products are analyzed by direct DNA sequencing using capillary gel electrophoresis and fluorescence detection. The percentage mutation is assessed by comparison of the VH region sequence to a germline gene sequence database (a sequence that differs by >2% is defined as Mutated).

Detection of the mutational status of IgVH genes in B-CLL for prognostic evaluation, i) Fluorescence-insitu hybridization (FISH) for 11q-, +12, 13q-, 17p- evaluation in B-CLL; ii) Zap70 detection.

Other Ig genes feature evaluations: evidence of antigen-driven selection, preferential usage of IgVH gene segments, usage of stereotyped IgVH-CDR3-IgVL combinations refining the prognostic assessment of CLL by identifying stereotyped BCR combinations associated with different prognosis, putative antigens responsible for neoplastic transformation or Ig combination to be used as targets for immunotherapy. p53 Mutation Analysis in CLL Over 10% of B-CLL patients have a dysfunctional p53 tumor suppressor gene on chromosome 17p13.1: p53 mutations, a p53 deletion or both, that predict a 6-31 month poor survival (Patients with normal karyotype: >100 months). Alkylating agents, purine analogs and some monoclonal therapies are ineffective in treating those CLL patients. Finally, chemotherapy can cause p53 gene alterations, such that even if not present at initial diagnosis, refractory CLL can exhibit new alterations of p53.

The p53 mutation assay incorporates PCR amplification and bidirectional sequencing of p53 exons 5-9 along with their respective flanking splice sites. The assay can detect a mutation present in at least 20% of a B-cell enriched sample. FISH analysis is also available to detect a deletion of the 17p chromosome.

Looking for Tech transfer processes in both pre-clinical and diagnostic activities through the obtaining of state-of-theart technologies for patient management. Collaborations with industrial partners aimed to:

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develop diagnostic kits ready-to-use for an advanced assessment of onco-hematological disease (genetic alterations). Commercialization and distribution; in vitro test of putative drugs for CCL therapy (evaluate the role, investigate the effect, analyze the distribution, other). Service procedures on blood samples incl. FISH testing.

More Information Valter Gattei, MD Clinical and Experimental Onco-Hematology Unit vgattei@cro.it Tel.: +39-0434-659-410