UPS - ACP, CUHK

Molecular Haematopathology

Test Description and Value

Haematolymphoid Maliganancies

PCR for BCL-1 translocation t(11;14)(q13;q32)

The IgH/BCL-1 t(11;14)(q13;q32) chromosomal translocation is found in 50-70% of mantle cell lymphoma, 10-20% of B-cell prolymphocytic leukaemia, plasma cell leukaemia, and splenic lymphoma with villous lymphocytes, as well as 2-5% of chronic lymphocytic leukaemia and multiple myeloma. Mantle cell lymphomas are typically more aggressive and have a worse prognosis than other low grade B-cell lymphomas. The test is employed to confirm the diagnosis of B-cell lymphomas, particularly mantle cell lymphoma. In addition, the BCL-1 translocation can be used as a tumor-specific marker for monitoring disease progression and treatment response.

References:

Raffeld M, Jaffe ES. bcl-1, t(11;14), and mantle cell-derived lymphomas. Blood. 1991; 78: 259-63.

Rimokh R, Berger F, Delsol G, Charrin C, Bertheas MF, Ffrench M, et al. Rearrangement and overexpression of the BCL-1/PRAD-1 gene in intermediate lymphocytic lymphomas and in t(11q13)-bearing leukemias. Blood. 1993; 81: 3063-7.

Jadayel D, Matutes E, Dyer MJ, Brito-Babapulle V, Khohkar MT, Oscier D, et al. Splenic lymphoma with villous lymphocytes: analysis of BCL-1 rearrangements and expression of the cyclin D1 gene. Blood. 1994; 83: 3664-71.

Kodet R, Mrhalova M, Krskova L, Soukup J, Campr V, Neskudla T, et al. Mantle cell lymphoma: improved diagnostics using a combined approach of immunohistochemistry and identification of t(11;14)(q13;q32) by polymerase chain reaction and fluorescence in situ hybridization. Virchows Arch. 2003; 442: 538-47.

PCR for BCL-2 translocation t(14;18)(q32;q21) – Major breakpoint region

The IgH/BCL-2 t(14;18)(q32;q21) chromosomal translocation is the most frequent karyotypic aberration in malignant lymphomas. About 65% of the BCL-2 breakpoints occur in the major breakpoint region (MBR). The translocation is found in 70-90% of follicular lymphoma, 50% of undifferentiated B-cell lymphoma, and 20-30% of diffuse large B-cell lymphoma. The test is useful in differentiating follicular lymphoma from other types of B-cell lymphomas that may have a similar morphological appearance (e.g. mantle cell lymphoma or lymphomas of mucosa associated lymphoid tissue [MALTomas]). Like BCL-1 translocation, the BCL-2 translocation can also be used as a tumor-specific marker for monitoring disease progression and treatment response.

References:

Cleary ML, Sklar J . Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint-cluster region near a transcriptionally active locus on chromosome 18. Proc Natl Acad Sci U S A. 1985; 82: 7439-43.

Ngan BY, Nourse J, Cleary ML. Detection of chromosomal translocation t(14;18) within the minor cluster region of bcl-2 by polymerase chain reaction and direct genomic sequencing of the enzymatically amplified DNA in follicular lymphomas. Blood. 1989; 73: 1759-62.

Gribben JG, Freedman A, Woo SD, Blake K, Shu RS, Freeman G, et al. All advanced stage non-Hodgkin's lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment. Blood. 1991; 78: 3275-80.

Zuckerman E, Zuckerman T, Sahar D, Streichman S, Attias D, Sabo E, et al. bcl-2 and immunoglobulin gene rearrangement in patients with hepatitis C virus infection. Br J Haematol. 2001; 112: 364-9.

IgH gene rearrangement (clonality assessment)

The IgH and TCR g / b gene rearrangement tests are requested for determination of clonality in suspected lymphoproliferative disorders. The tests are useful when malignant and reactive lymphoproliferations cannot be discriminated using immunohistology or immunophenotyping. In general, monoclonality correlates with neoplasia, whereas polyclonality correlates with benign reactive conditions. The clonal IgH/TCR g / b gene rearrangement (determined by nucleotide sequencing of the PCR product) can also be used as a tumor-specific marker for monitoring disease progression and treatment response.

References:

Trainor KJ, Brisco MJ, Wan JH, Neoh S, Grist S, Morley AA. Gene rearrangement in B- and T-lymphoproliferative disease detected by the polymerase chain reaction. Blood. 1991; 78: 192-6.

Deane M, McCarthy KP, Wiedemann LM, Norton JD. An improved method for detection of B-lymphoid clonality by polymerase chain reaction. Leukemia. 1991; 5: 726-30.

Cossman J, Zehnbauer B, Garrett CT, Smith LJ, Williams M, Jaffe ES, et al. Gene rearrangements in the diagnosis of lymphoma/leukemia. Guidelines for use based on a multiinstitutional study. Am J Clin Pathol. 1991; 95: 347-54.

van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia. 2003; 17: 2257-317.

T-cell receptor gamma / beta gene rearrangement (clonality assessment)

The IgH and TCR g / b gene rearrangement tests are indicated for determination of clonality in suspected lymphoproliferative disorders. The tests are useful when malignant and reactive lymphoproliferations cannot be discriminated using immunohistology or immunophenotyping. In general, monoclonality correlates with neoplasia, whereas polyclonality correlates with benign reactive conditions. The clonal IgH/TCR g / b gene rearrangement (determined by nucleotide sequencing of the PCR product) can also be used as a tumor-specific marker for monitoring disease progression and treatment response.

References:

Trainor KJ, Brisco MJ, Wan JH, Neoh S, Grist S, Morley AA. Gene rearrangement in B- and T-lymphoproliferative disease detected by the polymerase chain reaction. Blood. 1991; 78: 192-6.

Deane M, McCarthy KP, Wiedemann LM, Norton JD. An improved method for detection of B-lymphoid clonality by polymerase chain reaction. Leukemia. 1991; 5: 726-30.

BRAF V 600E Mutation Screening for Hairy Cell Leukaemia

The BRAF V600E mutation, caused by a T>A transversion in exon 15 of BRAF , has been identified as the disease-defining genetic event in hairy cell leukaemia (HCL). This mutation is found in all HCL cases but very rarely in other non-HCL chronic B-cell neoplasms. The test is thus useful in differentiating the diagnosis of HCL from other HCL-like disorders such as splenic marginal zone lymphoma and HCL variant. This is clinically important because current therapies with purine analogs (pentostatin and cladribine) are highly effective only in patients with HCL. In addition, BRAF -mutated HCL patients may be benefited from future therapeutic approaches using BRAF V600E inhibitors which have already shown remarkable activity in metastatic melanoma.

References:

Tiacci E , Trifonov V , Schiavoni G , Holmes A , Kern W , Martelli MP , et al. BRAF mutations in hairy-cell leukemia. N Engl J Med . 20 11 ; 364 : 2305-15 .

Arcaini L , Zibellini S , Boveri E , Riboni R , Rattotti S , Varettoni M , et al. The BRAF V600E mutation in hairy cell leukemia and other mature B-cell neoplasms. Blood . 20 12 ; 119 : 1 88 - 91 .

Tiacci E , Schiavoni G , Forconi F , Santi A , Trentin L , Ambrosetti A , et al. Simple genetic diagnosis of hairy cell leukemia by sensitive detection of the BRAF-V600E mutation. Blood . 2012;119:1 92-5 .

Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA , et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med . 20 10 ; 363 : 809-19 .

Myeloproligerative diseases

JAK2 tyrosine kinase gene Val617Phe Mutation Screening

The JAK2 V617F mutation, caused by a G>T transversion in exon 14 of JAK2, has been discovered by several teams using different approaches in the Philadelphia-negative myeloproliferative disorders. The test is requested for confirmation of the diagnosis of chronic myeloproliferative disorders and has been incorporated as a diagnostic criteria for polycythaemia vera, essential thrombocythaemia, and primary myelofibrosis by the World Health Organization. The mutation can also be used as a molecular marker for monitoring disease progression and treatment response. In addition, patients with the JAK2 mutation may be benefited from future therapeutic approaches using small-molecule JAK2 inhibitors.

References:

Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, et al . Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005; 365: 1054-61.

James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005; 434: 1144-8.

Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005; 352: 1779-90.

Pardanani A. JAK2 inhibitor therapy in myeloproliferative disorders: rationale, preclinical studies and ongoing clinical trials. Leukemia. 2008; 22: 23-30.

ZNF198-FGFR1 derived from Ph NEG myeloproliferative disorder with t(8;13)

The ZNF198-FGFR1 gene fusion derived from t(8;13)(p11;q12) is associated with an aggressive, atypical stem cell myeloproliferative disorder. The ZNF198-FGFR1 fusion protein is a constitutively activated tyrosine kinase that activates various downstream effectors and can transform murine haematopoietic cells to growth factor-independent growth. This fusion marker can be used for monitoring disease progression and treatment response. Patients with ZNF198-FGFR1 fusion may be targeted with small-molecule tyrosine kinase inhibitors like PKC412.

References:

Xiao S, Nalabolu SR, Aster JC, Ma J, Abruzzo L, Jaffe ES, et al. FGFR1 is fused with a novel zinc-finger gene, ZNF198, in the t(8;13) leukaemia/lymphoma syndrome. Nat Genet. 1998; 18: 84-7.

Still IH, Cowell JK. The t(8;13) atypical myeloproliferative disorder: further analysis of the ZNF198 gene and lack of evidence for multiple genes disrupted on chromosome 13. Blood. 1998; 92: 1456-8.

Smedley D, Hamoudi R, Clark J, Warren W, Abdul-Rauf M, Somers G, et al. The t(8;13)(p11;q11-12) rearrangement associated with an atypical myeloproliferative disorder fuses the fibroblast growth factor receptor 1 gene to a novel gene RAMP. Hum Mol Genet. 1998; 7: 637-42.

Chen J, Deangelo DJ, Kutok JL, Williams IR, Lee BH, Wadleigh M, et al. PKC412 inhibits the zinc finger 198-fibroblast growth factor receptor 1 fusion tyrosine kinase and is active in treatment of stem cell myeloproliferative disorder. Proc Natl Acad Sci U S A. 2004; 101: 14479-84.

Acute Lymphoblastic Leukaemia (ALL)

MLL-AF4 derived from ALL with t(4;11)(q21;q23)

The MLL-AF4 gene fusion is a hallmark of high-risk acute lymphoblastic leukaemia, with a particularly poor prognosis in infants. In addition, the fusion can be used as a molecular marker for monitoring disease progression and treatment response.

References:

Domer PH, Fakharzadeh SS, Chen CS, Jockel J, Johansen L, Silverman GA, et al. Acute mixed-lineage leukemia t(4;11)(q21;q23) generates an MLL-AF4 fusion product. Proc Natl Acad Sci U S A. 1993; 90: 7884-8.

Pallisgaard N, Hokland P, Riishoj DC, Pedersen B, Jorgensen P. Multiplex reverse transcription-polymerase chain reaction for simultaneous screening of 29 translocations and chromosomal aberrations in acute leukemia. Blood. 1998; 92: 574-88.

Ferrando AA, Look AT. Clinical implications of recurring chromosomal and associated molecular abnormalities in acute lymphoblastic leukemia. Semin Hematol. 2000; 37: 381-95.

Thomas M, Gessner A, Vornlocher HP, Hadwiger P, Greil J, Heidenreich O. Targeting MLL-AF4 with short interfering RNAs inhibits clonogenicity and engraftment of t(4;11)-positive human leukemic cells. Blood. 2005; 106: 3559-66.

E2A-PBX1 derived from ALL with t(1;19)(q23;p13)

The E2A-PBX1 gene fusion is found in about 25% of paediatric pre-B acute lymphoblastic leukaemia (ALL). Its presence is indicative of pre-B ALL and is generally associated with a less favourable prognosis. In addition, the fusion can be used as a molecular marker for monitoring disease progression and treatment response.

References:

Hunger SP, Galili N, Carroll AJ, Crist WM, Link MP, Cleary ML. The t(1;19)(q23;p13) results in consistent fusion of E2A and PBX1 coding sequences in acute lymphoblastic leukemias. Blood. 1991; 77: 687-93.

Ferrando AA, Look AT. Clinical implications of recurring chromosomal and associated molecular abnormalities in acute lymphoblastic leukemia. Semin Hematol. 2000; 37: 381-95.

Aspland SE, Bendall HH, Murre C. The role of E2A-PBX1 in leukemogenesis. Oncogene. 2001; 20: 5708-17.

TEL-AML1 derived from ALL with t(12;21)(p13;q22)

t(12;21) is often a cryptic translocation not readily detected by G-banding cytogenetic analysis. The resulting TEL-AML1 gene fusion is found in up to 25% of paediatric B-lineage acute lymphoblastic leukaemia but is considerably less prevalent in adult patients (~3%). Its presence is associated with a relatively favourable prognosis. In addition, the fusion can be used as a molecular marker for monitoring disease progression and treatment response.

References:

Golub TR, Barker GF, Bohlander SK, Hiebert SW, Ward DC, Bray-Ward P, et al. Fusion of the TEL gene on 12p13 to the AML1 gene on 21q22 in acute lymphoblastic leukemia. Proc Natl Acad Sci U S A. 1995; 92: 4917-21.

Ferrando AA, Look AT. Clinical implications of recurring chromosomal and associated molecular abnormalities in acute lymphoblastic leukemia. Semin Hematol. 2000; 37: 381-95.

Zelent A, Greaves M, Enver T. Role of the TEL-AML1 fusion gene in the molecular pathogenesis of childhood acute lymphoblastic leukaemia. Oncogene. 2004; 23: 4275-83.

BCR-ABL derived from ALL with t(9;22)(q34;q11)

The presence of BCR-ABL gene fusion (typically e1a2) in acute lymphoblastic leukaemia is associated with a worse prognosis and this fusion marker can be used for monitoring disease progression and treatment response. In addition, patients carrying this fusion may be targeted with BCR-ABL inhibitors such as imatinib and dasatinib.

References:

Kurzrock R, Shtalrid M, Romero P, Kloetzer WS, Talpas M, Trujillo JM, et al. A novel c-abl protein product in Philadelphia-positive acute lymphoblastic leukaemia. Nature. 1987; 325: 631-5.

Ferrando AA, Look AT. Clinical implications of recurring chromosomal and associated molecular abnormalities in acute lymphoblastic leukemia. Semin Hematol. 2000; 37: 381-95.

Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, Ford JM, et al. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med. 2001; 344: 1038-42.

Acute Myeloid Leukaemia (AML)

AML1-ETO derived from AML with t(8;21)(q22;q22)

The AML1-ETO gene fusion is found in about 12-15% of de novo acute myeloid leukaemia and up to 40% of the French-American-British (FAB) M2 subtype. Its presence is associated with a relatively favourable prognosis. In addition, the fusion can be used as a molecular marker for monitoring disease progression and treatment response.

References:

Erickson P, Gao J, Chang KS, Look T, Whisenant E, Raimondi S, et al. Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt. Blood. 1992; 80: 1825-31.

Downing JR, Head DR, Curcio-Brint AM, Hulshof MG, Motroni TA, Raimondi SC, et al. An AML1/ETO fusion transcript is consistently detected by RNA-based polymerase chain reaction in acute myelogenous leukemia containing the (8;21)(q22;q22) translocation. Blood. 1993; 81: 2860-5.

Peterson LF, Zhang DE. The 8;21 translocation in leukemogenesis. Oncogene. 2004 ; 23: 4255-62.

CBF b -MYH11 derived from AMLEso with inv(16)(p13;q22)

The CBF b -MYH11 gene fusion is associated with the French-American-British (FAB) M4 subtype with abnormal eosinophils and confers a relatively favourable prognosis. In addition, the fusion can be used as a molecular marker for monitoring disease progression and treatment response.

References:

Liu P, Tarle SA, Hajra A, Claxton DF, Marlton P, Freedman M, et al. Fusion between transcription factor CBF beta/PEBP2 beta and a myosin heavy chain in acute myeloid leukemia. Science. 1993; 261: 1041-4.

Claxton DF, Liu P, Hsu HB, Marlton P, Hester J, Collins F, et al. Detection of fusion transcripts generated by the inversion 16 chromosome in acute myelogenous leukemia. Blood. 1994; 83: 1750-6.

Shigesada K, van de Sluis B, Liu PP. Mechanism of leukemogenesis by the inv(16) chimeric gene CBFB/PEBP2B-MHY11. Oncogene. 2004; 23: 4297-307.

MLL-AF9 derived from AML with t(9;11)(p22;q23)

The MLL-AF9 gene fusion is often found in the French-American-British (FAB) M5 subtype and in therapy-related AML following treatment with topoisomerase II inhibitors. The presence of MLL rearrangement is generally associated with a worse prognosis as compared to other AML subtypes although it was reported that MLL-AF9 might have a favourable impact in childhood acute myeloid leukaemia. In addition, the fusion can be used as a molecular marker for monitoring disease progression and treatment response.

References:

Poirel H, Rack K, Delabesse E, Radford-Weiss I, Troussard X, Debert C, et al. Incidence and characterization of MLL gene (11q23) rearrangements in acute myeloid leukemia M1 and M5. Blood. 1996; 87: 2496-505.

Ayton PM, Cleary ML. Molecular mechanisms of leukemogenesis mediated by MLL fusion proteins. Oncogene. 2001; 20: 5695-707.

Rubnitz JE, Raimondi SC, Tong X, Srivastava DK, Razzouk BI, Shurtleff SA, et al. Favourable impact of the t(9;11) in childhood acute myeloid leukemia. J Clin Oncol. 2002; 20: 2302-9.

MLL-AF10 derived from AML with t(10;11)(p12;q23)

The MLL-AF10 gene fusion is often found in the French-American-British (FAB) M5 subtype and in therapy-related AML following treatment with topoisomerase II inhibitors. The prognosis of patients with MLL-AF10 fusion is poor. In addition, the fusion can be used as a molecular marker for monitoring disease progression and treatment response.

References:

Chaplin T, Bernard O, Beverloo HB, Saha V, Hagemeijer A, Berger R, et al. The t(10;11) translocation in acute myeloid leukemia (M5) consistently fuses the leucine zipper motif of AF10 onto the HRX gene. Blood. 1995; 86: 2073-6.

Ayton PM, Cleary ML. Molecular mechanisms of leukemogenesis mediated by MLL fusion proteins. Oncogene. 2001; 20: 5695-707.

Mirault ME, Boucher P, Tremblay A. Nucleotide-resolution mapping of topoisomerase-mediated and apoptotic DNA strand scissions at or near an MLL translocation hotspot. Am J Hum Genet. 2006; 79: 779-91.

PML-RAR a derived from AML with t(15;17)(q22;q21)

The PML-RAR a gene fusion is a hallmark of acute promyelocytic leukaemia (FAB M3 subtype) and predicts a beneficial response to all-trans retinoic acid treatment. In addition, the fusion can be used as a molecular marker for monitoring disease progression and treatment response.

References:

de The H, Lavau C, Marchio A, Chomienne C, Degos L, Dejean A. The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell. 1991; 66: 675-84.

Lin RJ, Sternsdorf T, Tini M, Evans RM. Transcriptional regulation in acute promyelocytic leukemia. Oncogene. 2001; 20: 7204-15.

Mistry AR, Pedersen EW, Solomon E, Grimwade D. The molecular pathogenesis of acute promyelocytic leukaemia: implications for the clinical management of the disease. Blood Rev. 2003; 17: 71-97.

Chronic Myeloid Leukaemia

BCR-ABL (b2a2 & b3a2) derived from CML with t(9;22)(q34;q11)

The BCR-ABL gene fusion (typically b2a2 and b3a2) is a diagnostic marker of chronic myeloid leukaemia and can be used for monitoring disease progression and treatment response. In addition, patients carrying this fusion may be targeted with BCR-ABL inhibitors such as imatinib and dasatinib.

References:

Teyssier JR, Bartram CR, Deville J, Potron G, Pigeon F. c-abl Oncogene and chromosome 22 "bcr" juxtaposition in chronic myelogenous leukemia. N Engl J Med. 1985; 312: 1393-4.

Ren R. Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia. Nat Rev Cancer. 2005; 5: 172-83.

Quantitative RT-PCR for BCR-ABL transcript (b2a2 & b3a2)

The test is requested for monitoring the treatment response of patients with chronic myeloid leukaemia to imatinib mesylate (or Gleevec). Serial monitoring of individual patients is recommended because a rising level of BCR-ABL transcripts is an early indication of loss of response and thus warrants alternative treatment regimens before the onset of relapse.

References:

Branford S, Hughes TP, Rudzki Z. Monitoring chronic myeloid leukaemia therapy by real-time quantitative PCR in blood is a reliable alternative to bone marrow cytogenetics. Br J Haematol. 1999; 107: 587-99.

Branford S, Rudzki Z, Parkinson I, Grigg A, Taylor K, Seymour JF, et al. Real-time quantitative PCR analysis can be used as a primary screen to identify patients with CML treated with imatinib who have BCR-ABL kinase domain mutations. Blood. 2004; 104: 2926-32.

Hughes T, Deininger M, Hochhaus A, Branford S, Radich J, Kaeda J, et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood. 2006; 108: 28-37.

Hughes T, Branford S. Molecular monitoring of BCR-ABL as a guide to clinical management in chronic myeloid leukaemia. Blood Rev. 2006; 20: 29-41.

Chronic Myelomonocytic Leukaemia & Myelodysplastic Syndrome

TEL-PDGFR b derived from CMML / MDS with t(5;12)(q33;p13)

The TEL-PDGFR b gene fusion is associated with chronic myelomonocytic leukaemia (CMML) and myelodysplastic syndrome (MDS). The TEL-PDGFR b is a novel transforming protein that self-associates and constitutively activates PDGFRbeta-dependent signaling pathways. Patients with this fusion may be targeted with the tyrosine kinase inhibitor imatinib mesylate and the fusion marker can be used for monitoring disease progression and treatment response.

References:

Golub TR, Barker GF, Lovett M, Gilliland DG. Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell. 1994; 77: 307-16.

Carroll M, Tomasson MH, Barker GF, Golub TR, Gilliland DG. The TEL/platelet-derived growth factor beta receptor (PDGF beta R) fusion in chronic myelomonocytic leukemia is a transforming protein that self-associates and activates PDGF beta R kinase-dependent signaling pathways. Proc Natl Acad Sci U S A. 1996; 93: 14845-50.

Apperley JF, Gardembas M, Melo JV, Russell-Jones R, Bain BJ, Baxter EJ, et al. Response to imatinib mesylate in patients with chronic myeloproliferative diseases with rearrangements of the platelet-derived growth factor receptor beta. N Engl J Med. 2002; 347: 481-7.

Stover EH, Chen J, Lee BH, Cools J, McDowell E, Adelsperger J, et al. The small molecule tyrosine kinase inhibitor AMN107 inhibits TEL-PDGFRbeta and FIP1L1-PDGFRalpha in vitro and in vivo. Blood. 2005; 106: 3206-13.

Chronic Eosinophilic Leukaemia

FIP1L1-PDGFRa derived from CEL with an interstitial deletion on chromosome 4q12

The FIP1L1-PDGFR a gene fusion is associated with eosinophilia-associated myeloproliferative disorders such as chronic eosinophilic leukaemia (CEL) and patients with this fusion may be targeted with the tyrosine kinase inhibitor imatinib mesylate. In addition, the fusion can be used as a molecular marker for monitoring disease progression and treatment response.

References:

Cools J, DeAngelo DJ, Gotlib J, Stover EH, Legare RD, Cortes J, et al. A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome. N Engl J Med. 2003; 348: 1201-14.

Cools J, Stover EH, Boulton CL, Gotlib J, Legare RD, Amaral SM, et al. PKC412 overcomes resistance to imatinib in a murine model of FIP1L1-PDGFRalpha-induced myeloproliferative disease. Cancer Cell. 2003; 3: 459-69.

Gotlib J, Cools J, Malone JM 3rd, Schrier SL, Gilliland DG, Coutre SE. The FIP1L1-PDGFRalpha fusion tyrosine kinase in hypereosinophilic syndrome and chronic eosinophilic leukemia: implications for diagnosis, classification, and management. Blood. 2004; 103: 2879-91.

Tyrosine kinase mutation screening

FLT3 D835

FMS-like receptor tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase with important roles in haematopoietic cell survival and proliferation. Activating FLT3 mutations including internal tandem duplication and D835 mutations are the most common genetic lesions in acute myeloid leukaemia and are associated with a poor prognosis. These mutations may be used as molecular markers for monitoring disease progression and treatment response. In addition, patients with mutant FLT3 may be benefited from small-molecule FLT3 inhibitors.

References:

Yamamoto Y, Kiyoi H, Nakano Y, Suzuki R, Kodera Y, Miyawaki S, et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood. 2001; 97: 2434-9.

Grundler R, Thiede C, Miething C, Steudel C, Peschel C, Duyster J. Sensitivity toward tyrosine kinase inhibitors varies between different activating mutations of the FLT3 receptor. Blood. 2003; 102: 646-51.

Yanada M, Matsuo K, Suzuki T, Kiyoi H, Naoe T. Prognostic significance of FLT3 internal tandem duplication and tyrosine kinase domain mutations for acute myeloid leukemia: a meta-analysis. Leukemia. 2005; 19: 1345-9.

Kiyoi H, Naoe T. Biology, clinical relevance, and molecularly targeted therapy in acute leukemia with FLT3 mutation. Int J Hematol. 2006; 83: 301-8.

FLT3 internal tandem duplication

References:

Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K, et al. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia. 1996; 10: 1911-8.

Zhang W, Konopleva M, Shi YX, McQueen T, Harris D, Ling X, et al. Mutant FLT3: a direct target of sorafenib in acute myelogenous leukemia. J Natl Cancer Inst. 2008; 100: 184-98.

c-Kit exon 8 and 17

Like FLT3, c-Kit belongs to a member of the class III receptor tyrosine kinase family. The presence of c-Kit exon 8 and 17 activating mutations confers a poor prognosis in acute myeloid leukaemia with the t(8;21) and inv(16) translocations. Thus, the screening has impacts in risk stratification of core-binding factor acute myeloid leukaemia. These mutations may be used as molecular markers for monitoring disease progression and treatment response. In addition, patients with mutant c-Kit may be targeted with novel tyrosine kinase inhibitors.

References:

Growney JD, Clark JJ, Adelsperger J, Stone R, Fabbro D, Griffin JD, et al. Activation mutations of human c-KIT resistant to imatinib mesylate are sensitive to the tyrosine kinase inhibitor PKC412. Blood. 2005; 106: 721-4.

Cammenga J, Horn S, Bergholz U, Sommer G, Besmer P, Fiedler W, et al. Extracellular KIT receptor mutants, commonly found in core binding factor AML, are constitutively active and respond to imatinib mesylate. Blood. 2005; 106: 3958-61.

Cairoli R, Beghini A, Grillo G, Nadali G, Elice F, Ripamonti CB, et al. Prognostic impact of c-KIT mutations in core binding factor leukemias: an Italian retrospective study. Blood. 2006; 107: 3463-8.

Paschka P, Marcucci G, Ruppert AS, Mrozek K, Chen H, Kittles RA, et al. Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;21): a Cancer and Leukemia Group B Study. J Clin Oncol. 2006; 24: 3904-11.

BCR-ABL tyrosine kinase domain

The test is requested for evaluation of the molecular mechanisms underlying imatinib resistance in CML patients. In addition, the presence of certain BCR-ABL tyrosine kinase mutations has important implications in patient management. For instance, the T315I mutation is also resistant to second-generation BCR-ABL inhibitors such as dasatinib and patients with this mutation may require alternative treatment strategies.

References:

Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN, et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science. 2001; 293: 876-80.

Gorre ME, Sawyers CL. Molecular mechanisms of resistance to STI571 in chronic myeloid leukemia. Curr Opin Hematol. 2002; 9: 303-7.

Quintas-Cardama A, Kantarjian H, Cortes J. Flying under the radar: the new wave of BCR-ABL inhibitors. Nat Rev Drug Discov. 2007; 6: 834-48.

Gontarewicz A, Balabanov S, Keller G, Colombo R, Graziano A, Pesenti E, et al. Simultaneous targeting of Aurora kinases and Bcr-Abl kinase by the small molecule inhibitor PHA-739358 is effective against Imatinib-resistant BCR-ABL mutations including T315I. Blood. 2008; in press.

Fluorescence In-situ Hybridization (FISH) detection of gene fusion

BCR-ABL

Interphase nuclei are treated and hybridized with the directly labeled locus specific identifier BCR and ABL DNA probe sequences homologous to specific gene sequences of chromosome 22q11.2 and chromosome 9q34, respectively. The hybridized BCR and ABL probes generate green and orange fluorescence in interphase nuclei and on metaphase chromosomes, respectively. Two green and two orange signals are observed in a normal nucleus, whereas one orange, one green and two orange/green fusion signals are displayed in an abnormal nucleus.

The test is indicative to chronic myelocytic leukaemia, acute lymphoblastic leukaemia and acute myeloblastic leukaemia with Philadelphia chromosome resulting from t(9;22) translocation. It is also informative to disease progression/regression by enumerating the clone size of nuclei with chimeric BCR-ABL rearrangement.

Reference:

Mohr B, Bornhauser M, Platzbecker U, Freiberg-Richter J, Naumann R, Prange-Krex G, et al. Problems with interphase fluorescence in situ hybridization in detecting BCR/ABL-positive cells in some patients using a novel technique with extra signals. Cancer Genet Cytogenet 2001; 127:111-7.

PML-RARα

Interphase nuclei are treated and hybridized with the directly labeled locus specific identifier PML / RARA DNA probe sequences homologous to specific gene sequences of chromosome 15q22 and chromosome 17q21, respectively. The hybridized PML and RARA probes generate orange and green fluorescence in interphase nuclei and on metaphase chromosomes, respectively. In a normal nucleus, two green and two orange signals are observed. In interphase nuclei with t(15;17), two of the probe signals (one orange and one green) will appear adjacent to one another, indicating the presence of the translocation t(15;17). In t(15;17) metaphase spreads, two of the probe signals (one orange and one green) will appear together on 15q, indicating the occurrence of the translocation.

The test is indicative to acute promyelocytic leukaemia with t(15;17) translocation. It is also informative to disease progression/regression by enumerating the clone size of nuclei with chimeric PML/rara rearrangement.

Reference:

Amare PS, Baisane C, Saikia T, Nair R, Gawade H, Advani S. Fluorescence in situ hybridization: a highly efficient technique of molecular diagnosis and predication for disease course in patients with myeloid leukemias. Cancer Genet Cytogenet 2001; 131:125-34.

AML1-ETO

The test is designed to detect the juxtaposition of the AML1 gene locus on chromosome 21q22 with the ETO gene locus on chromo0some 8q22. The translocation t(8;21) produces a fusion of the two genes on the derivative 8 chromosome that results in the novel chimeric gene, AML1-ETO. In normal cell without AML1-ETO fusion gene, two orange signals representing normal copies of ETO and two green signals representing normal copies of AML1 are observed. In a cell containing the AML1-ETO fusion gene, one orange (ETO), one green (AML1) and two orange/green (yellow) fusion signals are observed. The fusion signals represent the translocation of the two genes on the derivative 8 chromosome and derivative 21 chromosome. The t(8;21)(q22;q22) is found primarily in de novo acute myeloblastic leukaemia (AML) of FAB M2 subtype and in rare cases of AML M1 , AML M4 and therapy-related AML.

Reference:

C Godon C, Proffitt J, Dastugue N, Lafage-Pochitaloff M, Mozziconacci MJ, Talmant P, M Hackbarth M, Bataille R, Avet-Loiseau H. Large deletions 5' to the ETO breakpoint are recurrent events in patients with t(8;21) acute myeloid leukemia. Leukemia 2002; 16:1752-1754.

TEL-AML1

The test targets the TEL-AML1 gene fusion that occurs as a result of a cryptic translocation between chromosomes 12p13 and 21q22. Cytogenetically, the t(12;21) is a subtle abnormality and thus not easily detectable with standard cytogenetic banding techniques. In a normal nucleus, the expected pattern for a cell hybridized with the locus specific identifier TEL-AML1 ES Dual Color Translocation probe is the two orange (AML1), two green (TEL) signal pattern. The fusion (orange/green [yellow]) signal represents the translocation of the two genes. The test is useful to elucidate subtle translocation t(12;21) in B-lineage acute lymphoblastic leukaemia with apparently normal karyotype. TEL-AML1 is associated with a favorable prognosis.

Reference

Yehuda-Gafni O, Cividalli G, Abrahmov A, Weintrob M, Neriah SB, Cohen R, Abeliovich D. Fluorescence in situ hybridization analysis of the cryptic t(12;21) (p13;q22) in childhood B-lineage acute lymphoblastic leukemia. Cancer Genet Cytogenet. 2002; 132: 61-64.