Molecular Haematopathology

Test Description and Value

Neuroblastoma monitoring of residual disease

Tyrosine hydroxylase transcripts

Neuroblastoma originates from the neural crest and is characterized by the secretion of catecholamines. Tyrosine hydroxylase (TH) is the first enzyme involved in the metabolism of catecholamine synthesis. TH transcripts of neuroblastoma cells are investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) using primer-set complimentary to gene sequence of the TH gene. The detection of TH in bone marrow is indicative to neuroblastama stage 4. It is also informative in the assessment of dissemination of neuroblastoma to the haematopoietic stem cell graft.

Reference:

Tsang KS, Li CK, Tsoi WC, Leung Y, Shing MMK, Chik KW, et al. Detection of micrometastasis of neuroblastoma to bone marrow and tumor dissemination to hematopoietic autografts using flow cytometry and reverse transcriptase-polymerase chain reaction. Cancer 2003; 97:2887-97.

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.

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.

Giant Platelet Syndrome

MYH9 mutation screening (Exon 16, 26, 30, 38, 40)

Mutations of the MYH9 gene (encodes nonmuscle myosin heavy chain IIA[MYHIIA]) result in autosomal dominant platelet disorders including May-Hegglin anomaly (MHA), and Fechtner (FTNS) and Sebastian (SBS) syndromes, which are featured by thrombocytopenia, large platelets and characteristic leukocyte inclusions. FTNS has the additional clinical features of nephritis, deafness, and cataracts. In addition, MYH9 mutations also result in two other FTNS-like syndromes including Epstein syndrome (EPS) and Alport syndrome with macrothrombocytopenia (APSM). MYH9 mutations are clustered in conserved positions and critical regions in both the globular-head and the coiled-coil domains of MYHIIA and are predicted to result in altered assembly, dimerization, or stability of the quaternary myosin complex. The test is requested for evaluation of the underlying genetic mechanisms causing giant platelet syndrome.

References:

Seri M, Cusano R, Gangarossa S, Caridi G, Bordo D, Lo Nigro C, et al. Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome Consortium. Nat Genet. 2000; 26: 103-5.

Heath KE, Campos-Barros A, Toren A, Rozenfeld-Granot G, Carlsson LE, Savige J, et al. Nonmuscle myosin heavy chain IIA mutations define a spectrum of autosomal dominant macrothrombocytopenias: May-Hegglin anomaly and Fechtner, Sebastian, Epstein, and Alport-like syndromes. Am J Hum Genet. 2001; 69: 1033-45.

Dong F, Li S, Pujol-Moix N, Luban NL, Shin SW, Seo JH, et al. Genotype-phenotype correlation in MYH9-related thrombocytopenia. Br J Haematol. 2005; 130: 620-7.

Neuroblastoma monitoring of residual disease

Tyrosine hydroxylase transcripts

Neuroblastoma originates from the neural crest and is characterized by the secretion of catecholamines. Tyrosine hydroxylase (TH) is the first enzyme involved in the metabolism of catecholamine synthesis. TH transcripts of neuroblastoma cells are investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) using primer-set complimentary to gene sequence of the TH gene. The detection of TH in bone marrow is indicative to neuroblastama stage 4. It is also informative in the assessment of dissemination of neuroblastoma to the haematopoietic stem cell graft.

Reference:

Tsang KS, Li CK, Tsoi WC, Leung Y, Shing MMK, Chik KW, et al. Detection of micrometastasis of neuroblastoma to bone marrow and tumor dissemination to hematopoietic autografts using flow cytometry and reverse transcriptase-polymerase chain reaction. Cancer 2003; 97:2887-97.