Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia Is Separated into Two Subgroups Associated with Survival by BCR-ABL Fluorescence in situ Hybridization of Segmented Cell Nuclei: Report from a Single Institution.

Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) may include the lymphoid blast crisis of chronic myeloid leukemia (CML-BC). We applied fluorescence in situ hybridization (FISH) of the BCR-ABL fusion gene to peripheral blood and/or bone marrow smears to determine whether the fusion was restricted to mononuclear cell nuclei or if segmented cell nuclei representing mature neutrophils also carried the fusion (Seg-FISH). Among 20 patients with Ph+ ALL without a prior diagnosis of CML, 9 were Seg-FISH+ and 11 were Seg-FISH-. Seg-FISH+ cases were characterized by a higher rate of p210-type BCR-ABL transcripts, higher white cell and blast counts, and a higher rate of myeloid and T-lymphoid antigen expression than Seg-FISH- cases, in addition to 'major route' cytogenetic abnormalities associated with CML-BC. Eighteen patients were treated with tyrosine kinase inhibitors (TKIs) either alone or in combination with multiagent chemotherapy, and 7 underwent allogeneic hematopoietic stem cell transplantation. Progression-free and overall survivals were greater in the Seg-FISH+ group than in the Seg-FISH- group. These results suggest that the Seg-FISH+ group represents lymphoid CML-BC that occurs de novo, while the Seg-FISH- represents Ph+ ALL in the strict sense, and the two groups are associated with survival when treated with TKIs or TKI-combined therapy.

A unique subtype of diffuse large B-cell lymphoma primarily involving the bone marrow, spleen, and liver, defined by fluorodeoxyglucose-positron emission tomography combined with computed tomography.

We describe 10 cases of diffuse large B-cell lymphoma (DLBCL) confined to the bone marrow (BM), spleen, and liver, as evidenced by the uniformly increased uptake of fluorodeoxyglucose (FDG) on positron emission tomography combined with computed tomography (PET/CT). Ages ranged from 56 to 87. All, but one patient presented with 'B' symptoms, a poor performance status, and hepatosplenomegaly. All patients showed cytopenia and elevated lactate dehydrogenase levels and were classified into the high-risk category of the International Prognostic Index scoring. BM infiltration was diffuse, interstitial/intrasinusoidal, or mixed, and all showed the nongerminal center B immunophenotype. Five patients had a rearrangement involving 3q27/BCL6, while six had increased copies of MYC, BCL2, or BCL6. All patients were initially treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone, leading to complete responses in six out of eight evaluable patients. We propose BM, spleen, and liver-type DLBCL, which is defined by the findings of FDG-PET/CT.

Crizotinib resistance in acute myeloid leukemia with inv(2)(p23q13)/RAN binding protein 2 (RANBP2) anaplastic lymphoma kinase (ALK) fusion and monosomy 7.

This is the first report on the development of a p.G1269A mutation within the kinase domain (KD) of ALK after crizotinib treatment in RANBP2-ALK acute myeloid leukemia (AML). An elderly woman with AML with an inv(2)(p23q13)/RANBP2-ALK and monosomy 7 was treated with crizotinib. After a short-term hematological response and the restoration of normal hematopoiesis, she experienced a relapse of AML. Fluorescence in situ hybridization using the ALK break-apart probe confirmed the inv(2)(p23q13), while G-banded karyotyping revealed the deletion of a segment of the short arm of chromosome 1 [del(1)(p13p22)] after crizotinib therapy. The ALK gene carried a heterozygous mutation at the nucleotide position g.716751G>C within exon 25, causing the p.G1269A amino acid substitution within the ALK-KD. Reverse transcriptase PCR revealed that the mutated ALK allele was selectively transcribed and the mutation occurred in the ALK allele rearranged with RANBP2. As both the del(1)(p13p22) at the cytogenetic level and p.G1269A at the nucleotide level newly appeared after crizotinib treatment, it is likely that they were secondarily acquired alterations involved in crizotinib resistance. Although secondary genetic abnormalities in ALK are most frequently described in non-small cell lung cancers harboring an ALK alteration, this report suggests that an ALK-KD mutation can occur independently of the tumor cell type or fusion partner after crizotinib treatment.

[The monitoring of residual disease for chronic myelogenous leukemia patients treated with imatinib mesylate: detection of T315I mutation in ATP binding site of BCR/ABL gene].

Imatinib Mesylate, a specific inhibitor of BCR/ABL tyrosine kinase, was developed as a molecularly targeted drug for the treatment of patients with chronic myelogenous leukemia. We evaluated effectiveness of the drug on cytogenetic response for monitoring residual disease using fluorescence in situ hybridization(FISH), reverse transcription-nested-polymerase chain reaction(RT-nested-PCR) and competitive PCR strategy. Of 9 patients in chronic phase, 7 achieved major cytogenetic response(CR) and 2 achieved minor CR by FISH. In 3 out of 6 patients with complete CR, no BCR/ABL gene was detected by RT-nested-PCR in peripheral blood or bone marrow specimens. Of 4 patients in accelerated phase, 1 achieved complete CR but 3 developed blast crisis. Despite high efficacy of Imatinib, 5 out of 13 patients showed resistance to the drug. To clarify the mechanism of resistance, we have newly developed a method for investigating a point mutation of T315I in tyrosine kinase domain of BCR/ABL gene using RT-PCR restriction digested analysis. None of them showed T315I mutation. The sensitivity of the method was as low as 10 copies of mutant gene. The method is useful for screening the mutation when there are many clinical samples or low copy number of BCR/ABL gene. BCR/ABL value obtained by FISH was of use to predict cytogenetic response when residual disease was above 2 to 3%. Below this level, the routine use of RT-nested-PCR was suffices to monitor minimal residual disease.

Variant translocation partners of the anaplastic lymphoma kinase (ALK) gene in two cases of anaplastic large cell lymphoma, identified by inverse cDNA polymerase chain reaction.

In anaplastic large cell lymphoma (ALCL), the anaplastic lymphoma kinase (ALK) gene is rearranged with diverse partners due to variant translocations/inversions. Case 1 was a 39-year-old man who developed multiple tumors in the mediastinum, psoas muscle, lung, and lymph nodes. A biopsy specimen of the inguinal node was effaced by large tumor cells expressing CD30, epithelial membrane antigen, and cytoplasmic ALK, which led to a diagnosis of ALK(+) ALCL. Case 2 was a 51-year-old man who was initially diagnosed with undifferentiated carcinoma. He developed multiple skin tumors eight years after his initial presentation, and was finally diagnosed with ALK(+) ALCL. He died of therapy-related acute myeloid leukemia. G-banding and fluorescence in situ hybridization using an ALK break-apart probe revealed the rearrangement of ALK and suggested variant translocation in both cases. We applied an inverse cDNA polymerase chain reaction (PCR) strategy to identify the partner of ALK. Nucleotide sequencing of the PCR products and a database search revealed that the sequences of ATIC in case 1 and TRAF1 in case 2 appeared to follow those of ALK. We subsequently confirmed ATIC-ALK and TRAF1-ALK fusions by reverse transcriptase PCR and nucleotide sequencing. We successfully determined the partner gene of ALK in two cases of ALK(+) ALCL. ATIC is the second most common partner of variant ALK rearrangements, while the TRAF1-ALK fusion gene was first reported in 2013, and this is the second reported case of ALK(+) ALCL carrying TRAF1-ALK.

B-cell prolymphocytic leukemia carrying t(8;14)(q24;q32), associated with both autoimmune hemolytic anemia and pure red cell aplasia.

An 80-year-old man was referred to our department because of lymphocytosis. His white cell count was 17.1 × 10(3)/µL, with 64% prolymphocytes. He did not exhibit splenomegaly or lymphadenopathy. Prolymphocytes were CD5(+), CD10(-), CD19(+), CD20(+), CD21(+weak), CD22(+), CD23(-), and HLA-DR(+), and expressed µÎ´/λ cell-surface immunoglobulins. G-banding and fluorescence in situ hybridization using c-MYC and immunoglobulin heavy-chain (IgH) gene probe revealed that leukemia cells carried the t(8;14)(q24;q32)/c-MYC-IgH fusion gene, and breakage and reunion occurred within the non-coding region of c-MYC exon 1 as well as the α switch region of IgH. Nine months after the initial presentation, the patient's hemoglobin level fell to 5.7 g/dL. Coombs' test was positive and marked hypoplasia of erythroid precursors was detected in his bone marrow. The patient was treated with prednisolone followed by 4 weekly doses of rituximab, which led to resolution of the anemia and complete response of the underlying leukemia. The role of t(8;14)(q24;q32)/c-MYC-IgH in the pathogenesis of B-cell prolymphocytic leukemia (B-PLL) may not be identical to that in aggressive lymphoid neoplasms, and, in the present case, autoantibodies targeting both mature red cells and erythroid precursors may have been concurrently produced in the setting of B-PLL.

inv(2)(p23q13)/RAN-binding protein 2 (RANBP2)-ALK fusion gene in myeloid leukemia that developed in an elderly woman.

A 75-year-old woman presented with marked leukocytosis; the white cell count was 143.6 × 10³/µL with 38.6 % monocytes and 13.6 % immature granulocytes, including blasts. Bone marrow (BM) aspirate smears showed >90 % cellularity with hyperplasia of myeloid-lineage cells, 14.6 % monocytes, and 32.1 % blasts. The granulocyte series showed a range of dysplastic morphologies. The rate of peroxidase positivity was 51.5 %. CD36+ cells with monocytic differentiation comprised 64.6 % mononuclear cells. Metaphase spreads obtained from the BM revealed an aneuploid karyotype with -7 and a submetacentric marker chromosome derived from chromosome 2, which was determined to be inv(2)(p23q13) by fluorescence in situ hybridization using the Vysis ALK probe. RAN-binding protein 2 (RANBP2)-ALK fusion mRNA was confirmed by reverse transcriptase-mediated polymerase chain reaction and nucleotide sequencing. High-sensitivity anti-ALK immunohistochemistry of a BM biopsy specimen demonstrated nuclear membrane staining of leukemia cells. As the leukemia showed features of chronic myelomonocytic leukemia, the patient was treated with standard daunorubicin-cytarabine followed by azacitidine, leading to the durable suppression of leukemia progression. These findings suggest that inv(2)(p23q13)/RABBP2-ALK defines a small subset of myeloid leukemia characterized by differentiation to monocytes and sharing features of myelodysplastic syndrome/myeloproliferative neoplasm.