Comprehensive analysis of genetic alterations and their prognostic impacts in adult acute myeloid leukemia patients.

To clarify the cooperative roles of recurrently identified mutations and to establish a more precise risk classification system in acute myeloid leukemia (AML), we comprehensively analyzed mutations in 51 genes, as well as cytogenetics and 11 chimeric transcripts, in 197 adult patients with de novo AML who were registered in the Japan Adult Leukemia Study Group AML201 study. We identified a total of 505 mutations in 44 genes, while only five genes, FLT3, NPM1, CEBPA, DNMT3A and KIT, were mutated in more than 10% of the patients. Although several cooperative and exclusive mutation patterns were observed, the accumulated mutation number was higher in cytogenetically normal AML and lower in AML with RUNX1-RUNX1T1 and CBFB-MYH11, indicating a strong potential of these translocations for the initiation of AML. Furthermore, we evaluated the prognostic impacts of each sole mutation and the combinations of mutations and/or cytogenetics, and demonstrated that AML patients could be clearly stratified into five risk groups for overall survival by including the mutation status of DNMT3A, MLL-PTD and TP53 genes in the risk classification system of the European LeukemiaNet. These results indicate that the prognosis of AML could be stratified by the major mutation status in combination with cytogenetics.

Mutations in the receptor tyrosine kinase pathway are associated with clinical outcome in patients with acute myeloblastic leukemia harboring t(8;21)(q22;q22).

AML1-MTG8 generated by t(8;21) contributes to leukemic transformation, but additional events are required for full leukemogenesis. We examined whether mutations in the receptor tyrosine kinase (RTK) pathway could be the genetic events that cause acute myeloblastic leukemia (AML) harboring t(8;21). Mutations in the second tyrosine kinase domain, juxtamembrane (JM) domain and exon 8 of the C-KIT gene were observed in 10, one and three of 37 AML patients with t(8;21), respectively. Three patients showed an internal tandem duplication in the JM domain of the FLT3 gene. One patient had a mutation in the K-Ras gene at codon 12. As the occurrence of these mutations was mutually exclusive, a total of 18 (49%) patients showed mutations in the RTK pathway. These results suggest that activating mutations in the RTK pathway play a role in part as an additional event leading to the development of t(8;21) AML. The 6-year cumulative incidence of relapse in patients with RTK pathway mutations was 79.8%, compared with 13.5% in patients lacking such mutations (P=0.0029). Furthermore, the 6-year relapse-free survival in patients with mutations was 18% compared to 60% in those without mutations (P=0.0340), indicating that RTK mutations are associated with the clinical outcome in t(8;21) AML.

Nasal CD56 positive small round cell tumors. Differential diagnosis of hematological, neurogenic, and myogenic neoplasms.

CD56-positive nasal and nasal-type natural killer (NK)/T-cell lymphoma is now a well-defined disease entity. Rare cases of blastic NK-cell lymphoma positive for CD56 have been recently reported. However, CD56 expression is also identified in several types of non-hematopoietic small round cell tumors in which lymphoma is included as a differential consideration. Here, we present nine cases of CD56+ small round cell tumors of histological origin unrelated to nasal NK/T-cell lymphoma. Eight of the nine cases presented as solid tumors of the sinonasal region. Clinical, histological, ultrastructural, and immunohistochemical examination and gene analysis for T-cell receptor (TcR) and immunoglobulin heavy chain (IgH) genes and in situ hybridization (ISH) for Epstein-Barr virus (EBV) were performed. Two cases presented with features consistent with blastic NK-cell lymphoma or lymphoblastic lymphoma of NK-cell phenotype. These cases showed features of lymphoblastic lymphoma, phenotypes of sCD3-, cCD3+, CD45+, CD56+, TdT+, and human leukocyte antigen (HLA)-DR+, germline of IgH and TcR genes, and EBV negative reactivity. One case had myeloid/NK-precursor acute leukemia/lymphoma with a phenotype of CD13+, CD33+, CD34+, CD56+, and MPO-. Three cases were neurogenic, including one case of olfactory neuroblastoma and two of primitive neuroectodermal tumors (PNET). It was difficult to differentiate CD56+ PNET from blastic NK-cell lymphoma, especially when only paraffin-embedded sections were available. Myogenic markers, such as HHF35, alpha-sarcomeric actin, and desmin, were positive in three cases of rhabdomyosarcomas. Our findings suggest that as CD56 is used more routinely as a marker in immunohistochemical staining, the differential diagnosis of extranodal lymphohematological malignancies and small round cell tumors will become more complicated.

Long-term remission in an elderly patient with mantle cell leukemia treated with low-dose cyclophosphamide.

We present an elderly patient with mantle cell leukemia who was successfully treated with low-dose cyclophosphamide (CY). A 76-year-old female was diagnosed as mantle cell leukemia based on abnormal lymphocytosis and splenomegaly without lymphadenopathy. She was orally treated with 50 mg of CY daily and had continuous remission over 4 years. Rearrangements of BCL1 and immunoglobulin heavy chain genes in the peripheral blood lymphocytes were detected at diagnosis, but not 1 or 4 years later. Further studies are required to confirm the role of low-dose CY therapy for patients with mantle cell leukemia and lymphoma.

Biallelic and heterozygous point mutations in the runt domain of the AML1/PEBP2alphaB gene associated with myeloblastic leukemias.

The AML1 gene encoding the DNA-binding alpha-subunit in the Runt domain family of heterodimeric transcription factors has been noted for its frequent involvement in chromosomal translocations associated with leukemia. Using reverse transcriptase-polymerase chain reaction (RT-PCR) combined with nonisotopic RNase cleavage assay (NIRCA), we found point mutations of the AML1 gene in 8 of 160 leukemia patients: silent mutations, heterozygous missense mutations, and biallelic nonsense or frameshift mutations in 2, 4, and 2 cases, respectively. The mutations were all clustered within the Runt domain. Missense mutations identified in 3 patients showed neither DNA binding nor transactivation, although being active in heterodimerization. These defective missense mutants may be relevant to the predisposition or progression of leukemia. On the other hand, the biallelic nonsense mutants encoding truncated AML1 proteins lost almost all functions examined and may play a role in leukemogenesis leading to acute myeloblastic leukemia.

Acute myelomonoblastic leukemia carrying the PEBP2beta/MYH11 fusion gene.

As recurrent chromosome abnormalities in leukemia are highly associated with particular subtypes, the genetic events of specific chromosome alteration must be associated with leukemogenesis and characteristics of the disease. The chromosomal breakpoints involved in inv(16) and t(16;16) have been shown to generate the fusion gene PEBP2beta(CBFbeta)/MYH11. The PEBP2beta/MYH11 fusion transcripts in all 8 patients with M4Eo, 2 of 18 with M4, and one CML in the blastic phase were detected by using RT-PCR and Southern blotting. We demonstrated the marked expression of CD34 and c-KIT (CD117) antigens in myelomonoblastic leukemia cells from all patients carrying this fusion gene, which was in contrast to the patients with M4 but without the fusion gene. These results indicate that immunophenotypic analysis is useful for detection of leukemia with the fusion gene, and that the PEBP2beta/MYH11 fusion gene is involved in immature cells expressing CD34 and c-KIT antigens.

Mutation of CD95 (Fas/Apo-1) gene in adult T-cell leukemia cells.

CD95 antigen (also known as Fas or Apo-1) and Fas ligand play key roles in apoptosis of cells of the immune system, function as effector molecules of cytotoxic T lymphocytes, and function in the elimination of activated lymphocytes during the downregulation of the immune response. The critical roles of the Fas-Fas ligand system in apoptosis suggest that its inactivation may be involved in malignant transformation. We analyzed the expression of Fas antigen on adult T-cell leukemia (ATL) cells by flow cytometry and found that Fas antigen expression was absent in a case of ATL and markedly decreased in another case among 47 cases examined. Apoptosis could not be induced in the Fas-negative ATL cells by antibody against Fas antigen. Sequencing of reverse transcription-polymerase chain reaction products of the Fas genes in the Fas negative cells showed two types of aberrant transcripts: one had a 5-bp deletion and a 1-bp insertion in exon 2, and the other transcript lacked exon 4. These mutations caused the premature termination of both alleles, resulting in the loss of expression of surface Fas antigen. These aberrant transcripts were not detected in a nonleukemic B-cell line from the same patient. An RNase protection assay of the Fas gene showed mutations in 2 additional cases with Fas-positive ATL cells of 35 cases examined: 1 case lacked exon 4 and the other was a silent mutation. In the Fas antigen-negative case, leukemic cells were resistant to anticancer drugs in vivo, indicating that the loss of expression of Fas antigen may be associated with a poor response to anticancer drugs. Indeed, Fas-negative ATL cells were resistant to adriamycin-induced apoptosis in vitro, which is consistent with the finding that ATL in this case was resistant to chemotherapy. These findings indicate that mutation of the Fas gene may be associated with the progression of ATL and with resistance to anticancer drugs.

Analysis of prognostic factors in newly diagnosed acute promyelocytic leukemia treated with all-trans retinoic acid and chemotherapy. Japan Adult Leukemia Study Group.

PURPOSE: We conducted a multicenter study of differentiation therapy with all-trans retinoic acid (ATRA) followed by intensive chemotherapy in patients with newly diagnosed acute promyelocytic leukemia (APL) and analyzed the prognostic factors for predicting complete remission (CR), event-free survival (EFS), and disease-free survival (DFS). PATIENTS AND METHODS: All patients received ATRA until CR. If patients had an initial leukocyte count greater than 3.0 x 10(9)/L, they received daunorubicin (DNR) and behenoyl cytarabine (BHAC). During therapy, if patients showed blast and promyelocyte counts greater than 1.0 x 10(9)/L, they received additional DNR and BHAC. After achieving CR, patients received three courses of consolidation and six courses of maintenance/intensification chemotherapy. RESULTS: Of 198 registered, 196 were assessable (age range, 15 to 86 years; median, 46) and 173 (88%) achieved CR. Multivariate analysis showed that no or minor purpura at diagnosis (P = .0046) and age less than 30 years (P = .0076) were favorable factors for achievement of CR. Predicted 4-year overall survival and EFS rates were 74% and 54%, respectively, and the 4-year predicted DFS rate for 173 CR patients was 62%. Multivariate analysis showed that age less than 30 years (P = .0003) and initial leukocyte count less than 10 x 10(9)/L (P = .0296) were prognostic factors for longer EFS, and initial leukocyte count less than 10.0 x 10(9)/L was a sole significant prognostic factor for longer DFS (P = .0001). CONCLUSION: Our results show that age, hemorrhagic diathesis, and initial leukocyte count are prognostic factors for APL treated with ATRA followed by intensive chemotherapy.

TEL/AML1 fusion gene resulting from a cryptic t(12;21) is uncommon in adult patients with B-cell lineage ALL and CML lymphoblastic transformation.

TEL is a new member of the ETS-like family on chromosome 12 and forms fusion genes with several partners in leukemia. Among these fusion genes, the TEL/AML1 translocation resulting from t(12;21) is found in approximately one quarter of the childhood B-cell lineage acute lymphoblastic leukemia (ALL) cases and its prognosis is excellent. We examined 42 adult patients with B-cell lineage ALL and 13 adult patients with lymphoblastic transformation of chronic myeloid leukemia (CML) to detect TEL/AML1 fusion genes using the reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, but no translocation was detected. These findings indicate that absence of the TEL/AML1 fusion transcript partly correlates with the poorer outcome of adult B-cell lineage ALL as compared with childhood ALL and the TEL/AML1 fusion transcript is specific for pediatric B-cell lineage ALL.

Myelomonoblastic leukaemia cells carrying the PEBP2beta/MYH11 fusion gene are CD34, c-KIT+ immature cells.

To clarify the aspects affected by the PEBP2beta/MYH11 fusion gene involved in the inv(16), we analysed immunophenotypes in myelomonoblastic leukaemias. We found high expressions of CD34 and c-KIT antigens in myelomonoblastic cells from all patients carrying this fusion gene, including two with M4 and one CML blastic phase, in contrast to those with M4 without the fusion gene. These findings indicate that immunophenotyping is useful for detecting a leukaemia with the fusion gene in myelomonoblastic leukaemias and that the PEBP2beta/MYH11 gene is involved in immature cells expressing CD34 and c-KIT antigens.

A human T-cell lymphotropic virus type-I carrier with chronic renal failure, aplastic anemia, myelopathy, uveitis, Sjögren's syndrome and panniculitis.

A 53-year-old female infected with human T lymphotropic virus type-I (HTLV-I) suffered from chronic renal failure, aplastic anemia, myelopathy, uveitis, Sjögren's syndrome and Weber-Christian disease. Although HTLV-I antibody was negative in cerebrospinal fluid, she was diagnosed as HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) based on clinical and histological findings. Though to date there is no direct evidence, other complications have also been reported to be HTLV-I related diseases. This case provided the unique opportunity to observe various HTLV-I related diseases.

p53 gene mutation and loss of heterozygosity are associated with increased risk of disease progression in adult T cell leukemia.

Although the prototype of adult T cell leukemia (ATL) is an aggressive T cell neoplasm, ATL manifests four major clinical subtypes, acute, lymphoma, chronic, and smoldering. We studied the relationship between p53 gene alteration and clinical features in 34 patients with ATL, 14 acute type, 15 chronic type, and five crisis type transformed from chronic type. Using a polymerase chain reaction/single strand conformation polymorphism (PCR/SSCP) assay, followed by nucleotide sequencing, we detected mutations of the p53 gene in six of the 14 acute type patients, two of the five crisis type, and one of the 15 chronic type patients. Gene dosage studies, using PCR amplification and Southern blotting, showed loss of heterozygosity (LOH) of the p53 gene in four of the 14 acute type patients, two of the five crisis type, and one of 14 chronic type patients examined. These observations indicated that the frequency of p53 gene alterations in the acute and crisis types of ATL was markedly higher than that in chronic type, suggesting that p53 gene alteration plays a role in the disease progression of ATL.

Lack of the expression of EBNA-2 and LMP-1 in T-cell neoplasms possessing Epstein-Barr virus.

We investigated 34 cases of T-cell neoplasm [15 cases of T-cell granular lymphocytic leukemia (T-GLL), 10 cases of T-cell non-Hodgkin's lymphoma (T-NHL), six cases of T-cell chronic lymphocytic leukemia (T-CLL), and three cases of cutaneous T-cell lymphoma] to study their association with Epstein-Barr virus (EBV). In 4 (three T-NHL and one T-GLL) of 34 cases, EBV genome was detected in a single episomal form, while polyclonal EBV-DNA was detected in one (T-NHL) of the remaining cases. All three cases of T-NHL having monoclonal EBV episome showed histologically diffuse large-cell lymphoma and developed leukemic conversion. Phenotypic analysis showed that two of these four cases were CD4+, CD8-, and the remaining two cases were CD4-, CD8+. The cells from all four cases were confirmed to be in T-cell lineage by detecting the rearrangement of T-cell receptor (TCR) beta or gamma chain gene. By reverse transcription-polymerase chain reaction (RT-PCR), EBNA-1 was detected at low levels, and neither EBNA-2 nor LMP-1 were found in any of the three cases examined. Lack of the expression of EBNA-2 and LMP-1 was also confirmed by immunocytochemical staining. The cells of these four cases did not show rearrangement or overexpression of c-myc and bcl-2 genes by Southern and Northern blots, and the mutation of p53 gene was detected in only one patient. These results suggest that other latent gene products of EBV or other cellular oncogenes are involved in the development of Japanese T-cell neoplasm after EBV infection.

Smoldering gamma delta T-cell granular lymphocytic leukemia associated with pure red cell aplasia.

We present a case of granular lymphocytic leukemia (GLL) associated with pure red cell aplasia (PRCA). Twenty-seven percent of the peripheral blood mononuclear cells (PBMC) were positive for TCR delta 1, but not for delta TCS1 (V delta 2), and a rearrangement of the TCR chain (J delta 1) gene was detected. The PBMC and serum from the patient slightly suppressed CFU-E but not BFU-E colony formation in vitro. These findings suggest that monoclonal proliferation of TCR gamma delta-positive granular lymphocytes (GL) can be associated with PRCA even in a patient having a small proportion of leukemia GL, which we propose to designate as 'smoldering GLL'.

CD4+ CD8+ granular lymphocytic leukemia arising in a patient with acute myeloblastic leukemia.

A 59-year-old woman who had an 8-year history of acute myeloblastic leukemia (AML) developed granular lymphocytic leukemia (GLL). She had a small number of granular lymphocytes (GL) in her bone marrow (BM) at the onset of AML. The GL increased during complete remission (CR) of AML, but not at the relapse. During the third CR state of AML, GL increased to 4.0 x 10(9)/l in the peripheral blood (PB). The GL were T-cell receptor (TCR) alpha beta+ T cells and expressed both CD4 and CD8 antigens. Rearrangements of TCR beta and gamma chain genes were detected in the peripheral blood mononuclear cells (PBMNC), confirming that this patient had GLL. The PBMNC from the patient responded weakly to PHA or ConA, yet they responded to her own bone marrow mononuclear cells (BMMNC) or CD4-depleted BMMNC that contained AML cells stronger than her own PBMNC or normal PBMNC. These observations suggest that monoclonal proliferation of GL developed after the reactive proliferation of GL in response to AML cells.

Alcaptonuria: a case complicated with valvular heart disease and immunodeficiency.

A 75-year-old man was diagnosed with alcaptonuria by direct identification of homogentisic acid in the urine using gas chromatography-mass spectrometry. Complications included valvular heart disease with marked calcification detected by two-dimensional color-Doppler echocardiography and recurrent bacterial infection. Immunological analyses revealed a reduced number of T cells with compensatory expansion of CD56+, CD57+ natural killer (NK) cell population and impaired functions of cellular immunity such as phytohemagglutinin response, antibody-dependent cellular cytotoxicity, NK activity and interleukin-2 production. Humoral immunity and neutrophil functions were considered to be normal. This is the first reported case of alcaptonuria to our knowledge in which immunological abnormality was documented.