JAK2 V617F is a rare finding in de novo acute myeloid leukemia, but STAT3 activation is common and remains unexplained.

Signal transducer and activator of transcription (STAT) proteins are phosphorylated and activated by Janus kinases (JAKs). Recently, several groups identified a recurrent somatic point mutation constitutively activating the hematopoietic growth factor receptor-associated JAK2 tyrosine kinase in diverse chronic myeloid disorders - most commonly classic myeloproliferative disorders (MPD), especially polycythemia vera. We hypothesized that the JAK2 V617F mutation might also be present in samples from patients with acute myeloid leukemia (AML), especially erythroleukemia (AML-M6) or megakaryoblastic leukemia (AML-M7), where it might mimic erythropoietin or thrombopoietin signaling. First, we documented STAT3 activation by immunoblotting in AML-M6 and other AML subtypes. Immunoperoxidase staining confirmed phosphorylated STAT3 in malignant myeloblasts (21% of cases, including all AML-M3 samples tested). We then analyzed genomic DNA from 162 AML, 30 B-cell lymphoma, and 10 chronic lymphocytic leukemia (CLL) samples for JAK2 mutations, and assayed a subset for SOCS1 and FLT3 mutations. Janus kinase2 V617F was present in 13/162 AML samples (8%): 10/13 transformed MPD, and three apparent de novo AML (one of 12 AML-M6, one of 24 AML-M7, and one AML-M2 - all mixed clonality). FLT3 mutations were present in 5/32 (16%), while SOCS1 mutations were totally absent. Lymphoproliferative disorder samples were both JAK2 and SOCS1 wild type. Thus, while JAK2 V617F is uncommon in de novo AML and probably does not occur in lymphoid malignancy, unexplained STAT3 activation is common in AML. Janus kinase2 extrinsic regulators and other proteins in the JAK-STAT pathway should be interrogated to explain frequent STAT activation in AML.

Hereditary angio-oedema with normal C1 inhibitor in a family with affected women and men.

Recurrent angio-oedema is a sign of various acquired and inherited disease entities, including hereditary angio-oedema types I and II that result from a genetic deficiency of C1 inhibitor, and a recently described type of dominantly inherited angio-oedema, which does not show a deficiency of C1 inhibitor. Until now, this new type of hereditary angio-oedema, designated as hereditary angio-oedema type III, has been assumed to be a disorder specific to females. We now describe a four-generation family with dominantly inherited angio-oedema and normal C1 inhibitor in which, in contrast to all previous observations, not only five female but also three male family members were clinically affected. One male patient was mainly affected following the intake of angiotensin-converting enzyme inhibitors. Our current observation leads to new considerations about the classification of hereditary angio-oedema with normal C1 inhibitor. Either hereditary angio-oedema with normal C1 inhibitor can be an entity affecting females predominantly, but not exclusively; in that case, men appear to have a much reduced chance of clinical manifestations. Alternatively, our present observation of hereditary angio-oedema with normal C1 inhibitor affecting both sexes may represent a new disease entity, presumably with a different underlying defect.

FIP1L1-PDGFRA in eosinophilic disorders: prevalence in routine clinical practice, long-term experience with imatinib therapy, and a critical review of the literature.

We previously studied clinico-pathologic features of 89 consecutive adult patients with moderate-to-severe eosinophilia, and reported a FIP1L1-PDGFRA prevalence of 12%. In that series, all 11 FIP1L1-PDGFRA+ patients receiving imatinib achieved a complete response. We now extend our observations through a study of 741 unselected patients with eosinophilia for FIP1L1-PDGFRA, and present longer term follow up data for the imatinib-treated cohort. We also include data for three previously unreported FIP1L1-PDGFRA+ patients. Among the 741 requests, only 21 (3%) were found to carry the FIP1L1-PDGFRA mutation. While all 14 FIP1L1-PDGFRA+ patients receiving imatinib achieved a complete response, the 4 patients who attempted to discontinue imatinib all relapsed. We also find that it is possible to maintain patients in clinical remission with an empirically derived schedule of low-dose (50-100 mg), intermittent (once daily to once weekly) imatinib. Lastly, we present a comprehensive review of the literature pertaining to FIP1L1-PDGFRA in order to address several key aspects of this mutation from a clinical standpoint.

Interphase fluorescence in situ hybridization with an IGH probe is important in the evaluation of patients with a clinical diagnosis of chronic lymphocytic leukaemia.

Translocations involving IGH are common in some lymphoid malignancies but are believed to be rare in chronic lymphocytic leukaemia (CLL). To study the clinical utility of fluorescence in situ hybridization (FISH) for IGH translocations, we reviewed 1032 patients with a presumptive diagnosis of CLL. Seventy-six (7%) patients had IGH translocations. Pathology and clinical data were available for the 24 patients evaluated at the Mayo Clinic. Ten (42%) patients had IGH/cyclin D1 fusion and were diagnosed with mantle cell lymphoma (MCL). The immunophenotype was typical of MCL in three of these patients and atypical for MCL in seven patients. One patient had biclonal disease with typical MCL and CLL with IGH/BCL-2. Eleven (46%) patients had IGH/BCL-2 fusion including the patient with biclonal disease. Two of these patients had leukaemic phase follicular lymphoma and nine patients had CLL. The median progression-free survival of patients with CLL and IGH/BCL-2 translocation was 20.6 months. The two patients with IGH/BCL-3 fusion (one of these also had IGH/BCL-11a) had rapid disease progression. The IGH partner gene was not identified in two patients. We conclude that use of an IGH probe in FISH analysis of monoclonal B-cell lymphocytosis improves diagnostic precision and could have prognostic value in patients with CLL.

A surrogate marker profile for PML/RAR alpha expressing acute promyelocytic leukemia and the association of immunophenotypic markers with morphologic and molecular subtypes.

BACKGROUND: The availability of genotype-specific therapy for PML/RAR alpha(pos) acute promyelocytic leukemia (APL) requires that this disease be precisely diagnosed. Immunophenotypic characteristics heretofore proclaimed as reliably characterizing APL (HLA-DR(low), CD34(low), P-glycoprotein(low) myeloid phenotype) do not differentiate from APL-like immune profiles unassociated with the PML/RAR alpha fusion transcript. METHODS: To establish a surrogate marker profile for APL, we explored 19 potentially predictive markers compared with differentiated acute myeloid leukemia using the classification tree approach with recursive partitioning. RESULTS: In a test group of 58 APL patients, the most predictive immune profile was HLA-DR(low), CD11a(low) (alpha(L) subunit of the leukocyte integrin LFA-1), CD18(low) (beta(2) subunit of LFA-1). APL cells always expressed CD117 (c-kit) but lacked the progenitor antigen CD133 and the more mature myeloid antigen, CD11b (alpha(M) leukocyte integrin). This antigen pattern was validated in 90 additional APL patients. M3v APLs (n = 30) had more leukemic promyelocytes expressing the T-cell antigen, CD2 (P < 0.0001) or the stem cell marker, CD34 (P = 0.0003) and demonstrated higher fluorescence intensity for the binding of antibody to the common leukocyte antigen, CD45 (P = 0.0008) than M3 (n = 102). S-form APL (n = 45) had a higher percent of cells expressing CD2 or CD34 (P < 0.0001 for both) or the neural cell adhesion molecule CD56 (P = 0.001) than L-form APL (n = 66). CONCLUSIONS: PML/RAR alpha(pos) APL cells typically lack leukocyte integrins. HLA-DR(low), CD11a(low), CD18(low) is a reliable surrogate antigen expression profile for PML/RAR alpha(pos) APL, irrespective of morphology and transcript isoform.

Primary pulmonary MALT lymphomas show frequent and heterogeneous cytogenetic abnormalities, including aneuploidy and translocations involving API2 and MALT1 and IGH and MALT1.

API2-MALT1 fusion and aneuploidy are common chromosomal abnormalities in MALT lymphoma. In studying their incidence and relationship in primary pulmonary MALT lymphomas, a translocation involving MALT1 and IGH was also identified. In all, 28 primary pulmonary MALT lymphomas were studied by fluorescence in situ hybridization using an API2-MALT1 probe and multiple centromeric probes, as well as IGH-BCL2, IGH-MALT1, and MALT1 breakapart probes in selected cases. Seven (25%) had API2-MALT1 fusion; all seven lacked aneuploidy except for two with trisomy 3 in a small clone. Three (11%) had IGH-MALT1 fusion; two also showed trisomy 3 and 12. A total of 11 (39%) had aneuploidy only, with trisomy 3 and 18 being the most common. Ectopic nuclear bcl-10 expression, which has been previously associated with API2-MALT1, was seen by immunohistochemistry in 86% of API2-MALT1 fusion-positive cases, one IGH-MALT1 fusion-positive case, two aneuploidy-only cases, and two normal cases. In primary pulmonary MALT lymphomas, cytogenetic abnormalities are common (75%) and heterogeneous, encompassing API2-MALT1 and IGH-MALT1, which are mutually exclusive, as well as aneuploidy, which may be present in the latter but is rare in the former. Ectopic nuclear bcl-10 expression is associated with API2-MALT1 but may also be seen in IGH-MALT1 fusion-positive, aneuploidy-only, and normal cases.

Low expression of the myeloid differentiation antigen CD65s, a feature of poorly differentiated AML in older adults: study of 711 patients enrolled in ECOG trials.

CD65s appears when the progenitor antigen CD34 disappears, suggesting that this sialylated carbohydrate antigen marks a turning point in normal myeloid differentiation. We characterized acute myeloid leukemia (AML) with low CD65s expression (CD65s(low) AML) in 711 patients entered on seven Eastern Cooperative Oncology Group AML treatment trials (1986-1999). Of those, 198 (28%) qualified as having CD65s(low) AML. Morphologically, CD65s(low) AML was more common in FAB subgroups with minimal differentiation, M0/M1 (P=<0.0001). Early precursor antigens CD34, CD117 and terminal transferase were more frequent in CD65s(low) than CD65s(high) AML (P=<0.0001). Myeloperoxidase was present in fewer CD65s(low) myeloblasts, and the more mature myeloid antigens, CD15 and CD11b, were rarely detected (P=<0.0001). Yet, the two diagnoses did not differ in the distribution of cytogenetic prognostic groups or the occurrence of the multidrug-resistance mediator, P-glycoprotein. CD65s(low) AML patients were significantly older than CD65s(high) cases (P<0.0001). Furthermore, the incidence of CD65s(low) cases increased with age, from 20% in patients under the age of 50 years to 67% in patients older than 80 years (P<0.0001). Overall, complete remission (CR) rate and overall survival were comparable in CD65s(low) and CD65s(high) AML. However, among patients >55 years of age, CD65s(low) AML had a decreased CR rate of 33 vs 44% in CD65s(high) AML (P=0.055). Thus, CD65s(low) AML represents immunophenotypically undifferentiated disease and occurs predominantly in older adults. Although not statistically significant, the observed association between low CD65s expression and decreased CR rate only in patients over the age of 55 is intriguing.

Secondary myelodysplastic syndrome and acute myelogenous leukemia are significant complications following autologous stem cell transplantation for lymphoma.

Secondary myelodysplastic syndrome (sMDS) and acute myelogenous leukemia (AML) have been recognized with increasing frequency following autologous stem cell transplantation (ASCT). A retrospective analysis of 230 consecutive patients with Hodgkin's lymphoma (HL, 64) and non-Hodgkin's lymphoma (NHL, 166) who underwent ASCT was conducted to assess the incidence and risk factors for the development of sMDS/AML. At a median follow up of 41 months (range 0.1-177 months), 10 of 230 patients (4.3%) developed sMDS/AML. The 5-year-actuarial incidence of sMDS/AML was 13.1% and 5-year cumulative incidence by competing risk analysis was 4.2%. The median time to development of sMDS/AML was 39.9 months from the time of ASCT (range 12.1-62.0 months). Complex karyotypes at diagnosis of sMDS/AML included structural anomalies and/or loss of chromosome 5 (eight patients), 7 (five patients), 17 (two patients) and 20 (two patients). All patients subsequently died, at a median of 6.8 months (range 0-39.9) from diagnosis of sMDS/AML. Fluorescent in situ hybridization (FISH) analysis for -5/5q- and -7/7q- were normal in all six patients whose pre-ASCT bone marrow was available for testing. Five of the six had samples available for testing at diagnosis of sMDS/AML and all had abnormal FISH results. By univariate statistical analysis, male gender (P=0.01), prior alkylating agents (mechlorethamine for HL, P=0.001 and cyclophosphamide for NHL, P=0.05) and the number of prior treatment regimens (P=0.04) were significantly associated with the development of sMDS/AML. Given the relatively low incidence rate of sMDS/AML, these analyses are primarily exploratory in nature but provide some insight into relevant risk factors and illustrate the risk of developing sMDS/AML after myeloablative conditioning and ASCT for lymphoma.

Chromosome abnormalities clustering and its implications for pathogenesis and prognosis in myeloma.

The nonrandom recurrent nature of chromosome abnormalities in myeloma suggests a role for them in disease pathogenesis. We performed a careful cytogenetic analysis of patients with abnormal karyotypes (n = 254), to discern patterns of association, search for novel abnormalities and elucidate clinical implications. Patients with karyotypic abnormalities suggestive of myelodysplasia/acute leukemia were excluded. In this study we compared survival by abnormality only between patients with abnormal karyotypes. Patients with abnormalities were more likely to have features of aggressive disease as compared to all other patients without abnormalities entered into the myeloma database (lower hemoglobin, higher beta(2)-microglobulin, labeling-index and plasmocytosis; all P < 0.0001). Several groups of patients could be readily identified; hypodiploid (22%), pseudodiploid (36%), hyperdiploid (31%) and near-tetraploid (11%). Clustering associations were seen among several trisomies and monosomy of chromosome 13 and 14. Several monosomies (-2, -3, -13, -14 and -19), 1p translocations/ deletions, and hypodiploidy were associated with a significantly shorter survival. Trisomy of chromosome 13 was rare ( <2%). Even among patients with abnormal karyotypes, specific chromosome abnormalities can impart biologic variability in myeloma, including several monosomies, hypodiploidy and abnormalities of 1p.

B-CLL cells are capable of synthesis and secretion of both pro- and anti-angiogenic molecules.

Initial work has shown that clonal B cells from B-chronic lymphocytic leukemia (B-CLL) are able to synthesize pro-angiogenic molecules. In this study, our goal was to study the spectrum of angiogenic factors and receptors expressed in the CLL B cell. We used ELISA assays to determine the levels of basic fibroblast growth factors (bFGF), vascular endothelial growth factor (VEGF), endostatin, interferon-alpha (IFN-alpha) and thrombospondin-1 (TSP-1) secreted into culture medium by purified CLL B cells. These data demonstrated that CLL B cells spontaneously secrete a variety of pro- and anti-angiogenic factors, including bFGF (23.9 pg/ml +/- 7.9; mean +/- s.e.m.), VEGF (12.5 pg/ml +/- 2.3) and TSP-1 (1.9 ng/ml +/- 0.3). Out of these three factors, CLL B cells consistently secreted bFGF and TSP-1, while VEGF was expressed in approximately two-thirds of CLL patients. Of interest, hypoxic conditions dramatically upregulated VEGF expression at both the mRNA and protein levels. We also employed ribonuclease protection assays to assay CLL B cell expression of a variety of other angiogenesis-related molecules. These analyses revealed that CLL B cells consistently express mRNA for VEGF receptor 1 (VEGFR1), thrombin receptor, endoglin, and angiopoietin. Further analysis of VEGFR expression by RT-PCR revealed that CLL B cells expressed both VEGFR1 mRNA and VEGFR2 mRNA. In summary, these data collectively indicate that CLL B cells express both pro- and anti-angiogenic molecules and several vascular factor receptors. Because of the co-expression of angiogenic molecules and receptors for some of these molecules, these data suggest that the biology of the leukemic cells may also be directly impacted by angiogenic factors as a result of autocrine pathways of stimulation.

Agnogenic myeloid metaplasia associated with Klinefelter syndrome: a case report.

Klinefelter syndrome is the most commonly diagnosed sex chromosome disorder among males. It is usually associated with 47 chromosomes, including two Xs and one Y. The formal cytogenetic designation for Klinefelter syndrome is 47, XXY; the extra sex chromosome is due to meiotic chromosomal nondisjunction. Increased risk of various malignant diseases has been recognized among patients with different congenital chromosomal abnormalities. Since the early 1960s, numerous reports have appeared of an increased risk of malignant neoplasms among patients with Klinefelter syndrome. Evidence suggests a correlation with increased incidences of germ cell tumors and breast cancers. Whether these patients are at an increased risk of hematologic malignant disease, especially acute leukemia, is still uncertain. This report describes a patient with agnogenic myeloid metaplasia and Klinefelter syndrome, an association not previously reported.

Secondary acute myelogenous leukemia with MLL gene rearrangement following radioimmunotherapy (RAIT) for non-Hodgkin's lymphoma.

Targeted therapy with conjugated and unconjugated monoclonal antibodies for non-Hodgkin's lymphoma has revolutionized the approach to this disease. The efficacy and low toxicity of these agents have allowed introduction of this strategy in the early stages of therapy. Longer follow-up is needed before validating the safety of these agents. Since monoclonal antibodies are being given as front-line therapy, it is important to identify all potential adverse events. We report a case of secondary acute myelogenous leukemia (AML) with 11q23 cytogenetic abnormality and mixed lymphoid leukemia (MLL) gene expression in a patient treated with Y90 labeled anti-CD20 antibody (Zevalin). The patient was not exposed to topoisomerase II inhibitors. Our observations suggest a relationship between 11q23 leukemia and radioimmunotherapy (RAIT) and further studies are needed.

Comparison of peripheral blood interphase cytogenetics with bone marrow karyotype analysis in myelofibrosis with myeloid metaplasia.

In a prospective study of 42 patients with myelofibrosis with myeloid metaplasia (MMM), peripheral blood (PB) and bone marrow (BM) interphase cytogenetics and PB CD34 enumeration were performed concomitantly with BM karyotype analysis. Interphase cytogenetics was performed with a panel of fluorescence in situ hybridization (FISH) probes that were capable of detecting most of the known recurrent cytogenetic lesions in MMM. There was a close concordance in the results of interphase cytogenetics between PB and BM, regardless of the PB CD34 count. In general, FISH-detectable abnormalities were also detected by BM karyotype. Although complementary, interphase cytogenetics may not always provide the necessary karyotypic information in MMM.

Pulmonary atresia with ventricular septal defect and persistent airway hyperresponsiveness.

OBJECTIVE: We and others have observed significant hyperinflation and airflow obstruction after the surgical repair of pulmonary atresia and ventricular septal defect. This study sought to objectively characterize this problem and determine the prevalence of airway hyperresponsiveness in these patients. METHODS: We performed a prospective study of children and young adults with pulmonary atresia and ventricular septal defect between June 1996 and December 1998. The participants were stratified into 2 distinct molecular genotypes on the basis of chromosome 22q11.2 microdeletion. A clinical diagnosis of asthma and an objective assessment of airway hyperresponsiveness were determined by means of an asthma inventory scale and methacholine challenge testing, respectively. Thirty-three patients were enrolled. Thirteen had velocardiofacial syndrome, each with chromosome 22q11.2 microdeletion. RESULTS: None of the nonsyndromic patients had evidence for haploinsufficiency. Overall, 66.7% (22/33) met criteria for a clinical diagnosis of airway hyperresponsiveness: 62% (8/13) from the microdeletion genotype and 70% (14/20) from the nonsyndromic group. CONCLUSIONS: We have identified an extremely strong association between pulmonary atresia and ventricular septal defect and persistent airway hyperresponsiveness. Haploinsufficiency at chromosome 22q11.2 did not contribute to this predilection for airway hyperresponsiveness. These results provide a basis to anticipate persistent respiratory difficulties after operations in patients with pulmonary atresia and ventricular septal defect. Moreover, this at-risk patient population may yield unique insights into fundamental mechanisms involved in the pathogenesis of airway hyperresponsiveness.

Phase II study of combination human recombinant GM-CSF with intermediate-dose cytarabine and mitoxantrone chemotherapy in patients with high-risk myelodysplastic syndromes (RAEB, RAEBT, and CMML): an Eastern Cooperative Oncology Group Study.

A Phase II study of GM-CSF with intermediate-dose cytarabine and mitoxantrone was conducted in patients with high-risk myelodysplastic syndrome. It was designed to evaluate if priming with growth factor could increase the efficiency of chemotherapy. In this older population only two of 10 patients achieved a bone marrow CR, including one patient whose leukemic blasts had an "S" phase increase of 2.55x at 48 hr. Unexpected hepatotoxicity was noted. This regimen cannot be recommended for this elderly population of patients.

Deletions of chromosome 13 in multiple myeloma identified by interphase FISH usually denote large deletions of the q arm or monosomy.

Deletions of the long arm of chromosome 13 (13q-) are observed in patients with multiple myeloma (MM), are rarely observed in the monoclonal gammopathy of undetermined significance (MGUS) and have been associated with a worsened prognosis in MM. However, no minimally deleted region in the 13q arm has been defined at 13q, and consequently no tumor suppressor genes have yet been identified that are important for disease pathogenesis. We attempted to characterize these chromosome 13q deletions at the molecular cytogenetic level. We studied 351 newly diagnosed patients, entered into the E9486/E9487 clinical study of the Eastern Cooperative Oncology Group. Fluorescent in situ hybridization (FISH) combined with immune fluorescent detection (cIg-FISH) of clonal plasma cells (PC) and cytomorphology were used to analyze interphase, bone marrow (BM) cell, cytospin slides. We simultaneously used DNA probes for the following locus specific probes (LSI); LSI 13 (Rb) and D13S319, which hybridize to 13q14. We subsequently studied distal deletions using the D13S25 probe (13q14.3) and a subtelomeric probe (13qSTP) for the 13q-arm (D13S327) in 40 cases with documented LSI 13 (Rb)/D13S319 deletion and 40 without deletion of these loci. Of 325 evaluable patients, we found 13q deletions in 176 (54%) using LSI 13 (Rb) and D13S319 probes. Of 40 patients with LSI 13 (Rb)/D13S319 deletions, 34 (85%) had coexistent deletion of both D13S25/13qSTP. These results indicate that chromosome 13 deletions in MM involve loss of most if not all of the 13q arm perhaps even indicating monosomy. In six cases the 13qSTP signal was conserved, but D13S25 was lost indicating large interstitial deletions involving 13q14. In 39 of the 40 cases without LSI 13 (Rb)/D13S319 deletions, the normal pattern of two pairs of signals was observed for D13S25/13qSTP. Deletions involving 13q14 are very common in MM as detected by cIg-FISH. These deletions appear to predominantly involve loss of large segments of the 13q arm or monosomy 13, and only occasionally represent an interstitial deletion.

Cytogenetic findings and their clinical relevance in myelofibrosis with myeloid metaplasia.

The prognostic significance of bone marrow cytogenetic lesions in myelofibrosis with myeloid metaplasia (MMM) was investigated in a retrospective series of 165 patients. An abnormal karyotype was demonstrated in 57% of patients. At diagnosis (n = 92), 48% of the patients had detectable cytogenetic abnormalities, and clonal evolution was frequently demonstrated in sequential studies. More than 90% of the anomalies were represented by 20q-, 13q-, +8, +9, 12p-, and abnormalities of chromosomes 1 and 7. Of these, 20q-, 13q- and +8 were the most frequent sole abnormalities, each occurring in 15-25% of the abnormal cases. Trisomy 9 and abnormalities of chromosomes 1 and 7 were equally prevalent but were usually associated with additional cytogenetic lesions. Chromosome 5 abnormalities were infrequent but were over-represented in the group of patients exposed to genotoxic therapy. In a multivariate analysis that incorporated other clinical and laboratory variables, the presence of an abnormal karyotype did not carry an adverse prognosis. Instead, +8, 12p-, advanced age and anaemia were independent prognostic determinants of inferior survival. In particular, survival was not adversely affected by the presence of either 20q- or 13q-.