Prokineticin signaling is required for the maintenance of a de novo population of c-KIT⁺ cells to sustain neuroblastoma progression.

High cellular heterogeneity within neuroblastomas (NBs) may account for the non-uniform response to treatment. c-KIT(+) cells are frequently detected in NB, but how they influence NB behavior still remains elusive. Here, we used NB tumor-initiating cells to reconstitute NB development and demonstrated that c-KIT(+) cells are de novo generated and dynamically maintained within the tumors to sustain tumor progression. c-KIT(+) NB cells express higher levels of neural crest and stem cell markers (SLUG, SOX2 and NANOG) and are endowed with high clonogenic capacity, differentiation plasticity and are refractory to drugs. With serial transplantation assays, we found that c-KIT expression is not required for tumor formation, but c-KIT(+) cells are more aggressive and can induce tumors ninefold more efficiently than c-KIT(-/low) cells. Intriguingly, c-KIT(+) cells exhibited a long-term in vivo self-renewal capacity to sustain the formation of secondary and tertiary tumors in mice. In addition, we showed that Prokineticin signaling and mitogen-activated protein kinase pathways are crucial for the maintenance of c-KIT(+) cells in tumor to promote NB progression. Our results highlight the importance of this de novo population of NB cells in sustainable growth of NB and reveal specific signaling pathways that may provide targets leading to more effective NB therapies.

Methylation of TET2, CBL and CEBPA in Ph-negative myeloproliferative neoplasms.

A loss-of-function mutation of TET2, CBL and CEBPA has been implicated in the pathogenesis or leukaemic transformation of myeloproliferative neoplasm. As tumour suppressor genes may potentially be inactivated by promoter hypermethylation, the authors studied the methylation status of these genes in three cell lines and diagnostic marrow samples from 45 patients with myeloproliferative neoplasm (MPN) (essential thrombocythaemia, N=34; polycythaemia vera, N=7 and primary myelofibrosis, N=4) by methylation-specific PCR. TET2 was heterozygously methylated in MEG-01 and K562 but completely unmethylated in HEL. On the other hand, both CBL and CEBPA were completely unmethylated in all three cell lines. In the primary marrow samples, methylation of TET2 occurred in two (5.9%) patients with essential thrombocythaemia (4.4% of all patients), both without JAK2 V617 mutation, but not in polycythaemia vera or primary myelofibrosis. There was no association between TET2 methylation with the type of MPN (p=0.713). Hypermethylation of CBL or CEBPA was not detected in any patients. In summary, methylation of TET2, CBL and CEBPA is infrequent in MPN at diagnosis. The role of methylation of these genes at the time of leukaemic transformation warrants further study.

Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting potential.

Aldehyde dehydrogenase (ALDH) activity is used to define normal hematopoietic stem cell (HSC), but its link to leukemic stem cells (LSC) in acute myeloid leukemia (AML) is currently unknown. We hypothesize that ALDH activity in AML might be correlated with the presence of LSC. Fifty-eight bone marrow (BM) samples were collected from AML (n=43), acute lymphoblastic leukemia (ALL) (n=8) and normal cases (n=7). In 14 AML cases, a high SSC(lo)ALDH(br) cell population was identified (ALDH(+)AML) (median: 14.89%, range: 5.65-48.01%), with the majority of the SSC(lo)ALDH(br) cells coexpressing CD34(+). In another 29 cases, there was undetectable (n=23) or rare (< or =5%) (n=6) SSC(lo)ALDH(br) population (ALDH(-)AML). Among other clinicopathologic variables, ALDH(+)AML was significantly associated with adverse cytogenetic abnormalities. CD34(+) BM cells from ALDH(+)AML engrafted significantly better in NOD/SCID mice (ALDH(+)AML: injected bone 21.11+/-9.07%; uninjected bone 1.52+/-0.75% vs ALDH(-)AML: injected bone 1.77+/-1.66% (P=0.05); uninjected bone 0.23+/-0.23% (P=0.03)) with the engrafting cells showing molecular and cytogenetic aberrations identical to the original clones. Normal BM contained a small SSC(lo)ALDH(br) population (median: 2.92%, range: 0.92-5.79%), but none of the ALL cases showed this fraction. In conclusion, SSC(lo)ALDH(br) cells in ALDH(+)AML might denote primitive LSC and confer an inferior prognosis in patients.

Amplification and overexpression of aurora kinase A (AURKA) in immortalized human ovarian epithelial (HOSE) cells.

Immortalization is an early and essential step of human carcinogenesis. Amplification of chromosome 20q has been shown to be a common event in immortalized cells and cancers. We have previously reported that gain and amplification of chromosome 20q is a non-random and common event in immortalized human ovarian surface epithelial (HOSE) cells. The chromosome 20q harbors genes including TGIF2 (20q11.2-q12), AIB1 (20q12), PTPN1 (20q13.1), ZNF217 (20q13.2), and AURKA (20q13.2-q13.3), which were previously reported to be amplified and overexpressed in ovarian cancers. Some of these genes may be involved in immortalization of HOSE cells and represent crucial premalignant changes in ovarian surface epithelium. Investigation of the involvement of these genes was examined in four pairs of pre-crisis (preimmortalized) and post-crisis (immortalized) HOSE cells. Overexpression of AURKA (Aurora kinase A), also known as BTAK and STK15, by both real time-quantitative polymerase chain reaction (RT-QPCR) and Western blotting was detected in all the four immortalized HOSE cells examined while overexpression of AIB1 and ZNF217 was observed in two of four immortalized HOSE cells examined. Overexpression of TGIF2 and PTPN1 was not significant in our immortalized HOSE cell systems. The degree of overexpression of AURKA was shown to be closely associated with the amplification of chromosome 20q in immortalized HOSE cells. Fluorescence in situ hybridization (FISH) with labeled P1 artificial clone (PAC) confirmed the amplification of the chromosomal region (20q13.2-13.3) where AURKA resides. DNA amplification of AURKA was also confirmed using semi-quantitative PCR. Our study showed that amplification and overexpression of AURKA is a common and significant event during immortalization of HOSE cells and may represent an important premalignant change in ovarian carcinogenesis.

Pathogenesis of jumping translocations: a molecular cytogenetics study.

BACKGROUND/AIMS: Jumping translocations are rare cytogenetic aberrations in haematological malignancies, the pathogenesis of which remains to be fully characterised. We investigated the mechanism of formation of jumping translocations in a case of adult common acute lymphoblastic leukaemia (ALL) positive for the Ph translocation. METHODS: Interphase and metaphase fluorescence in situ hybridisation (FISH) was performed using several probe systems. Results were correlated with findings on conventional cytogenetics. Granulocytes, T-cells and leukaemic B-cells in peripheral blood were sorted by immunomagnetic method and the terminal restriction fragment (TRF) length of these cellular populations was determined by Southern blot analysis. RESULTS: Duplicated BCR-ABL fusion signals were found on a dic(14;22)der(22)t(9;22) chromosome. Clonal jumping translocations, existing as evolutionary changes, involved the donor chromosomal segment distal to 1q12 jumping onto the telomere ends of 11q, 15p, 19p and 20p. Telomere length was decreased in the neoplastic B-cell population and contributed to the formation of the dicentric chromosome that showed absence of telomere repeats at fusion ends. Subsequent pericentromeric heterochromatin decondensation of chromosome 1q occurred, and this donor segment was randomly fused to the shortened telomere ends of non-homologous chromosomes. CONCLUSIONS: Both telomere shortening and pericentromeric heterochromatin decondensation contribute to the formation of jumping translocations, which is most probably a multi-stage process.

Transformation of diffuse large B-cell lymphoma into pre-B acute lymphoblastic leukemia: clinicopathologic features and clonal relationship.

A patient with fibrosing alveolitis developed a diffuse large B-cell (DLBC) lymphoma that expressed CD20 and CD30. After an initial response, the lymphoma relapsed and was salvaged with further chemotherapy. After another remission of 3 years, a pre-B-cell acute lymphoblastic leukemia (ALL), which expressed CD10, CD19, CD22, CD79a, CD34 and terminal deoxyribonucleotidyl transferase, developed and led to death. Molecular analysis of the immunoglobulin heavy-chain gene showed that the initial lymphoma and its relapse were clonally related. At leukemic relapse, 2 clones related to the initial and relapsed lymphoma clones were present. DLBC lymphomas arise from post-follicle center B cells, whereas ALL arises from pregerminal B cells. Therefore, a direct transformation of DLBC lymphoma to ALL appears unlikely. The overall features suggest instead separate lymphoma and leukemic evolution from a common mutated B-cell precursor rather than transformation of DLBC lymphoma to ALL.

Chronic myeloid leukemia in an adolescent with Ollier's disease after intensive X-ray exposure.

We report a 19-year-old man with Ollier's disease with multiple orthopedic procedures performed for leg length discrepancy; who developed chronic myeloid leukemia presenting with intramuscular hematoma. His symptoms resolved with cytoreductive treatment by hydroxyurea. Cytogenetic and molecular investigations showed a complex Philadelphia translocation t(9;22;13) (q34;q11.2;q12), with predominance of ela2 BCR/ABL splicing and deletion of reciprocal der(9) ABL/BCR locus, all suggesting poor prognosis. The cumulative X-ray exposure from repeated operations from the age of 7 to 12 years was estimated to be around 16 mSv, approximately the dose of 720 chest X rays. Literature review showed two other cases of leukemia occurring in patients with multiple enchondromatosis. Although the development of CML in this young patient might be related partly to genetic defects, the repeated radiation exposure, especially at young age and directly on the marrow tissue in the long bones, might also be an important pathogenetic factor.

Derivative chromosome 9 deletions in chronic myeloid leukaemia: interpretation of atypical D-FISH pattern.

BACKGROUND/AIMS: New molecular cytogenetic techniques are increasingly applied as a routine investigative tool in haematological malignancies, both at diagnosis and subsequent monitoring. This report describes the interpretation of atypical signal patterns encountered using BCR-ABL dual colour dual fusion fluorescence in situ hybridisation (D-FISH) translocation probes in chronic myeloid leukaemia (CML). METHODS: Interphase FISH experiments were carried out using BCR-ABL D-FISH probes in 46 patients with CML at diagnosis and during subsequent disease monitoring. Atypical hybridisation signal patterns were characterised by molecular cytogenetic techniques and correlated with conventional karyotyping. RESULTS: Two patients showed atypical interphase D-FISH patterns with one orange, one green, and one fusion (1O1G1F) signal. The presence of BCR-ABL gene fusion was documented by a dual colour single fusion (S-FISH) probe. The submicroscopic deletion of the ABL-BCR fusion gene on the derivative chromosome 9 in these cases was subsequently characterised by metaphase FISH on relocated G banded metaphases. CONCLUSIONS: Atypical interphase D-FISH patterns should not be interpreted in isolation and should be considered in conjunction with other cytogenetic or molecular genetic investigations.

Concurrent mediastinal B cell lymphoma and chronic myeloid leukemia with an unusually favorable response to chemotherapy.

A 67-year-old Chinese woman presented with mediastinal B cell lymphoma in 1992 with incidental leukocytosis. Bone marrow and peripheral blood findings confirmed the diagnosis of chronic myeloid leukemia (CML). After combination chemotherapy and radiotherapy for lymphoma, her peripheral blood counts remained normal, and she refused further treatment for nearly six years. Frank hematologic relapse occurred in 1998 and low dose hydroxyurea was used, which was stopped after six months owing to cytopenia. She remained well without treatment at 12-year follow up. Retrospective Southern blot analysis confirmed BCR gene rearrangement in marrow in 1992 and 1998, but not in the lymphoma or the latest peripheral blood. Fluorescence in-situ hybridzation analysis showed no Philadelphia chromosome positive (Ph+) cells in the peripheral blood at last (FISH) follow-up, but BCR/ABL remained detectable. The relevance of the concomitant occurrence of CML and lymphoma and the unusually favorable response of CML to chemotherapy to the pathogenesis of CML is discussed.

Aberrant p15 gene promoter methylation in therapy-related myelodysplastic syndrome and acute myeloid leukaemia: clinicopathological and karyotypic associations.

Seventeen patients with therapy-related myelodysplastic syndrome/acute myeloid leukaemia (t-MDS/AML) were examined for aberrant p15 gene methylation by methylation-specific polymerase chain reaction. Ten patients (58%) showed p15 methylation, which was significantly related to monosomy/deletion of chromosome 7q, but not to antecedent chemotherapy, blast count, leukaemic evolution or survival. In three of six patients with marrow samples obtained prior to the diagnosis of t-MDS/AML, p15 methylation predated disease development by up to 2 years. Bone marrow transplantation led to the disappearance of p15 methylation in one patient. These results showed that p15 methylation was an early event in the evolution of some t-MDS/AML patients.

Trisomy 21 and other chromosomal abnormalities in acute promyelocytic leukemia.

We describe a case of acute promyelocytic leukemia (APL) with t(15;17)(q22;q12) and trisomy 21 as an additional change in a patient who died at relapse after achieving complete remission (CR) for the duration of 20 months. A survey of 42 cases of APL with cytogenetic study performed at our institutionover the past 10 years showed 12 cases (28.6%) having chromosomal changes in addition to t(15;17). Trisomy 8 and trisomy 21 as additional changes were noted in 4 and 2 cases, respectively, with one patient showing both trisomies simultaneously. Two cases showed t(15;17) in hyperdiploid clones. Among the 10 patients with follow-up data, all eventually relapsed and none achieved continuous complete remission 1. Survival analysis performed in APL patients with adequate follow-up data showed no significant difference in overall and disease free survival between those with and without additional cytogenetic changes. After excluding cases with one induction death, the overall survival was significantly in favor of the group without additional cytogenetic abnormalities (P = 0.022). Late relapses may therefore be significantly more common in APL patients with additional cytogenetic abnormalities, and may not be reflected by analysis focused at three-year survival only. As APL is now considered a curable disease, any confirmed long-term survival impact of additional cytogenetic changes is expected to have important management implications.

Clonal marrow abnormalities after azathioprine and sulfasalazine exposure in Crohn's disease: a cautionary tale.

We report a patient with longstanding Crohn's disease (CD) developing recurrent sepsis and impaired neutrophil function tests. His inflammatory bowel disease was controlled with local steroids and sulfasalazine with only short exposure to azathioprine. His blood counts remained within normal range, but the marrow showed mild dysplasia. Repeated cytogenetic examinations revealed trisomies 8 and 9, which are typical for therapy related myelodysplasia. Fluorescent in situ hybridization (FISH) study showed stable persistent trisomies, confined to the myeloid lineage, one year after discontinuation of sulfasalazine. The long-term use of immunodulating agents in patients with CD is not without risks, and early therapy related myelodysplasia might not be easily detected by blood count and morphology assessment alone.

Tyrosine kinase inhibitor STI571 in the treatment of Philadelphia chromosome-positive leukaemia failing myeloablative stem cell transplantation.

Eight patients with Philadelphia chromosome-positive (Ph(+)) leukaemia relapsing from stem cell transplantation (SCT) (one syngeneic and seven allogeneic) were treated with the tyrosine kinase inhibitor STI571. Five patients relapsing as chronic myeloid leukaemia (CML) in chronic phase achieved a complete haematological response, with complete and major cytogenetic responses occurring in four and one cases, respectively. One patient became negative for BCR/ABL in the bone marrow. Three patients relapsed as acute leukaemia (two CML in myeloblastic crisis and one Ph(+) acute lymphoblastic leukaemia), all of whom achieved haematological and cytogenetic responses. One patient also became BCR/ABL negative. However, pancytopenia and graft-versus-host disease led to cessation of treatment in the remaining two cases, which was followed by disease recurrence refractory to further STI treatment. Our results showed that Ph(+) leukaemic relapses after SCT might respond well to STI571 therapy.