Treatment Efficacy and Resistance Mechanisms Using the Second-Generation ALK Inhibitor AP26113 in Human NPM-ALK-Positive Anaplastic Large Cell Lymphoma.

UNLABELLED: ALK is a tyrosine kinase receptor involved in a broad range of solid and hematologic tumors. Among 70% to 80% of ALK(+) anaplastic large cell lymphomas (ALCL) are caused by the aberrant oncogenic fusion protein NPM-ALK. Crizotinib was the first clinically relevant ALK inhibitor, now approved for the treatment of late-stage and metastatic cases of lung cancer. However, patients frequently develop drug resistance to Crizotinib, mainly due to the appearance of point mutations located in the ALK kinase domain. Fortunately, other inhibitors are available and in clinical trial, suggesting the potential for second-line therapies to overcome Crizotinib resistance. This study focuses on the ongoing phase I/II trial small-molecule tyrosine kinase inhibitor (TKI) AP26113, by Ariad Pharmaceuticals, which targets both ALK and EGFR. Two NPM-ALK(+) human cell lines, KARPAS-299 and SUP-M2, were grown in the presence of increasing concentrations of AP26113, and eight lines were selected that demonstrated resistance. All lines show IC50 values higher (130 to 1,000-fold) than the parental line. Mechanistically, KARPAS-299 populations resistant to AP26113 show NPM-ALK overexpression, whereas SUP-M2-resistant cells harbor several point mutations spanning the entire ALK kinase domain. In particular, amino acid substitutions: L1196M, S1206C, the double F1174V+L1198F and L1122V+L1196M mutations were identified. The knowledge of the possible appearance of new clinically relevant mechanisms of drug resistance is a useful tool for the management of new TKI-resistant cases. IMPLICATIONS: This work defines reliable ALCL model systems of AP26113 resistance and provides a valuable tool in the management of all cases of relapse upon NPM-ALK-targeted therapy.

Bacterial sepsis.

Bacterial sepsis remain a major cause of mortality and morbidity in the newborn. The severe outcome of neonatal sepsis, despite the advances in perinatal and neonatal care and use of potent antibiotics, is related to the neonatal reduced immune defenses and the complex interactions between the infecting microorganism and the host responses. An early diagnosis, based on the clinical picture, the isolation of microorganisms and the positivity of inflammatory indexed, is mandatory. A timely treatment should be aimed to the elimination of pathogens with antimicrobials. Intravenous immunoglobulin and hematopoietic growth factors may be considered to improve the disturbed immune homeostases.

Surgical management of renal cell carcinoma during the second trimester of pregnancy.

Renal cell carcinoma is rarely diagnosed during pregnancy and its management represents a real challenge. Pregnancy demands special consideration in terms of diagnostic evaluation and management, particularly during the second trimester. We report a patient undergoing left radical nephrectomy using a thoracoretroperitoneal approach at 22 weeks' gestation. Histological analysis revealed a pT2R0G2 chromophobic renal cell carcinoma. Furthermore, we review the sparse literature available.

The first ambulatory screening on thromboembolism: a multicentre, cross-sectional, observational study on risk factors for venous thromboembolism.

OBJECTIVES: To assess the prevalence of risk factors for venous thromboembolism (VTE) and the prevalence of recent (<1 year) VTE [including superficial vein thrombosis (SVT), deep vein thrombosis (DVT) and pulmonary embolism (PE)] amongst patients attending general practitioner (GP) surgeries. DESIGN: Multicentre, cross-sectional, observational study. SETTING: A total of 1536 GP surgeries. PARTICIPANTS: A total of 15 180 adult, co-operative subjects, who had consulted their GP for a health disorder and signed the informed consent form. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Prevalence of known VTE risk factors graded according to importance and prevalence of recent (<1 year) VTE events (including SVT), based on interviews. RESULTS: About 1:5 patients had at least one strong risk factor and about 1:20 had at least two risk factors, with no difference between sexes. The prevalence of strong risk factors increased with age. Most were related to medical conditions: history of SVT and/or DVT/PE, heart failure and malignancy. About 3:4 women and 2:3 men had at least one moderate to weak risk factor; nearly 1:2 women and 1:3 men had at least two moderate to weak risk factors. The most common were: history of VTE, smoking, history of miscarriage, estrogen therapy, obesity, and varicose veins. Overall, 80% women and 67% men had at least one risk factor, and 50% women and 35% men had at least two risk factors. The prevalence of recent (<1 year) VTE was 3.4% in women and 2.4% in men, and increased with age. The majority of cases were SVT in both sexes (2.5% in women and 1.5% in men). CONCLUSIONS: The prevalence of risk factors for VTE amongst patients attending GP surgeries is high. GPs should bear this in mind during their daily practice.

The paired box domain gene PAX5 is fused to ETV6/TEL in an acute lymphoblastic leukemia case.

The PAX5 gene, encoding the B-cell-specific activator protein, is a critical determinant of commitment to the B-lymphocyte pathway. This gene, mapped at 9p13, is juxtaposed to the immunoglobulin heavy chain (IgH) gene as a result of the t(9;14)(p13;q32), a rare but recurring translocation found in a subset of B-cell non-Hodgkin's lymphoma cases. In all of these, this translocation results in deregulated expression of the gene product because of the proximity of IgH. We present here the molecular characterization of a previously reported acute lymphoblastic leukemia case carrying a t(9;12)(q11;p13) translocation. Using 5' rapid amplification of cDNA ends PCR, a novel chimeric transcript was identified that contained the NH(2)-terminal region of PAX5 and most of the ETV6/TEL gene on 12p13. According to the fusion transcript, the resulting chimeric protein would retain the PAX5 paired-box domain and both the helix-loop-helix and DNA binding domains of TEL. Thus, it is reasonable to hypothesize that this protein could act as an aberrant transcription factor. This is the first report of PAX5 rearrangement in a human malignancy resulting in a chimeric transcript.

Familial partial monosomy 7 and myelodysplasia: different parental origin of the monosomy 7 suggests action of a mutator gene.

Two sisters are reported, both with a myelodysplastic syndrome (MDS) associated with partial monosomy 7. A trisomy 8 was also present in one of them, who later developed an acute myeloid leukemia (AML) of the M0 FAB-type and died, whereas the other died with no evolution into AML. Besides FISH studies, microsatellite analysis was performed on both sisters to gather information on the parental origin of the chromosome 7 involved in partial monosomy and of the extra chromosome 8. The chromosomes 7 involved were of different parental origin in the two sisters, thus confirming that familial monosomy 7 is not explained by a germ-line mutation of a putative tumor-suppressor gene. Similar results were obtained in two other families out of the 12 reported in the literature. Noteworthy is the association with a mendelian disease in 3 out of 12 monosomy 7 families, which suggest that a mutator gene, capable of inducing both karyotype instability and a mendelian disorder, might act to induce chromosome 7 anomalies in the marrow. We postulate that, in fact, an inherited mutation in any of a group of mutator genes causes familial monosomy 7 also in the absence of a recognized mendelian disease, and that marrow chromosome 7 anomalies, in turn, lead to MDS/AML.

The tyrosine kinase abl-related gene ARG is fused to ETV6 in an AML-M4Eo patient with a t(1;12)(q25;p13): molecular cloning of both reciprocal transcripts.

The Ets variant gene 6 (ETV6/TEL) gene is rearranged in the majority of patients with 12p13 translocations fused to a number of different partners. We present here a case of acute myeloid leukemia M4 with eosinophilia (AML-M4Eo) positive for the CBFb/MYH11 rearrangement and carrying a t(1;12)(q25;p13) that involves the ETV6 gene at 12p13. By 3'rapid amplification of cDNA ends-polymerase chain reaction (3'RACE-PCR), a novel fusion transcript was identified between the ETV6 and the Abelson-related gene (ARG) at 1q25, resulting in a chimeric protein consisting of the HLH oligomerization domain of ETV6 and the SH2, SH3, and protein tyrosine kinase (PTK) domains of ARG. The reciprocal transcript ARG-ETV6 was also detected in the patient RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), although at a lower expression level. The ARG gene encodes for a nonreceptor tyrosine kinase characterized by high homology with c-Abl in the TK, SH2, and SH3 domains. This is the first report on ARG involvement in a human malignancy.

Characterization of the human myeloid leukemia-derived cell line GF-D8 by multiplex fluorescence in situ hybridization, subtelomeric probes, and comparative genomic hybridization.

The human myeloid leukemia cell line GF-D8 was established from the peripheral blood blasts of a patient with acute myeloid leukemia FAB subtype MI (AML-MI). The karyotype, which has not changed significantly over several years of culture, was described initially as 44,XY,-5,del(7q),inv(7q),add(8q),add(11q),del(12p),-15,-17,+mar. With the advent of multicolor fluorescence in situ hybridization (FISH) techniques, the prospect of accurately characterizing this complex karyotype became feasible. In the present study, we applied 24-color whole-chromosome painting and analyzed the results using a filter-based detection system and proprietary software for multiplex FISH (M-FISH). This resulted in the refinement of the karyotype and the identification of hitherto unsuspected chromosome rearrangements. M-FISH identified the origin of the add(8q) and add(11q) as well as the small marker chromosome. Both the del(7q) and del(12p) were redefined as unbalanced translocations and an apparently normal chromosome 11 was shown to be t(11;17). Importantly, the del(12p) was shown to be a der(12)t(7;12). Single-color whole-chromosome painting studies confirmed these findings, but also identified a cryptic t(Y;12) not seen in the original M-FISH analysis. We then carried out a FISH screening assay using a complete set of chromosome-specific subtelomeric probes. This allowed the identification of p and q subtelomeric regions involved in the translocations and indicated amplification of the 8q subtelomeric region. Comparative genomic hybridization (CGH) revealed a highly unbalanced karyotype, as deletions accompanied the majority of translocations, and identified the regions of amplification as 8q22.3-qter and 11q21-qter. Finally, conventional FISH with centromeric and unique sequence probes was necessary to elucidate all of the rearrangements.

Delineation of multiple deleted regions in 7q in myeloid disorders.

Loss of chromosome material due to deletions of the long arm of chromosome 7, del(7q), is a consistent finding in all types of myeloid disorders, invariably associated with a poor prognosis. Two different segments, 7q22 and 7q32-q33, have been implicated as critical regions of gene loss associated with these disorders. In the present study, we used fluorescence in situ hybridization (FISH) to characterize the 7q22 breakpoint of an apparently balanced t(7;7)(p13;q22) in an acute myeloid leukemia patient. FISH analysis on bone marrow metaphases from this patient revealed that the sequence corresponding to a series of three ordered cosmids from 7q22 was deleted from one of the der(7) chromosomes. These cosmids contain the human homologue of the Drosophila homeobox gene cut (CUTL1) and span a region of approximately 150 kb. Although the proximal boundary of the deleted segment could not be exactly defined, we estimate the size of this deletion to be approximately 500 kb. Subsequently, we carried out FISH studies using the CUTL1 cosmids on a further 16 patients with deletions of 7q and myeloid disorders. The sequence corresponding to at least two of the cosmids was deleted from the del(7q) in 11 out of 14 cases with a proximal breakpoint within 7q22. Further detailed FISH mapping in this series of 17 patients has identified two other nonoverlapping commonly deleted segments at 7q31-q32 and 7q33, respectively. These data confirm and refine other studies, implying that several different genes on 7q may be involved in the pathogenesis of myeloid diseases. Genes Chromosomes Cancer 25:384-392, 1999.

Identification of a novel molecular partner of the E2A gene in childhood leukemia.

The 'promiscuous' E2A gene, at 19p13.3, is fused with two different molecular partners, PBX1 and HLF, following two chromosome translocations recurrent in childhood pre-B ALL. We have identified a novel gene, FB1, by virtue of its fusion with E2A and by a combination of molecular techniques. FB1 was localized on 19q13.4, suggesting that the novel chimera originated by a cryptic rearrangement of chromosome 19. Two FB1 transcripts, of 1.2 kb and 1.1 kb, are differentially expressed at low level in a variety of human tissues, including hemopoietic cell lines from different lineages. Accordingly, FB1 cDNA displays high homology with a number of cDNA clones from different human tissues. High homology was found also with cDNA clones from mouse and rat, suggesting that the sequence might be conserved at least among mammals. The function of the putative FB1 protein, however, is currently unknown as database sequence comparisons have failed to reveal strong homology with known proteins. The E2A/FB1 fusion appears to be a recurrent feature of pre-B ALLs, suggesting that it might have a role in the development and/or progression of leukemogenesis.

Identification of new partner chromosomes involved in fusions with the ETV6 (TEL) gene in hematologic malignancies.

Several partner genes on different chromosomes have been reported to be fused with the ETV6 gene (located in chromosome band 12p13), with different breakpoints and different frequencies, in various hematologic malignancies, particularly acute myeloid and lymphoid leukemias and myelodysplastic syndromes. By using FISH and molecular analyses, we have analyzed five different pediatric and adult patients carrying cytogenetic abnormalities involving 12p13. Our findings demonstrate that ETV6 was rearranged in all the cases analyzed. In particular, ETV6 was disrupted by translocations with chromosomal bands 7q22, 7q36, 9q11, and 13q12, not previously described as partners of ETV6 in translocations, thus extending its promiscuity in rearranging with different partner genes.

Unbalanced t(3;12) in a case of juvenile myelomonocytic leukemia (JMML) results in partial trisomy of 3q as defined by FISH.

Juvenile myelomonocytic leukemia (JMML) is a rare disorder of early childhood, to which no recurrent chromosome rearrangement has been yet associated. We report a case where leukemic cells harbored a 46,XX,der(12)t(3;12) (q21 approximately 22;p13.33) karyotype, resulting in partial trisomy of 3q. The origin of chromosome material translocated to chromosome 12 was assessed by chromosome painting using a whole chromosome 3-specific probe. The breakpoint regions were defined by FISH using YAC probes from 3q and 12p chromosomal regions. Interestingly, partial trisomy of 3q has been detected in a previously reported JMML case, consequent to the presence of a der(15)t(3;15)(q13.1;q26). The involvement of a similar chromosome 3 rearrangement in these two JMML cases suggests the hypothesis that either the resulting duplication of some gene/s on 3q or the loss of heterozygosity (LOH) of some gene/s on 3p may be involved in one of the steps leading to JMML. On the other hand, it cannot be ruled out that the relevant mutation in our case might be consequent to the particular breakpoints at bands 3q21 approximately 22 and 12p13.3, that may alter the structure and/or expression of the involved gene/s.

Detection of the breakpoint cluster region-ABL fusion in chronic myeloid leukemia with variant Philadelphia chromosome translocations by in situ hybridization.

Fluorescence in situ hybridization (FISH) technique has been successfully used to detect the BCR-ABL gene fusion in chronic myeloid leukemia (CML) with the classic form of the Philadelphia chromosome (Ph). We applied FISH to study three CML patients showing variant Ph chromosome (either complex or simple type). The results demonstrate that the use of a yeast artificial chromosome (YAC)-derived probe (D107F9) and a cosmid probe (cos-abl 8), specific for BCR and ABL genes respectively, allows also the detection of the BCR-ABL fusion in CML patients with variant Ph.

Molecular diagnosis and clinical relevance of t(9;22), t(4;11) and t(1 ;19) chromosome abnormalities in a consecutive group of 141 adult patients with acute lymphoblastic leukemia.

Over a time period of five years leukemic blast samples from 141 consecutive patients with adult ALL were referred to our laboratory, for molecular evaluation of chromosome abnormalities. The t(9;22), t(4;11) and t(1;19) which are most commonly found in adult ALL with a B-precursor phenotype were molecularly analyzed by similar RT-PCR based protocols. BCR-ABL transcripts generated by the t(9;22) translocation were demonstrated in 36 patients (25%) and were restricted to the 109 patients with B precursor ALL (33% of this group). Of 83 patients showing a, common phenotype (CD10+), 34 were BCR-ABL positive (41%) whereas only 2 out of 26 with Null ALL (HLADr+, CD19+, CD10) were positive. Interestingly, the percent of BCR-ABL positive CD1O+ ALL increases significantly with age being 20% in patients less than 30 years old and more than 50% in older patients. None of the T-ALL (24 patients) and B-ALL (8 patients) were positive. The majority of cases (67%) showed the p190 gene subtype. The cytogenetic diagnosis of Philadelphia chromosome was always confirmed by the molecular analysis and this approach allowed for the detection of the presence of the BCR-ABL rearrangement in 26 patients when a negative result or no metaphases were obtained. The complete remission rate was similar among BCR-ABL positive and negative patients but a shorter remission duration was observed in those showing molecular evidence of t(9;22) and this finding was significantly evident in CD1O+ ALL patients. By means of comparison, in most of the same adult ALL patients, we analyzed the yet unrecognized prevalence of the t(4;11) and t(1;19) translocations by the molecular analysis of their chromosomal breakpoints. Rearrangements of the ALL-1 gene on 11q23 band and ALL- l1AF.4 fusion transcripts specific for the t(4;11) were demonstrated in 7 out of the 21 Null ALL investigated, with no additional positive cases found among the other ALL subgroups. Overall the clinical behavior of t(4; 11) positive patients was dismal with a very short CR duration. Chimeric E2A-PBX1 transcripts generated by the t(1;19) were found in only two of the 87 B-precursor ALL analyzed. The presented results provide further evidence for the utility of RT-PCR based methods for the molecular diagnosis of chromosome translocations in ALL. The identification of such abnormalities can significantly contribute to the identification of more appropriate therapeutic options for standard and high risk ALL patients

Reverse transcriptase/polymerase chain reaction follow-up and minimal residual disease detection in t(1;19)-positive acute lymphoblastic leukaemia.

The t(1;19) is the most frequent recurring chromosomal translocation in childhood acute lymphoblastic leukaemia (ALL). In most cases typical chimaeric E21-PBX1 transcripts are expressed as a consequence of this rearrangement, allowing the molecular detection of the t(1;19) at the RNA level. This translocations has been associated with a poor clinical outcome, although intensified chemotherapy has been reported to nullify its adverse prognostic impact. We therefore used reverse transcriptase/polymerase chain reaction (RT-PCR) to detect residual leukaemic cells at successive times during treatment and to monitor the response to chemotherapy in six t(1;19)-positive ALL pediatric patients. Five of these patients rapidly achieved molecular remission and no evidence of minimal residual disease (MRD) was found in the remission bone marrows beyond the third month of treatment. One patient still displayed residual leukaemic cells at the end of therapy, although she has been in continuous complete clinical remission (CCR) for 84 months. However, this patient is peculiar in our series in that two different types of chimaeric E2A-PBX1 transcripts were expressed in her leukaemic cells, only one being detectable in remission.

Establishment of human acute myelogenous leukemia lines secreting interleukin-1 beta in SCID mice.

A reproducible in vivo model of human acute myelogenous leukemia (AML) was established in severe combined immunodeficient (SCID) mice. The AML-CL and AML-PS lines were originated from leukemic blasts purified respectively from the peripheral blood of a 27-year-old woman with previously untreated hyperleukocytotic AML and from the bone marrow of a 61-year-old man during the third leukemic relapse. The 2 lines were maintained and serially transplanted i.p. in SCID mice. AML-PS and AML-CL produced ascitogenous gross tumors after approximately 4 and 6 weeks, respectively, and all mice died within 6-8 weeks. Microscopic evaluation of different organs at autopsy showed massive involvement of bone marrow, liver and spleen, though with differences in the tumor burdens for the 2 lines (AML-CL > AML-PS). Flow cytometric analysis documented the spread of leukemic cells to bone marrow, peripheral blood and spleen. AML-PS and AML-CL cells show an immunophenotypic profile (CD13+, CD33+) and cytogenetic findings similar to freshly isolated blasts. Interleukin-1 beta (IL-1 beta) gene expression was observed by Northern blot analysis in leukemic cells from AML-CL and AML-PS SCID mice. After 24 hr of culture both lines released IL-1 beta in culture supernatants. High levels of circulating IL-1 beta were secreted in plasma of tumor-bearing mice. This AML-SCID murine model could contribute to an understanding of the mechanisms of AML growth in vivo and the possible role of the autocrine production of IL-1 beta in promoting cell growth.

Reciprocal translocation t(12;13)(p13;q14) in acute nonlymphoblastic leukemia: report and cytogenetic analysis of two cases.

Two cases of acute nonlymphoblastic leukemia with a reciprocal translocation t(12;13)(p13;q14) are described. Both patients were male adults with a diagnosis of M0 FAB type. Beside standard cytogenetic analysis, we applied fluorescence in situ hybridization (FISH) in order to investigate the position of the RB gene with respect to the breakpoint at 13q14. Our results showed that the RB gene was proximal to the breakpoint, but, apparently, not split in either case.