ALBERT

Description: Platelet thrombospondin interacts with plasminogen in a specific and saturable manner. Thrombospondin was found to specifically bind to plasminogen and the nonenzyme chain of plasmin. Preincubation of 125I- labeled thrombospondin with 30 mmol/L lysine was without effect in the binding of thrombospondin to immobilized plasminogen; preincubation of 125I-labeled plasminogen with 30 mmol/L lysine, on the other hand, significantly reduced the binding of plasminogen to immobilized thrombospondin, suggesting that the interaction of thrombospondin with plasminogen is not the direct result of the lysine binding sites of plasminogen. Arginine and benzamidine, ligands known to specifically bind to the kringle 5 domain of plasminogen, blocked the binding of thrombospondin to plasminogen. Limited elastase proteolysis of plasminogen and plasmin resulted in the generation of two distinct thrombospondin binding domains, one of which was retained on lysine- agarose. The isolation and amino-terminal analysis of these domains following elastase proteolysis of plasminogen identified them, respectively, as a domain containing kringle structures 4 and 5 and plasmin and the other domain consisting of kringle 5-plasmin. A 16- residue synthetic peptide, which represents the amino acids linking kringle 4 to kringle 5 (residues 435–450 of native plasminogen), was without effect in either binding to thrombospondin or blocking the binding of thrombospondin to plasminogen. Plasminogen, therefore, possesses a single thrombospondin interactive site that is independent of, but influenced by, the lysine binding site containing kringle structures and most likely is located within the kringle 5 domain.

Description: Idiopathic hypereosinophilic syndrome (HES) is an infrequent hematological disorder characterized by persistent eosinophilia with organ involvement, often presenting to other medical specialists. We studied 89 primary HES defined as a peripheral blood eosinophilia greater than 1,500 cells/μL for longer than 6 months, absence of other apparent aetiologies for eosinophilia and symptoms of organ involvement. All patients were negative by molecular analysis for TEL-PDGFRB, and/or BCR-ABL transcripts, frequently associated with HES/CMML/MDS syndromes. We also sought for the recently reported involvement of PDGFRalpha, cryptically fused with FIP1L1 in some HES patients responsive to Imatinib therapy: 34 patients were positive for the FIP1L1-PDGFRA rearrangement and all of them showed previously unreported, abnormal-sized fusion transcripts. A specific RT-PCR assay has been set up for this analysis and a quantitative Q-RT-PCR Taqman assay is in development. Of note, all FIP1L1-PDGFRA positive patients were male. 56 out of 89 (62%) primary HES patients, including 31/34 FIP1L1-PDGFRA positive, were sensitive to imatinib mesylate (100 to 400 mg/daily). Median follow up of treatment was 10 months (range 2–28). Rapid and complete haematological responses (CHR) to imatinib therapy were recorded in all FIP1L1-PDGFRA positive patients (91%) after one month of therapy and partial response in nine cases with HES but without FIP1L1-PDGFRA fusion transcript. Complete molecular response without evidence of FIP1L1-PDGFRA transcript by qualitative RT-PCR was also recorded in the majority of responding patients after median 2 months of therapy, however in few cases the complete molecular response could be recognised only after more than 18 months of imatinib therapy. We conclude that FIP1L1-PDGFRA rearrangement is a useful molecular marker of myeloproliferative HES, a predictor of imatinib-responsiveness and as a means to follow therapy in this subgroup of patients.

Description: We report here the cloning of the cDNA coding for platelet connective tissue-activating peptide-III (CTAP-III) from a lambda gt11 expression library prepared using messenger RNA (mRNA) isolated from human platelets. The open reading frame of the clone coded for a protein with 128 amino acid residues. Since the precursor of CTAP-III, platelet basic protein (PBP is 94 amino acids long, the 5′-translated region of the cDNA codes for a leader sequence 34 amino acids long. This leader sequence, like the sequence of mature CTAP-III, shows significant homology to the sequence of platelet factor 4 (PF4), the only other platelet specific alpha-granule protein cloned until now, from a human erythroleukemic (HEL) cell line-derived cDNA library. These leader sequences are probably critical for targeting such proteins to the alpha-granule. Northern blot hybridization with platelet and megakaryocyte mRNA shows a single species mRNA of approximately 0.8 kb, suggesting that the corresponding cDNA is full length. The cloning of platelet specific CTAP-III provides additional evidence for the platelet specificity of the cDNA library used.

Description: The FIP1L1-PDGFRA fusion gene results from a cytogenetically invisible interstitial chromosomal deletion on chromosome 4q12 and was recently identified as a recurrent molecular abnormality in patients with chronic eosinophilic leukemia (CEL) and systemic mastocytosis with eosinophilia (SME). The pathogenesis of FIP1L1-PDGFRA positive CEL/SME is similar to BCR-ABL positive chronic myeloid leukemia (CML) with constitutively increased tyrosine kinase activity of the fusion protein and excellent response to treatment with imatinib. The breakpoints within FIP1L1 are variable and a number of different exons are fused to a truncated PDGFRA exon 12. However, the numbers of sequenced fusion transcripts in single reports have been too small for a more detailed analysis of the anatomy and relative frequency of the different fusion transcripts. We therefore sought to collect data from FIP1L1-PDGFRA positive patients from several laboratories across Europe (France, Germany, Italy, UK) in a collaborative study within the workpackage "Minimal residual disease" of the European LeukemiaNet. A total number of 43 FIP1L1-PDGFRA positive cases were identified by RT-PCR. For yet unknown reasons a considerable number of cases were only found to be positive after nested RT-PCR despite adequate sample quality, high leukocyte counts and marked eosinophilia. Possible reasons might be a relatively low proportion of FIP1L1-PDGFRA positive cells, relatively low expression of the fusion transcript and/or relatively rapid fusion transcript degradation; although FIP1L1-PDGFRA was found to be expressed at a level comparable to the ABL control gene in RQ-PCR analysis of EOL-1 cell line. Sequence analysis revealed that all PDGFRA breakpoints fell exclusively within exon 12, thus retaining the entire kinase domain of PDGFRA in all cases. The truncated PDGFRA (p) exon 12 was fused to FIP1L1 (f) exons 9 to 13 (formerly described as 7a, 8, 8a, 9 and 10 - Cools et al., NEJM. 2003;348: 1201–1214): f9p12 (n=1; 2%), f10p12 (n = 10; 22%), f11p12 (n=15; 33%), f12p12 (n=7; 15%), f13p12 (n=10; 22%). An insertion of additional sequences of up to 107 bp was found in 24 patients (56%) leading to an open reading frame in all cases. These sequences were derived from introns of FIP1L1 in 14 cases, from FIP1L1 exon 13 in one case and from 4q33 in one case, the latter indicating a more complex rearrangement. Eight inserts of 2 – 6 bp could not be matched to known sequences because they were too short. An entirely identical fusion transcript resulting from an identical PDGFRA sequence fused to the same FIP1L1 exon without insert was found in 6 patients with a f12p12 fusion transcript and 4 patients with a f10p12 fusion transcript. We conclude that the combination of a great variability of breakpoints within FIP1L1 and PDGFRA plus the insertion of sequences which are variable in length and origin lead to unique FIP1L1-PDGFRA fusion sequences in the majority of patients. This carries important implications for strategies for molecular detection and development of RQ-PCR assays to determine response to imatinib or alternative tyrosine kinase inhibitors.

Description: We have identified a novel, recurrent t(8;9)(p21–22;p23–24) in six patients with diverse hematological malignancies: atypical CML (n=4), secondary AML following idiopathic myelofibrosis (n=1) and pre B-ALL (n=1). Because of the involvement of several different tyrosine kinases in atypical CML, we focused our analysis on this class of gene. Initial FISH studies of one patient indicated that the janus kinase 2 gene (JAK2), located at 9p24, was disrupted. RACE-PCR was then used to identify the 8p21 partner gene as PCM1, a large centrosomal protein that contains multiple coiled-coil domains. RT-PCR and FISH analysis confirmed the fusion in this case, and also identified PCM1-JAK2 in the five other t(8;9) patients (RT-PCR and FISH, n=4; RT-PCR only, n=1; FISH only, n=1). Four different types of in-frame mRNA junction were identified, but in all cases the chimeric mRNA is predicted to encode a protein that retains several of the predicted coiled-coil domains from PCM1 and the entire tyrosine kinase domain of JAK2. Reciprocal JAK2-PCM1 mRNA could not be amplified in any patient. Clinically, 4 patients displayed CML-like hyperplasia with variable degrees of myelofibrosis and eosinophilia. Similar to typical CML, the clinical course of these patients was variable: one is alive 11 months after allogeneic stem cell transplantation, one transformed to acute leukemia 5 years after diagnosis, one died 4 days after presentation and one achieved a major cytogenetic response with interferon but died due to neurodegenerative disease and pneumonia at 7.5 years. Of the two remaining patients, one presented with secondary AML following idiopathic myelofibrosis and remains in remission 15 years after diagnosis following intensive chemotherapy and maintenance with interferon. The final patient died shortly after induction therapy for pre B-ALL. In conclusion, PCM1-JAK2 is a novel recurrent fusion gene in hematological malignancies that is likely to deregulate hemopoiesis in a manner similar to BCR-ABL. Patients with PCM1-JAK2 disease are attractive targets for signal transduction therapy.

Description: Platelet thrombospondin interacts with plasminogen in a specific and saturable manner. Thrombospondin was found to specifically bind to plasminogen and the nonenzyme chain of plasmin. Preincubation of 125I- labeled thrombospondin with 30 mmol/L lysine was without effect in the binding of thrombospondin to immobilized plasminogen; preincubation of 125I-labeled plasminogen with 30 mmol/L lysine, on the other hand, significantly reduced the binding of plasminogen to immobilized thrombospondin, suggesting that the interaction of thrombospondin with plasminogen is not the direct result of the lysine binding sites of plasminogen. Arginine and benzamidine, ligands known to specifically bind to the kringle 5 domain of plasminogen, blocked the binding of thrombospondin to plasminogen. Limited elastase proteolysis of plasminogen and plasmin resulted in the generation of two distinct thrombospondin binding domains, one of which was retained on lysine- agarose. The isolation and amino-terminal analysis of these domains following elastase proteolysis of plasminogen identified them, respectively, as a domain containing kringle structures 4 and 5 and plasmin and the other domain consisting of kringle 5-plasmin. A 16- residue synthetic peptide, which represents the amino acids linking kringle 4 to kringle 5 (residues 435–450 of native plasminogen), was without effect in either binding to thrombospondin or blocking the binding of thrombospondin to plasminogen. Plasminogen, therefore, possesses a single thrombospondin interactive site that is independent of, but influenced by, the lysine binding site containing kringle structures and most likely is located within the kringle 5 domain.

Description: We report here the cloning of the cDNA coding for platelet connective tissue-activating peptide-III (CTAP-III) from a lambda gt11 expression library prepared using messenger RNA (mRNA) isolated from human platelets. The open reading frame of the clone coded for a protein with 128 amino acid residues. Since the precursor of CTAP-III, platelet basic protein (PBP is 94 amino acids long, the 5′-translated region of the cDNA codes for a leader sequence 34 amino acids long. This leader sequence, like the sequence of mature CTAP-III, shows significant homology to the sequence of platelet factor 4 (PF4), the only other platelet specific alpha-granule protein cloned until now, from a human erythroleukemic (HEL) cell line-derived cDNA library. These leader sequences are probably critical for targeting such proteins to the alpha-granule. Northern blot hybridization with platelet and megakaryocyte mRNA shows a single species mRNA of approximately 0.8 kb, suggesting that the corresponding cDNA is full length. The cloning of platelet specific CTAP-III provides additional evidence for the platelet specificity of the cDNA library used.