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Interactions of malignant multiple myeloma (MM) plasma cells with the microenvironment control MM plasma-cell growth, survival, drug-resistance and dissemination. As microvascular density increases in the bone marrow in MM, we investigated whether bone marrow MM endothelial cells control disease progression via the junctional adhesion molecule-A (JAM-A). Membrane and cytoplasmic JAM-A levels were upregulated in MM endothelial cells in 111 patients with newly diagnosed MM and in 201 with relapsed/refractory MM compared to the levels in patients with monoclonal gammopathy of undetermined significance and healthy controls. Elevated membrane expression of JAM-A on MM endothelial cells predicted poor clinical outcome. Mechanistically, addition of recombinant JAM-A to MM endothelial cells increased angiogenesis, whereas inhibition of this adhesion molecule impaired angiogenesis and MM growth in two-dimensional and three-dimensional in vitro cell cultures and chorioallantoic membrane assays. To corroborate these findings, we treated MM-bearing mice with a JAM-A-blocking monoclonal antibody and demonstrated impaired MM progression, corresponding to decreased MM-related vascularity. These findings support the concept that JAM-A is an important mediator of MM progression through facilitating MM-associated angiogenesis. Elevated JAM-A expression on bone marrow endothelial cells is an independent prognostic factor for the survival of both patients with newly diagnosed MM and those with relapsed/refractory MM. Blocking JAM-A restricts angiogenesis in vitro, in utero and in vivo and represents a suitable druggable molecule to halt neo-angiogenesis and MM progression.

The karyotypically silent t(4;14)(p16.3;q32) translocation can be found in approximately 15-20% of multiple myeloma (MM) patients and results in the ectopic expression of fibroblast growth factor receptor 3 (FGFR3) from der4. Point mutations in specific FGFR3 domains can be found in the translocated allele, and have been recently proven to be oncogenic. These mutations produce a constitutively activated receptor, which shows dimerization and autophosphorylation even in the absence of ligand. We investigated the presence of FGFR3 expression and activating mutations in a series of newly diagnosed MM patients.We validated a new sensitive and specific Taqman real-time reverse-transcription polymerase-chain-reaction (RT-PCR) set up to evaluate FGFR3 mRNA expression, and applied it to 78 newly diagnosed patients; in positive cases, FGFR3 mRNA transcripts were sequenced. Fluorescence in situ hybridization (FISH) was done in 32 cases with sufficient material.Real-time RT-PCR revealed FGFR3 mRNA expression in 10/78 (13%) patients. In two cases, sequence analysis revealed novel FGFR3 mutations. In a patient with FISH evidence of the t(4;14), a CGC to TGC transition was detected in codon 248. In a patient without the t(4;14), three additional, abnormal-sized transcripts were detected, corresponding to truncated transcripts originating from cryptic splice donor sites located within exon 7.We describe a novel FGFR3 mutation (with a demonstrated deregulatory mechanism), as well as a case of alternative splicing in the absence of t(4;14), detected in newly diagnosed MM patients overexpressing FGFR3. This implies that FGFR3 mutation can occur at an early stage of myelomagenesis and even in the absence of the t(4;14).

We previously described a novel type of the chimeric bcr-abl mRNA transcript in a patient with a Philadelphia chromosome positive chronic myeloid leukemia. A similar bcr-abl transcript has also been described by others.Sequence analysis of the fusion region showed a join between part of exon e8 of the bcr gene and an intronic sequence of abl intron 1b spliced on exon a2 of the abl gene, giving rise to an in-frame e8-int-a2 bcr-abl transcript, translated into a 197.5 kDa BCR-ABL protein of 1804 amino acid residues, which we named p200 BCR-ABL.In this work, employing protein comparison analysis (pFAM) we show that these novel bcr-abl transcripts retain the DBL homology (DH) domain and the recently recognized CDC24 homology domain, but not the pleckstrin homology (PH) domain of the bcr gene.This observation, along with the myeloid immunophenotype of the tumor and, at least in one case, the patient's correspondingly good response to alpha-interferon therapy, suggests that p200 BCR-ABL is more similar to p210 BCR-ABL, in which the DH, CDC24 and PH domains are all maintained, than to p185, in which these domains are all lost.

We further characterized a novel type of chimeric BCR-ABL mRNA transcript detected in a patient with Philadelphia chromosome positive (Ph+) chronic myeloid leukemia (CML).We used reverse-transcription polymerase chain reaction (RT-PCR) and sequence analysis of the fusion region of the amplified cDNA fragment. Western analysis was performed on total protein.Part of exon e8 of the BCR gene was joined to an intronic sequence of ABL intron Ib spliced on exon a2 of the ABL gene, giving rise to an in-frame e8-int-a2 BCR-ABL transcript. Only part of exon 8 of the BCR gene (e8) (intra-exonic break) was retained. The consequent BCR-int-ABL transcript was translated into a BCR-ABL protein of 1804 amino acid residues with a molecular mass of 197.5 kilodaltons (kDa) called p200 BCR-ABL. The 3' part of bcr exon 8 recombined within or alongside Alu elements at the additional sites. Sequence motifs similar to consensus binding sites of the lymphoid-associated TRAX and translisin proteins were present on both participating strands at 22q11 and 9q34 recombination sites, respectively. No differences in clinical or laboratory findings at diagnosis were found between this patient and CML patients with bcr-abl fusion.The presence of Alu sequences and of the translisin binding motif on both sides of the breaks in this novel translocation suggests a possible general mechanism of molecular recombination in CML patients.

Capillary electrophoresis (CE) has become an attractive alternative to SLAB gel analysis for direct and accurate detection of amplified product, and a few cycles of polymerase chain reactions (PCRs) could be sufficient for both quantitative and qualitative analysis. We try to assess: 1) whether CE could be a practical, non-isotopic method for direct detection of the presence of amplified bcr-abl obtained by a reverse transcription (RT)-PCR (qualitative analysis) and 2) whether it is possible to quantify PCR products using a competitive RT-PCR measuring peak areas of CE electropherograms (quantitative analysis).The two types of bcr-abl chronic myelogenous leukemia (CML) associated transcript products were generated by RT-PCR (qualitative analysis) from 1 microgram of total RNA extracted from bone marrow samples of 34 CML patients at diagnosis (median age 47.5; range 18-65; median Sokal's score 0.9; range 0.53-2.78). The PCR products were analyzed by SLAB-gel electrophoresis (SGE) on 2% agarose gels and by CE (128 runs; median 3.3 times for each sample). Furthermore, we assessed the amount of PCR product (quantitative analysis) by a competitive RT-PCR approach and by CE (bcr-abl transcripts were expressed as transcript per microgram of total RNA examined).CE separation of PCR products obtained by qualitative RT-PCR showed baseline resolution for the two peaks corresponding to the two types of bcr-abl junctions: the b2-a2 type (343 base pairs, 10 patients) was revealed at 9.33 min [standard deviation (SD) = 0.1] and the b3-a2 type (418 base pair, 24 patients) at 10.03 min (SD = 0.25). By quantitative analysis we found that there is great interpatient variability in bcr-abl expression at diagnosis: the median value of the amount of bcr-abl transcript was 78,000 bcr-abl transcript/microgram total RNA ranging from 17,300 to 750,000. The amount of bcr-abl transcript at diagnosis was related to the number of blast cells (mean value 128,859 vs. 331,722 in patients with 0% blast cells and1% blast cells, respectively; p = 0.004) and Sokal's score (mean value 156,865 vs. 408,800 in patients with Sokal's score0.8 and1.2, respectively; p = 0.003).Our results confirm that CE analysis offers greater resolution and enhanced sensitivity for detection and quantification of bcr-abl PCR product in the study of this leukemia. Qualitative analysis by CE of bcr-abl product provides a rapid technique (less than 20 min) for the analysis of subnanogram amounts of DNA fragments. CE run times are short, the capillary can be re-used and full automation may be feasible with data acquisition by a computer-controlled step. Competitive/quantitative analysis of bcr-abl as analyzed by CE allowed fewer reactions and more precise quantification.

Traditional gel electrophoresis of PCR amplification products of regions from T-cell receptor genes often does not differentiate monoclonal from polyclonal rearrangements. We used polymerase chain reaction (PCR) and heteroduplex analysis on polyacrylamide gels to improve the detection of monoclonal rearrangements.We investigated heteroduplex analysis of the amplified V gamma-J gamma junctions of the rearranged T-cell receptor gamma (TcR-gamma) gene by electrophoretic separation on non-denaturing polyacrylamide gel (PAGE) in 8 T-cell acute lymphoblastic leukemia (T-ALL) patients analyzed at diagnosis.Clonal homoduplex and heteroduplex bands were present only in the T-ALL samples and not in controls. We confirmed clonality by direct sequencing of the V gamma-J gamma junction. In 2 instances the analysis was performed on samples obtained from the same patient at diagnosis and at relapse, respectively; the presence of the same clonal TcR-gamma rearranged cell remained detectable during clinical progression of the disease.Our heteroduplex analysis showed that separation of the PCR product by electrophoresis on non-denaturing PAGE is a rapid and convenient method for the detection of clonal TcR-gamma rearrangements in T-ALL.

After allogeneic bone marrow transplantation (BMT) it is important to be able to distinguish between the host and donor origin of cells in order to monitor the engraftment process. However, identifying whether the hematopoietic stem cells are of donor or recipient origin may be a difficult task. DNA studies using Southern blotting techniques or the amplification by PCR of regions in the human genome with high polymorphic neutral sequence variation showing Mendelian inheritance as variable number of tandem repeats (VNTR) can detect the origin of host bone marrow after BMT. We have tried to apply these sensitive systems of detection to the early stages of BMT when small numbers of regenerated cells are available for analysis.We used in vitro polymerase chain reaction (PCR) amplification of three single-locus simple repetitive DNA sequences, all of which vary extensively in their repeat number among different individuals (VNTR D1S80, ApoB, and D17S5), to evaluate post transplant engraftment in six patients who showed no signs of peripheral blood engraftment at 2-3 weeks after transplant. We tested 2 patients with chronic myelogenous leukemia (CML), 2 with B-acute lymphoblastic leukemia (B-ALL), 1 with T-acute lymphoblastic leukemia (T-ALL), and 1 with aplastic anemia (SAA), all in prolonged aplasia following allogeneic bone marrow transplantation (BMT).In a sequential analysis protocol with the different loci, the donor was distinguishable from the recipient in all pairs with at least one of the three markers used. After 16 days (median 16.2; range 15 to 20 days) we found that complete chimerism was present in 5 patients: 4 of donor origin (= engraftment) and one of host origin (= rejection), this last case being one of mixed chimerism. In the 2 cases in which the presence (one complete and one partial) of host DNA was detected, rejection of the donor bone marrow followed, and in 1 patient a second BMT was necessary. The other 4 patients with complete chimerism of donor origin achieved hematological reconstitution and we documented complete engraftment of donor bone marrow a few months later.Utilizing PCR to document early post-transplant engraftment and chimerism in the first month after BMT has the advantage over Southern blotting of being more sensitive and requiring small amounts of sample. It may also be useful for guiding subsequent therapeutic decisions.

HIV-seropositive patients who belong to the three major acquired immunodeficiency syndrome (AIDS) risk groups may develop an idiopathic thrombocytopenic purpura (ITP) which is related to the HIV infection. HIV-associated ITP clinically resembles classic ITP but, in spite of very low platelet numbers, bleeding is rarely severe, and moderate splenomegaly and lymphadenomegalies are seldom present. Treatment is the same as that given for classic ITP because the pathogenesis is in many ways similar. Immunosuppressors can be dangerous in the case of retrovirosis, and splenectomy may lead to AIDS. High doses of immunoglobulins often give an improved platelet count but this tends to be short-lived, and long-term periodical infusions usually lose therapeutical effect. Alpha interferon gives conflicting results and Danatrol is not usually effective. Specific anti-D immunoglobulins produce a high percentage of positive results and may be administered for long-term maintenance without side effects. Zidovudine (AZT) may produce a good platelet increase in a large number of patients, but there is no consensus for the use of this anti-retroviral drug in otherwise asymptomatic HIV-positive patients. In conclusion, since it is very unusual for bleeding to occur, moderate thrombocytopenia is best left untreated because a spontaneous increase in platelet count is possible. But if the platelet count is very low, or if bleeding is present, treatment is mandatory and must produce a rapid platelet increment with minimal side effects.