The genetic hallmark of MDS is a gain or loss of chromosomal loci identified in bone marrow (BM) cells in ∼50% of pts at diagnosis. We previously demonstrated in longitudinal chromosome study a modulating effect of chronic AZA C therapy on the MDS clone which subdivided patients into five distinct cytogenetic groups with statistically significant differences in survival (P=0.0003) (Najfeld et al, ASH 2004). Our goal was, therefore, to investigate whether PB cells can be used in substitute of BM cells for detection of genomic defects to monitor the clone during AzaC-based therapy. Chromosomal and interphase (I-) FISH studies were performed using BM and PB cells at baseline and following AzaC-based therapy. I-FISH studies were evaluated with a panel of six probes [EGR1 (5q31), D7S522 (7q31), D8Z2 (8p11.1-q11.1), MLL (11q23), Rb1 (13q14), D20S108 (20q12)]. Of the 47 pts studied, 25 (53%) had a normal karyotype and disomy for the MDS panel of six probes. The other 22 pts (47%) were cytogenetically abnormal, showing concordant results in 16 of 22 pts (73%) for cytogenetic and I-FISH genomic defect in BM cells (showing ≤20% frequency difference). The remaining 6 pts had a mean of 77% (range 37.5–100%) of abnormal metaphase cells and a mean of 42% (range 1.8–73.3%) of abnormal BM interphase nuclei. The 35% difference in frequency seen between metaphase and interphase BM cells is attributed to the proliferative advantage of metaphase cells with a complex karyotype. The frequency of the abnormal MDS-marked clone in BM and PB cells was concordant in 13 of 20 pts (65%). Hematological response of these pts was PR=1, hematological improvement=3, stable disease=2, too early for evaluation=6, and 1 patient had no hematological response after 10 months of AZA C treatment. The remaining 7 pts had a mean of 59% (range 48–68%) of abnormal BM metaphase cells and a mean of 21% (range 7–41%) of abnormal PB interphase nuclei. The MDS abnormal clone was detectable in more than two-fold higher frequency in BM compared to PB prior to treatment in 35% of pts. Preliminary sequential studies in discordant pts revealed a transition between the BM and PB cells to concordant frequencies within 4–5 months after AzaC-based therapy. These early observations suggest that monitoring AzaC-based therapy can be achieved using peripheral blood cells in 80% of pts. Remarkably, one pt with monosomy 7, a notoriously poor IPSS indicator, demonstrated a hematological improvement, full cytogenetic and 97% FISH remission, both in the BM and PB after 4 months of AzaC-based therapy. To examine AzaC’s response on cell lineage involvement in MDS, purified BM and PB cells were subjected to FISH analysis before and during treatment from patients who were cytogenetically abnormal at baseline. Similar frequencies of genomic imbalances were seen in purified BM and PB derived CD34+ (97% vs. 96%) and CD15+ cells (94% vs. 98%), indicating that these cell populations had a similar response to AZAC therapy, as monitored in PB or BM, during the initial treatment period (4–5 months). Purified BM and PB-derived T-lymphocytes (CD3+/4+/8+) had normal disomic patterns before and during AzaC-based therapy, indicating that T-cells were not involved in the MDS clone in these pts. In contrast, BM and PB derived B-cells (CD19+) had slightly discordant results, showing a mean of 83% vs. 56% respectively of MDS-marked clone, indicating a trend towards greater response in PB- derived B-cells when compared to BM-derived CD19+ cells. In summary, our results demonstrates that although the MDS abnormal clone may be detected in both the BM and PB at the start of therapy, due to the proliferative advantage of the abnormal clone the optimal tissue should be bone marrow. This is the first study demonstrating that tracking the MDS-marked clone during the AZA-C therapy is feasible in peripheral blood cells.
Objective: To evaluate the clinical manifestations, type and persistence of antiphospholipid (aPL) antibodies, and outcomes of patients evaluated in a hematology practice clinic for the diagnosis of antiphospholipid syndrome (APLS). Methods: We systematically reviewed the medical records of all patients referred to our Hemostasis and Thrombosis Practice Clinic for evaluation of APLS from January 1995 to July 2005. APLS was defined by the Sapporo criteria. 180 patients were identified who either had APLS as defined by the Sapporo criteria or had documented aPL antibodies on 2 separate occasions at least 6 weeks apart without any clinical manifestation for APLS. Data collected included demographics, clinical manifestations, type of aPL antibodies, presence or absence of lupus anticoagulant (LAC), persistence or fluctuation of the aPL antibodies, presence of other thrombophilias, antithrombotic therapy, and outcomes. Results: Of the 180 patients, 141 were females and 39 were males. The age range was 21 to 89 yr. 119 patients (66%) fulfilled the clinical criteria for APLS and 61(34%) did not. Among the 119 patients with APLS, the clinical manifestations included arterial thromboembolism (ATE) in 53 (46 idiopathic, 7 secondary), venous thromboembolism (VTE) in 44 (31 idiopathic, 13 secondary), both ATE and VTE in 7 and pregnancy losses (PL) in 30. Among the 30 patients with PL, 19 had recurrent PL before the 10th week of gestation and 11 had PL after the 10th week of gestation. 5 patients with PL also had ATE and/or VTE. 94 patients had anticardiolipin (aCL) antibody (medium titer IgG isotype), 57 had aCL antibody (high titer IgG), 29 had antiphosphatidylserine (aPS) antibody (medium titer IgG), 27 had aPS antibody (high titer IgG), 11 had anti-β2 glycoprotein I (anti-β2GPI) antibody (medium titer), 13 had anti-β2GPI antibody (high titer), 47 had aCL antibody (medium titer IgM), 15 had aCL antibody (high titer IgM), 74 had aPS antibody (medium titer IgM), 43 had aPS antibody (high titer IgM), 20 had anti-β2GPI medium titer IgM, 12 had anti-β2GPI high titer IgM, 10 had anti-β2GPI medium titer IgA isotype, 6 had anti-β2GPI high titer IgA isotype and 36 had LAC. 115 patients had persistent aPLA and 65 had fluctuating aPLA. 33 patients had other thrombophilias: factor V Leiden (n=5), prothrombin gene mutation (G20210A) (n=6), protein S deficiency (n=4), increased homocysteine level (>12 mcmol/L) (n=9), hyperfibrinogenemia (n=3), elevated factor VIII (n=4) and factor XI (n=1) and plasminogen deficiency (n=1). Antithrombotic therapy included warfarin in 71 patients, aspirin in 85, clopidogrel in 3, and LMWH in 36 (2 on chronic therapy and 34 during pregnancy). 21 patients were on no antithrombotic medications. Of the 180 patients, 110 patients had succesful outcomes defined as either absence of recurrent thrombosis or pregnancy losses. 7 patients had recurrent PL while 10 had recurrent thrombosis. 1 patient died. 52 patients were lost to follow-up. Conclusions: The majority of our patients with APLS were women. The most common clinical manifestations were ATE, followed by VTE and PL. The most prevalent aPL antibody was aCL medium titer IgG isotype and the least common was anti-β2GPI high titer IgA. Antithrombotic therapy resulted in successful outcomes in approximately 2/3 of patients.
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