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Routine sequencing of the BCR-ABL kinase domain for the identification of resistance-associated point mutations is now a critical aspect of the management of chronic myeloid leukemia patients showing suboptimal response to imatinib. The recent correspondence between Lee and Santamaria ([1][1]–[3][

Abstract 2257 Poster Board II-234 Introduction: Infertility is common following allogeneic stem cell transplant (allo-SCT). Very little data are available on the use and success of artificial reproductive techniques in patients who have undergone allo-SCT as treatment for cancer. Patients and Methods: We have performed a retrospective survey to assess the uptake of gamete/embryo storage prior to SCT, their use following SCT and the success rate of these techniques. Two hundred patients (95 female) who received allo-SCT between 1979 and 2007 responded to a questionnaire at a median of 12 years (range 2-30 years) post transplant. The median age at SCT was 35 years (range 17-61 years). Results: Ninety-four men (90%) recalled being counselled about post transplant infertility. Seventy-two recalled being offered sperm storage and 36 men stored sperm prior to allo-SCT. Of the 68 men who did not store sperm, 43 did not wish to have children after SCT; 37/43 had completed their families or did not want children, 4/43 considered themselves too old and 2/43 were homosexual. Of the remainder the reasons were various; no semen storage available in country of residence (n=1), pre-pubertal at first allo-SCT (n=1), previous chemotherapy had induced infertility (n=4), sperm of insufficient quality to permit storage (n=7), insufficient time pre-transplant to arrange storage (n=5), unknown (n=8). Of the 36 men who stored sperm, 21 attempted pregnancy post SCT. 14 men fathered a total of 24 children (17 successful pregnancies including 7 twin pregnancies). In these men intracytoplasmic sperm injection (ICSI) was used in 12 pregnancies and inter-uterine insemination was used in 2 pregnancies. The method was not specified in 3 pregnancies. The median number of attempts for a successful pregnancy was 1 (range 1-10). The median number of attempts for men who were unsuccessful was 2 (1-4). In addition, three men fathered children with donated sperm and one man conceived naturally post transplant. Fifty-nine women (63%) recalled being counselled about post transplant infertility. Of these, 32 were offered storage of gametes/embryos. Ten patients undertook storage; oocytes (n=1), ovarian tissue (n=1), embryos (n=6) or a combination of these (n=2). The women who did not store gametes/embryos most had either completed their families (n=28), were too old (n=17) or there was insufficient time prior to transplant (n=13). In 6 cases, the technology was not available or insufficiently advanced to be successful at the time of SCT. Other given reasons included chemotherapy induced infertility, concerns about medical co-morbidity and worries about contamination of the stored tissue with malignant cells. Three women attempted to become pregnant with stored embryos. One was successful on her second attempt after a spontaneous abortion. One had 3 attempts and although succeeded in becoming pregnant unfortunately miscarried at 12 weeks. The third woman had one failed attempt. Of the other patients, one woman had 2 successful pregnancies with donated eggs and one woman adopted her child. Two women delayed their transplant in order to have children. Conclusions: The uptake of gamete storage was relatively high for male patients. Over half of men who had not already completed their families elected to store sperm. Subsequently two thirds of these men were successful in fathering children with stored sperm. Unfortunately the potential for women to store gametes/embryos is much lower. Less than a fifth of young women who had not completed their families undertook gamete / embryo storage prior to SCT and only one patient proceeded to have a successful pregnancy. Disclosures: No relevant conflicts of interest to declare.

Abstract 3248 Poster Board III-1 The polyspecific organic cation transporter OCT1 (SLC22A1) exhibits broad substrate specificity and facilitates the intracellular uptake of imatinib mesylate (IM). The contribution of OCT1 to the clinical outcome of CML patients treated with IM remains controversial. The aim of this study was to compare OCT1 transcript levels in different normal cell populations, to examine the effect of BCR-ABL1 oncoprotein and IM on OCT1 expression, to investigate the predictive value of OCT1 levels and to determine whether OCT1 SNPs in CML patients correlate with altered IM response and sensitivity. We found that OCT1 mRNA expression was higher in normal PMNs than in normal MNCs (p Disclosures: Novartis: Consultancy, Research Funding.

Abstract 3290 Poster Board III-1 There is a great variability in the degree of molecular responses achieved by chronic myeloid leukemia (CML) patients treated with imatinib. These different levels of molecular response could reflect different degrees of adherence to therapy. We measured the adherence to imatinib therapy in 87 consecutive CML chronic phase patients who had received imatinib 400 mg day as first line therapy for a median of 59.7 months before enrolment (range 25–104) and therefore all them were in complete cytogenetic response. Adherence levels were monitored during a 3-month period using microelectronic monitoring devices (MEMS) and were related to levels of molecular response. MEMS consist of an electronic device fitted in the cap of a normal looking medication bottle that automatically records each time the bottle is opened. MEMS are considered as the ‘gold standard' for measuring adherence. We also measured the imatinib plasma level, the presence of TKD mutations and the following prognostic factors measured at diagnosis: hOCT1 transcripts level, polymorphism 1236C>T in ABCB1, Sokal risk group, hemoglobin, leukocytes , BCR-ABL1 transcript type and BCR1-ABL1 ratio and demographic data. The study protocol was approved by the Research Ethics Committee and patients gave written informed consent to participate. The median adherence rate was 97.6% (range 22.6–103.8%). In 23 (26.4%) patients adherence was ≤90% (median 76%) and in 12 (13.8%) ≤80% (median 63%). We found a strong association between adherence rate (≤90% or >90%) and the 6-year probability of major molecular response (MMR) (28.4% vs 94.5%, p Disclosures: Mahon: Novartis: Consultancy, Research Funding. Apperley:Novartis: Consultancy, Honoraria. Rezvani:Novartis: Consultancy, Honoraria, Research Funding. Marin:Novartis: Consultancy, Research Funding.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is no longer the first treatment option for patients with chronic myelogenous leukemia (CML) but there is a considerable debate about its use as a second line therapy. When used in this indication the second-generation tyrosine kinase inhibitors (2G-TKI) induce complete cytogenetic responses (CCyR) in 40–50% of patients in chronic phase but those without CCyR are unlikely to benefit in long term. It is therefore important to identify groups of patients with a good outcome after transplantation so that this may be offered as second line therapy where appropriate. The outcome of allo-SCT has improved over time so we restricted our analysis to the most recent 8 years to coincide with the introduction of imatinib into clinical practice. 131 patients received myeloablative transplants from January 2000 till December 2007. 67 patients were transplanted in chronic phase (14 in second and 2 in third chronic phase), 46 in accelerated phase and 2 in blastic phase. Forty-nine patients received imatinib at some point prior to transplantation and 30 of these experienced failure of imatinib therapy (as defined by European LeukemiaNet criteria). Conditioning consisted of cyclophosphamide and total body irradiation for 51 recipients of sibling stem cells. In addition in vivo T cell depletion with anti CD52 antibody (Campath 1H) was used for 80 unrelated donor transplants. The median age of the patients was 33.4 (15 to 56) years and the median disease duration at transplant was 13 (2 to 105) months. The probability of overall survival (OS) at 3 and 5 years was 64.8% and 62.6% respectively. We confirmed the prognostic value of the EBMT risk assessment score (Gratwohl) and pretransplant level of the C-reactive protein (CRP) and developed a combined additive pretransplant scoring system based on these predictive factors (EBMT risk assessment score plus 0 for CRP 10 mg/L). This identified 5 prognostic groups (Figure 1) with 3yr probabilities of survival of 92.6% (N= 27, score 0–1), 86.2% (N=29, score 2), 58.2% (N=29, score 3), 47.5% (N=20, score 4) and 30.8% (N=26, score 5 or more). The patients who failed imatinib (N=30) had significantly higher prognostic scores on the above described pre-transplant scoring system compared to the rest of patients transplanted (p=0.001). However, in a multivariate analysis adjusted for prognostic scores, their OS was significantly better (p=0.032). The OS in the best prognostic group is comparable with that of unselected patients treated with imatinib and it is possible that their long-term survival might be better. Allogeneic transplantation is unlikely to be preferred as the first line therapy even in selected patients due to its higher early mortality but our data support its use as second line therapy in patients in chronic phase who failed imatinib and have poor pre-2G-TKI predictive factors for CCyR as determined previously at our institution (namely Sokal risk score at diagnosis, the best cytogenetic response obtained on imatinib, G-CSF requirement during imatinib therapy and time from detection of imatinib failure to onset of 2G-TKI therapy) but achieved good score on the pre-transplant scoring system. It should also be used for those whose disease is more advanced where the 2G-TKI do not offer durable remissions. Figure 1 Figure 1.

Autologous stem cell transplantation (ASCT) remains the standard consolidation therapy for patients with multiple myeloma (MM) and chemosensitive relapsed lymphoma (r-Ly). Peripheral blood as a source of stem cells (PBSC) has largely replaced marrow and has the advantage of improved engraftment rates. PBSC are routinely collected following administration of chemotherapy in combination with GCSF. However, the resultant pancytopenia poses a significant risk to patients and additional chemotherapy prior to ASCT may lead to increased end organ damage potentially precluding future therapies (including ASCT). Novel agents can achieve PBSC mobilisation without the use of cytotoxics. In the advent of such drugs, we reviewed the efficacy of, and complications experienced by patients during PBSC mobilisation. We also analysed the cost implications of adverse events. Of 151 consecutive attempts, 13.2% of patients failed to reach our criteria in order to attempt pheresis (1 × 104 CD34 cells/ml). Of those achieving target and undergoing pheresis, 6% did not achieve an adequate cell dose for future ASCT (2 × 106CD34+cells/kg) giving an overall failure rate of 19.2%. Furthermore 17.9% failed to harvest our ideal of 4 × 106/kg (permitting >1 ASCT procedure). Factors contributing to failure in achieving target CD34+ve PB count on univariate analysis were; >2 lines of previous chemotherapy and occurrence of neutropenic sepsis (NS (p=0.002, and 0.005 respectively). These factors remained significant on multivariate analysis (RR: 4.4 and 6.2). These same factors also affected CD34+ cell yield on both univariate and multivariate analysis (RR: 3.3 and 4.6). No differences were seen between MM and r-Ly. Overall, the complication rate was 34.4%, with 24.1% of patients suffering NS requiring admission. The mortality rate was 1.3% (NS and intra-cranial bleed). Of those developing NS, only 52% eventually harvested sufficient cells, but with a median delay of 3 days. The median cost of PBSC collection was $17,381.46 ($1,978.97–$39,355.73). NS significantly increased the cost of mobilisation at a median cost of $25,532.95 vs $16,4921) (p= Conclusion: Our results suggest that patients who are potential candidates for ASCT should be harvested as soon as they achieve remission to prevent failure following additional therapy upon relapse. One fifth of patients will fail. The risks associated with current mobilisation protocols are substantial, and also impact greatly on cost, particularly relevant in the current climate of economic probity. Therefore these data suggests that transplant centres should consider the use of non-myelosuppressive agents either in place of, or as a dose reduction strategy for autologous stem cell procurement.

Second generation tyrosine kinase inhibitors (2G-TKI) have displaced allogeneic stem cell transplant as the preferred therapy for patients with CML in chronic phase (CP) who fail imatinib. However a significant proportion of patients still fail to respond to 2G-TKI and may benefit from alternative therapy (including stem cell transplant). We have performed univariate and multivariate analyses in our cohort of 80 patients treated with dasatinib (n=67) or nilotinib (n=13) while still in first CP after imatinib failure in order to identify those patients who will benefit most with these therapies. The median age was 50 years and 46% were male; 72 patients were resistant to imatinib (2 primary haematological resistance, 40 primary cytogenetic, 32 secondary cytogenetic and 25 developed secondary hematologic resistance) and 8 were intolerant. 20 had developed kinase domain mutations while on imatinib therapy. 31 and 29 patients received maximal doses of imatinib 600 and 800 mg per day respectively. The median follow up was 28.3 months (range 6–42). The 3-year cumulative incidence of CCyR was 52.6%. The multivariate analysis identified four pre-2G-TKI independent predictive factors for CCyR, namely low Sokal risk score at diagnosis, the best cytogenetic response obtained on imatinib, G-CSF requirement during imatinib therapy and time from detection of imatinib failure (as defined by European LeukemiaNet criteria) to onset of second 2G-TKI therapy. Using these factors we devised a scoring system that could be used to predict the probability of achieving CCyR on 2G-TKI therapy. The score was calculated by allocating one point when any one of the following four features was present: (1) intermediate or high Sokal risk group, (2) need of G-CSF support during imatinib therapy, (3) institution of 2G-TKI more than 18 months after imatinib failure, and (4) failure to achieve a cytogenetic response on imatinib (≥95% Ph-pos). The 3-year cumulative incidence of CCyR for patients with 0–1 points was 95.6%, with 2 points 50% and with 3–4 points 18.7% (p For the 80 patients the probability of 3-year survival was 89.6%. We performed a 3-month landmark analysis to study the relationship between molecular response and subsequent outcome. The 44 patients with a BCR-ABL1/ABL ratio less than 15% at 3 months had a 3-year overall survival of 100% while the 36 patients with a ratio >15% had a survival of 77.4% (p=0.003, Figure 2). We performed a multivariate analysis including all relevant variables defined at the start of 2G-TKI and the 3-month transcript level. The 3-month transcript level was the only independent predictor for survival. Similarly we performed a 6-month landmark analysis where we explored the relationship between cytogenetic response and outcome. Patients who had achieved a McyR (n=38) or a CCyR (n=32) had a significantly better survival than those with lower levels of cytogenetic response (100% vs. 79.2% (p=0.006) and 100% vs 82.6 (p=0.02)) respectively. We also performed a multivariate analysis including the variables defined at the initiation of therapy, the 3-month transcript levels and the cytogenetic response at 6 months. Interestingly the 3-month molecular response was the only independent variable predicting for survival. Similar results were found for progression-free survival (data not shown). We conclude that factors measureable before starting treatment with 2G-TKI may be valuable for predicting response; molecular responses at 3-months and cytogenetic responses at 6 months provide further information about the value of continuing treatment with 2G-TKI. Figure 1 Figure 1. Figure 2 Figure 2.

We analysed outcome for 211 CML patients treated with imatinib in chronic phase (CP) (99 newly diagnosed and 112 late chronic phase) who were screened for BCR-ABL kinase domain (KD) mutations using direct sequencing regardless of the response status. When a mutation was found all available previous cDNA samples were analysed by pyrosequencing to establish the date of its first occurrence and subsequent kinetics. The median age of patients was 47.4 years. The Sokal risk score was ‘low’ in 57 patients, ‘intermediate’ in 82 and ‘high’ in 72. The median follow up from starting imatinib was 45 months (rage 6 to 89 months). A mutation was detected in 34 of the 211 patients (16%) at a median time of 27 months from starting imatinib. Twenty-two different mutations were identified, the most frequent being M244V (n=6) and F359V (n=3). When studied serially by pyrosequencing the size of the mutant subclone never exceeded 50% of total BCR-ABL transcripts in 8 patients, while in 17 patients it exceeded 90% on at least one occasion. 48 patients discontinued imatinib while still in CP and received either dasatinib, nilotinib or an allograft. The overall progression-free survival (absence of advanced phase) at 5 years was 73%. Major (MCyR) and complete (CCyR) cytogenetic responses were achieved by 153 and 123 patients respectively; 56 patients achieved major molecular response. 24% of the patient with up front cytogenetic resistance had a mutation while 40% of the patients with acquired cytogenetic resistance develop a mutation. In an-intention-to-treat analysis, patients harboring a mutant clone had a poorer PFS at 4 years (78% versus 57%, p=0.0014). The various mutations had no differential effects based on their known imatinib IC50. By multivariate analysis, factors associated with worse PFS were the presence of a KD mutation and failure to achieve CCyR (relative risks for PFS 2.6 and 8.7 respectively, p=0.002). Interestingly, the adverse effect of the presence of a KD mutation was restricted to the patients who achieved a MCyR (PFS 91% versus 62% at 5 years, p = 0.0006); it had no adverse impact on patients who failed to achieve a MCyR (PFS 42% and 49%, p=0.73). Similar results were found when the analysis was repeated according to the achievement of CCyR (data not shown). Surprisingly patients with a continuously low percentage (≤50%) of mutated vs wild type (>50%) clones fared worse than patients in whom the mutated clone became the predominant population (PFS 14% vs 69% respectively, p=0.0005). Comparable results were obtained when the patients were censored at the point of discontinuing imatinib, correcting for the effects of subsequent treatment, ie allografting (data not shown). The fact that the adverse effect of a mutation seems to be restricted to patients who had achieved cytogenetic response, the fact that mutations present at low level seemed to have a remarkable adverse effect and the fact that the in-vitro level of resistance to imatinib of the specific mutation did not affect the PFS could all be explained if the development of a mutation is only a reflection of the genomic instability of the disease that leads to secondary resistance to imatinib and eventually to transformation. Thus genomic instability may be less important in explaining primary resistance to imatinib and eventual transformation in patients with up-front resistance.

Imatinib is remarkably effective in treating patients with newly diagnosed chronic myeloid leukemia (CML) in chronic phase (CP) but somewhat less effective in treating patients who have previously received interferon-alfa (IFN), most of whom can be classified as being in ‘late CP’. For such patients who have failed IFN, a proportion achieve complete cytogenetic responses (CCyR) and subsequently major molecular responses (MMolR) on imatinib, but the durability of these responses is not yet established. We analyzed long-term outcomes for 216 consecutive CML-CP patients who started on imatinib after failing IFN at our institution. Their Sokal risk score was ‘low’ in 58 (27%) patients, ‘intermediate’ in 75 (35%) and ‘high’ in 83 (38%). At the time of starting imatinib 73 (34%) patients were IFN intolerant, 40 (18%) were hematologically resistant to IFN and 103 (48%) were cytogenetically resistant; of this last group 58 (27%) had primary resistance and 45 (21%) secondary resistance. The median interval between diagnosis and start of imatinib was 38 months (range 6 to 217). Ninety-two patients (42.6%; 95CI, 36.0–49.5%) achieved CCyR during follow-up; the estimated cumulative incidence of CCyR at 5 years was 46.8% (95CI 40.0–53.7%). Forty-five patients (20.8%; 95CI, 15.6–26.9%) achieved a MMolR; the estimated cumulative incidence of MMolR at 5 years was 23.7% (95CI 16.6–32.8%). The independent factors predicting achievement of MMolR were prior response to interferon and favorable Sokal category: the relative risks (RR) for achievement of a MMolR response were 3.34 for patients with secondary cytogenetic resistance to IFN (p< 0.0001) and 0.5 and 0.2 for the Sokal intermediate and high risk groups respectively (p=0.012). For the 45 patients who achieved a MMolR the median follow-up was 68 months (range, 32–85 months); 24 (53%) patients achieved a 4-log reduction in the BCR-ABL transcript level, and in 10 (22%) cases the transcripts became undetectable. By intention-to-treat analysis the estimated progression-free survival (PFS, defined as loss of complete hematologic response or progression to advanced phase) for this group at 72 months was 100%. At latest follow-up 7 patients (16%) had lost their MMolR but only 2 (4%) of these had lost their CCyR. When comparing those 45 patients with 76 CML-CP patients who received front-line imatinib and achieved a MMolR (out of 207 patients at our institution), we found no differences in terms of cumulative loss of CCyR (p=0.60) or of MMolR (p= 0.94), suggesting a comparable durability of the responses in these two patient groups. In conclusion, although patients who receive imatinib in late CP generally fare worse than patients starting imatinib soon after diagnosis, patients in the two groups who achieve a MMolR have equivalent outcomes.

Pleural effusions (PE) are a relatively common side effect of dasatinib (an oral multi-targeted kinase inhibitor) and have been reported in 20–30% of CML patients. The underlying mechanism is unclear but may be related to inhibition of the PDGFR gene. We report a series of 43 patients, who received dasatinib while in first chronic phase, after imatinib resistance (n=31) or intolerance (n=12). Twenty two patients had previously received interferon. Patients were treated with dasatinib 70 mg twice daily. The median follow-up was 149 days (range 21–730). Of the 43 patients, 14 developed PE at a median of 150 days (range 21–698) after starting dasatinib. The dasatinib was interrupted in all cases and not resumed until the PE had resolved completely, which occurred in all cases. Diuretics were administered in some cases. The median duration of discontinuation was 14 days (range 7–65). In 8 of the 14 patients the drug was re-started at reduced dose following the first episode of PE. PE recurred in 4 patients (3 patients had two and 1 patient 5 episodes). The dose was further reduced in these cases and eventually abandoned in 2 patients. We performed univariate and multivariate analysis to identify prognostic factors for the development of PE. Significant variables were: prior skin rash on imatinib therapy, [21% non PE vs 57% PE (p=0.035)], skin rash on dasatinib [(before the development of PE vs any time during the follow up) 4% non PE vs 47% PE (p=0.002)], and previous history of autoimmune disease, [non PE 7% vs 50% PE (p=0.006)]. The documented autoimmune diseases were: hyperthyroidism (n=1), hypothyroidism (n=3), systemic lupus erythematosus (n=1), Sweets syndrome (n=1) and auto-immune hepatitis (n=1). We did not find correlation between the previous history of autoimmune disorders and having been treated with interferon. The dose of dasatinib prior to the onset of PE was compared with the dose in patients who did not develop PE at 6 months; patients still receiving 70 mg twice a day were more likely to develop PE than those for whom the dose had been reduced, with a relative risk (RR) for the development of PE of 4.7 (95CI 1.2–18.4, p=0.02). The only variables that were predictive in the multivariate analysis were a history of autoimmune disease and the dose of dasatinib [RR 13 (95CI 1.6–103) and RR 7.4 (95CI 1.2–44.3) respectively]. Interestingly the presence of generalised fluid retention was not found to be significant (p=0.54) nor the previous therapy with interferon (p=1.0). Our results are in agreement with recent reports which suggest that dasatinib-induced PE does not correlate with generalised fluid retention and may be mediated by an immune mechanism. Moreover dasatinib-induced PE are usually exudates and are believed to respond to steroids. In summary in this series PE occurred more frequently in patients with a previous history of autoimmunity and imatinib-related dermatological side effects; both observations support the notion that PE secondary to dasatinib therapy may have an auto-immune pathogenesis.

Imatinib is remarkably effective as single-agent treatment of newly diagnosed chronic myeloid leukemia (CML) in chronic phase (CP) but most of the published data are based on a single multicenter study (acronym IRIS) carried out under the supervision of the manufacturer. We report here an independent validation of the IRIS results. At our institution from June 2000 and August 2006 207 consecutive newly diagnosed CML-CP patients were started on imatinib 400mg within 6 months from diagnosis, of whom 17 were included in the IRIS study. Their median age was 46.3 years. Their Sokal risk score was ‘low’ in 59 (28.5%) patients, ‘intermediate’ in 73 (35.3%) and ‘high’ in 54 (26.1%). The median follow-up was 33 months (range 12–85). By intention to treat analysis the estimated overall survival (OS) and progression-free survival (PFS, defined by progression to accelerated or blastic phase) at 48 months were respectively 90.5% (95CI, 82.1–95.2%) and 90.2% (95CI, 84.5–94.0%) (data censored at the time of allografting). 34 (16%) patients discontinued imatinib at some point while still in CP and received a second generation TK inhibitor or an allograft. The cumulative incidence of complete cytogenetic response (CCyR) and major molecular response (MMolR) were 59.1% (95CI, 52.0–65.9%) and 11.9% (95CI, 8.0–17.4%) by 12 months, and 86.7% (95CI, 79.4–91.7%) and 58.4% (95CI, 47.6–68.5%) by 48 months respectively (data censored at the time of changing to another treatment). The cumulative incidence of loss of CCyR at 48 months was 17.5% (95CI, 9.9–29.1%). The imatinib dosage was increased in 70 (33.8%) patients to 600 mg/day and in 19 (11.6%) patients the dose was further increased to 800 mg/day during follow-up; in 5(2%) patients the dose of imatinib was directly escalated to 800mg/day. The only pre-therapy factor found to be predictive for PFS was the Sokal (and also Euro) score. A 12-month landmark analysis showed that patients who achieved a CCyR had significantly better OS and PFS at 48 months (OS - 95.2% vs 87.1%, p=0.026; PFS - 97.8% vs 87.3%, p=0.007 respectively), while the achievement of a MMolR by 18 months had no significant impact on OS or PFS. Nevertheless patients in CCyR who achieved a MMolR at any time during the follow-up were significantly less likely to lose their CCyR than CCyR patients who maintained higher BCR-ABL transcript levels (RR=0.08, p=0.0003). No plateau was seen either in the OS or in the PFS curves; however when analysis was restricted to patients who were on CCyR at 12 months the OS and PFS curves did achieve plateaux. Five patients (2.4%) achieved a complete molecular response (CMolR) during the follow up. When restricting the analysis to the 31 patients who had at least 4 years of follow-up only 2 patients (6.5%) had a sustained CMolR while on imatinib 400mg/day. In summary our findings confirm the results reported in the recent IRIS update, notably the high rates of CCyR and high rates OS and PFS at 48 months. Moreover, our results suggest that achieving a CCyR before 12 months is strongly associated with better OS, which accords with the recommendations published recently by the European LeukemiaNet. We showed moreover that once a patient has achieved CCyR, a further reduction in the transcript levels to MMolR is still clinically relevant, although the time-frame is not yet clear.

Neoplastic cells bearing fusion genes that express an activated tyrosine kinase may set the scene for accumulation of genetic lesions by dysregulating DNA damage repair mechanisms and causing genetic instability. The observation that the BCR-ABL fusion gene alters pre-mRNA splicing in a variety of other genes including Ikaros and PYK2 supports this hypothesis. However, the only current evidence for acquired genetic change in the BCR-ABL gene itself is limited to finding mutations in the BCR-ABL kinase domain in patients treated with imatinib mesylate (IM). Here we report the observation that some patients with CML have abnormally small BCR-ABL transcripts both before and during treatment. Patients with sub-optimal response to IM are screened for mutations specifically within the BCR-ABL kinase domain by performing nested PCR, thereby excluding amplification of the non-translocated ABL allele. In the first round PCR amplification is performed across the fusion and the amplicons generated are subjected to a second round to yield an expected 863 bp (containing ABL exons 4 through 9 and thus the entire BCR-ABL kinase domain) PCR fragment. Smaller amplicons were observed in 49 (9.9%) of the 494 CML patients investigated. There was marked variation in the mRNA species when the abnormally small amplicons were subjected to direct sequencing; we found exon skipping, intra-exon splicing and insertion of intronic sequences. Similarly, in some cases the open reading frame was maintained whilst in others there were frame shifts leading to premature stop codons. The commonest finding, (22 of the 49 patients) was skipping of ABL exon 7 from codons 362 to 424, which includes the activation loop of the kinase domain. The smaller amplicons persisted even after the first round products had been diluted to 1:160. We also noted that the normal 863 bp fragment was present in some cases but was not detectable in others; its absence could reflect preferential amplification of the smaller transcripts. In a number of cases the same abnormally short amplicons were identified in the same patient studied serially on three or more separate occasions. We subsequently performed a second round of nested PCR with primers designed to amplify across the BCR-ABL junction such that the product included sequences from BCR 13 to ABL exon 9. With these new primers the detection frequency of abnormally small transcripts was increased. Furthermore, we observed the smaller transcripts in all of the 12 patients tested prior to beginning IM therapy. We then sought to determine if the normal ABL allele was involved; in order to avoid amplifying the BCR-ABL kinase domain, we performed a single round of PCR and restricted the analysis to patients in complete cytogenetic remission (CCyR). Only the expected 863 bp amplicon was observed in cDNA samples from 19 CML patients in CCyR whose BCR-ABL/ABL ratios ranged from 0.01 to 0.98. Furthermore, the smaller amplicons were not observed in cDNA samples from 20 normal individuals. We conclude that these abnormalities may result either from aberrant alternative splicing or from spontaneous deletions, or from a combination of both mechanisms. They may be a manifestation of the genetic instability believed to be an integral feature of CML.

Budd-Chiari Syndrome (BCS) is a group of disorders resulting from obstruction to hepatic venous outflow; myeloproliferative disorder (MPD) accounts for 10–40% of cases. A number of BCS cases labelled as ‘idiopathic’ do not fulfil the accepted diagnostic criteria for MPD but have features suggestive of a latent form of MPD based on hyperplastic bone marrow and spontaneous erythroid colony progenitor cell culture studies; these cases may subsequently develop overt MPD. A clonal mutation in JAK2 tyrosine kinase (JAK2V617F) occurs in a high proportion of patients with MPD and is of potential use in the characterization of latent MPD in BCS. We studied 44 patients with BCS (female n=27, mean age 36.1 years, SD 13) presenting between 1985 and 2005. Genomic DNA obtained from archived bone marrow films was screened by allele-specific PCR for the JAK2V617F tyrosine kinase mutation. JAK2V617F was detected in 27/44 (61.4%) subjects. Fulminant hepatic failure was the presenting feature in 86.4%. 3/38 subjects had previously diagnosed Polycythemia Vera and all of these were JAK2V617F positive. Analysis for hereditary thrombophilia in 32/44 cases showed Factor V Leiden (FVL) heterozygosity (n=2) and Protein C deficiency (n=1); JAK2V617F was detected in one of the FVL cases. Screening for antiphospholipid antibodies and paroxysmal nocturnal hemoglobinuria in 31 and 30 out of 44 cases respectively proved negative. 22/44 cases had splenomegaly of which 72.2% had JAK2V617F detected. Mean hemoglobin concentration was higher in patients with JAK2V617F (12.4 g/dL, SD 2.8) compared to the wild-type allele (10.2 g/dL, SD 2.1)(p=0.005). Mean neutrophil count was similar (p=0.26) in both groups (JAK2V617F 7.8 x109/L, SD 5.5; wild-type 6.1 x109/L, SD 4.5). Mean platelet count was higher (p=0.01) in subjects with JAK2V617F (289 x109/L, SD 192) compared to wild-type (180 x109/L, SD 158). The bone marrow (BM) was hyperplastic in all lineages in 19/44 subjects of which 15 (78.9%) contained the JAK2V617F mutation. Importantly, in 25/44 subjects without hyperplastic BM, 48% had JAK2V617F detected. Clonal cytogenetic abnormalities occurred in 6/44 subjects all of which carried JAK2V617F. In 35 evaluable subjects, 10/35 (28.6%) showed endogenous erythroid colony (EEC) formation (8/10 JAK2V617F positive), and the remainder (25/35) showed no EEC formation (14/25 JAK2V617F positive). Treatment of BCS included anticoagulation (n=44), porto-systemic shunt insertion (n=26) and orthotopic liver transplantation (OLT) (n=15). Overall survival was 81.8% (median 32.5 months, range 2 days-240 months). In the 8 non-survivors, JAK2V617F was detected in 50%. 15/44 subjects underwent OLT (11/15 JAK2V617F positive) of which 14/15 are alive at a median of 36 months post transplant (1–240 months). 14/44 subjects were treated with cytoreductive treatment following diagnosis of BCS and in 92.9% of these JAK2V617F was detected. 5/15 subjects went on to receive cytoreductive treatment following OLT of which all were JAK2V617F positive. We have found a high prevalence of JAK2V617F in patients with BCS. Latent MPD was missed in 12/25 subjects on BM morphology and 14/25 subjects on BM progenitor culture. We suggest that JAK2V617F analysis should be performed in all new cases of BCS. Given their age, these patients are possible candidates for JAK2 targeted therapy.

VEGF is a potent angiogenic regulator implicated in increased angiogenesis, which is characteristic of CML. VEGF interacts with its two tyrosine kinase (TK) receptors, Flt-1 (VEGFR-1) and KDR (Flk-1/VEGFR-2). CML therapy with imatinib mesylate, which targets BCR-ABL TK activity, induces haematological remission in 95% and complete cytogenetic remission (CCR)/ major response (MR) in 60% of cases. We describe the serial evaluation of patient bone marrow (BM) trephines and the analysis of the impact of imatinib on VEGF in all stages of CML during therapy. Consecutive CML (n=38) patients (25 males, 13 females, median age of 56 years (19 to 81) were sequentially analysed during the course of imatinib therapy. Chronic phase (CP) n=24, accelerated phase (AP) n=11 and blast crisis (BC) n=3 prior to imatinib therapy. BM examination was performed at diagnosis, at 1 month, then 3 monthly following imatinib therapy. Immunohistochemical analysis determined expression of VEGF, VEGFR1/Flt-1 or VEGFR2/KDR. Results were correlated with cytogenetic response. In normal BM cellular VEGF and its receptors were expressed in megakaryocytes and macrophages, but rarely in myeloid cells. VEGF was not expressed in erythroblasts, lymphocytes or plasma cells. A weak VEGFR1 signal was observed in monocytes/histiocytes and VEGFR2 in histiocytes only. VEGF expression in CML was detected as a diffuse cytoplasmic pattern in myeloid and monocyte precursors, classified as 4+ (very intense staining), 3+ (strong), 2+ (moderate), 1+ (weak), or 0 (completely negative) throughout. The intensity of staining varied and did not correlate with the stage of disease, but VEGF was strongly expressed in the megakaryocytes. Both VEGFR1/Flt-1 and VEGFR2/KDR were expressed in monocytic/myeloid population and in megakaryocytes. The staining intensity varied from borderline detectable to a very strong staining intensity. 14/38 (37%) of patients showed a reduction in VEGF expression, which was particularly marked with respect to VEGFR2 receptor staining. Of these, 10/14 patients (71%) achieved CCR, one MR (major response) and 3 minimal/NR (no response). All patients in this group had a reduction in BM cellularity, median 30% (15–70%). No change in VEGF receptor expression was detected in 10/38 (26%). Only 1 patient achieved CCR, 3 MR, with a minor/NR in the rest (60%), and a median BM cellularity of 30% (10–100%). Increasing VEGF expression was observed in 14/38 patients (37%). Here 6/14 (43%) patients showed NR (2 minor/minimal responses) with CCR in only 4 of the evaluable patients. The median BM cellularity was 25% (10–95%). 3/14 patients went on to develop progressive disease In summary, reduced expression of VEGF and its receptors, particularly KDR, can predict a favourable response to imatinib and correlates with a reduced BM cellularity and cytogenetic response. In patients with an increase or no change in VEGF expression there is a greater incidence of a poor cytogenetic response and a higher tendency to relapse despite a reduction in BM trephine cellularity, but a longer follow-up may be necessary.

Reduced-intensity or “nonmyeloablative” conditioning (RIC) regimens, which include Alemtuzumab, results in stable donor hematopoietic and lymphoid engraftment with minimal graft versus host disease. At our institution 182 patients with myeloid and lymphoid malignancies have undergone RIC transplantation between March 1998 and March 2004. In order to ascertain whether the inclusion of Alemtuzumab affects post transplant immune reconstitution, we have assessed the incidence of clonal gammopathies and organ specific autoantibodies in all patients in whom information was available. We used 44 patients who had conventional nommyeloablative HSCT as our control group. In the RIC HSCT group 40(22%) developed clonal gammopathies. The median age at RIC HSCT was 55 years (range 24–66); the underlying dignosis was MDS(n=15), AML(n=15), NHL(n=8) and CML(n=2). A gammopathy was detected at a median of 19 weeks post RIC HSCT (range 8–44). Bone marrow aspiration and biopsy did not show increased numbers of plasma cells. Chimerism studies (unfractionated bone marrow and CD3+, CD15+, CD19+ and CD56+ peripheral blood fractions) showed that 18(45%) were fully donor, 18(45%) had mixed chimerism and it was not available for 4(10 %). By contrast clonal gammopathies were detected in only 4 of 44 patients who had conventional nonmyeloablative conditioning. There was a significant difference between two groups (P=0.05). 9 of 182 (5%) patients (AML=6, MDS=1, MCL=1) developed immune cytopenias at a median of 149 days(range 24–238 days). Those comprised of ITP(n=4), AIHA(n=3), Evans’ syndrome(n=2), red cell and megakaryocytic aplasia(n=1). Chimerism studies (unfractionated bone marrow and CD3+, CD15+, CD19+ and CD56+ peripheral blood fractions) showed that 6(66%) patients were fully donor, 1(11%) was mixed chimeric and for 2(23%) patients chimerism studies were not available. One patient did not need any treatment; one patient with AIHA had a complete response to prednisolone. 2 patients (1 ITP and 1 Evans’ syndrome) responded to IVIG. The five non-responders to IVIG (2 AIHA, 2 ITP, and 1 red cell aplasia and megakaryocytic aplasia) resonded to treatment with Rituximab 375mg/m2 weekly for 4 weeks with a maximal response at a mean of 30 days from commencing the treatment. 3 of 182(1.6%) patients developed autoimmune thyroid dysfunction. All patients had normal thyroid function tests and no antibodies to thyrotropin receptor before RIC HSCT. None of the patients, who had clonal gammopathy, immune cytopenia and autoimmune thyroid dysfunction, developed PTLD. We hypothesize that the RIC regimens incorporating monoclonal antibody Alemtuzumab causes clonal gammopathies, immune cytopenias and autoimmune thyroid dysfunction due to dysregulated B cell proliferation secondary to prolonged T cell recovery time.

Increasing numbers of allogeneic hematopoietic stem cell transplantation (allo-SCT) are being performed for patients who have failed a previous allogeneic or autologous SCT. We investigated whether the EBMT risk score could predict outcome after a subsequent allo-SCT. We analyzed prognostic factors in 124 consecutive patients who underwent a second transplantation using an allogeneic donor at our institution. Patients with either a first autologous (N = 64) or first allogeneic (N = 60) SCT were included. Age, disease stage, time interval from diagnosis to transplantation, donor type, and donor-recipient sex combination were used to establish a score from 0 to 7 points, from which 3 groups were identified. The 5-year survival probability decreased from 51.7% for risk scores 0-3 (low, n = 25), to 29.3% for risk score 4 (intermediate, n = 42), and only 10.4% for risk scores 5-7 (high, n = 57), P = .001. We propose that the EBMT risk score can identify patients most likely to benefit from a second transplantation.