ALBERT

Description: Background: The Revised International Prognostic Scoring System (R-IPSS) stratifies MDS patients better than the original IPSS scoring system. Although RBC-transfusion dependency (RBC-TD) is associated with poor prognosis, it is not included in the R-IPSS. Another limitation of R-IPSS is that it is designed to assess the prognosis of patients only at the time of diagnosis; it does not provide prognostic guidance during the disease course. We hypothesise that the use of RBC-transfusion dependency status as a time-varying covariate improves R-IPSS. Aim: To assess the impact of RBC-TD as a time-varying covariate in addition to R-IPSS in predicting survival outcome of MDS patients. Materials and Methods: To match the patient selection criteria as in R-IPSS, primary MDS patients, AML (blast 20-30%) and CMML (WBC≤12x109/L) not treated with disease modifying agents or stem cell transplantation were included. RBC-TD was defined as RBC transfusion of at least 1 unit/8 weeks for at least 4 months (Malcovati et al; JCO 2007). For the statistical analysis of overall survival (OS) measured in months since diagnosis, the Akaike Information Criterion (AIC) was used to assess the goodness-of-fit of a model. Landmark analyses at 6, 12 and 24 months after the diagnosis were also conducted; individuals who experienced the event (i.e. death) before the landmark time point were excluded. The remaining patients were then classified into two groups – RBC-TD noted at or before the landmark time point and transfusion independent at the landmark time point. Results: In our study, 295 patients met the inclusion criteria for analysis, their median age was 75 years (21-97 years) and 66% patients were male. The majority of patients were RCMD, RAEB1 and RAEB2. R-IPSS improved the risk stratification of MDS patients, predominantly for the IPSS-intermediate group (Table I). The median OS in R-IPSS Very Low, Low, Intermediate, High and Very High risk group was 87, 62, 28, 13 and 12 months respectively (p

Description: Key Points Imatinib achieves deep and durable remissions in patients with myeloid neoplasms bearing PDGFRB. Allogeneic stem cell transplantation is no longer indicated for patients with chronic myeloproliferative neoplasm bearing PDGFRB who respond to imatinib.

Description: Aim: To develop and evaluate a DNA-based method for monitoring of MRD in CML Background: At present, monitoring of MRD in CML uses RNA and reverse transcription (RT-PCR). This leads to a number of disadvantages, including the potential for RNA degradation, requirement for reverse transcription, difficulty in standardisation and only an indirect relationship between assay result and cell number. Methods: A highly multiplexed, short-range PCR using six BCR primers and a pool of 282 ABL primers was used to amplify across the BCR-ABL translocation breakpoint and was followed by 2–3 rounds of “bottleneck PCR”, a technique recently-developed in our laboratory, which adjusts primer concentration so as to minimise non-specific amplification and facilitate highly multiplexed PCRs. The breakpoint was isolated and sequenced, patient-specific primers were synthesised, and MRD was quantified using 10 μg of DNA and a 3 round nested quantitative PCR incorporating a Taqman probe in the third round. Samples from patients on treatment were divided and assayed both by this technique and by RT-PCR. 10 μg of DNA from a normal individual was used in each assay as a control for non-specificity. Results: The BCR-ABL breakpoint was successfully isolated and sequenced in 28 of the 29 patients studied and MRD was assayed in 38 samples from 24 patients. Follow-up samples from 4 patients were unavailable MRD was detected and measured by both methods in 22 samples, detected and measured only by DNA-PCR in 10 samples and not detected by either method in 6 samples. The limit of detection of RT-PCR was in accord with previous results which indicated it to be a mean decrease of 4.5-logs below baseline. The mean limit of detection of DNA-PCR was an MRD of 7.2 x 10−7. Assay precision was determined by performing independent replicate assays on different days and by different individuals on the 32 samples with MRD detectable by DNA-PCR. The median SD of a single assay was 0.15 log units with the range being 0.00 – 0.64 log units. Figure Figure Conclusions Monitoring of MRD by DNA-PCR is feasible in the great majority of patients with CML. In terms of possible clinical benefits, DNA-PCR is more sensitive than RT-PCR, and provides a direct measure of leukemic cell number in the individual patient. Use of DNA rather than RNA simplifies specimen collection and transport. In terms of laboratory benefits, DNA-PCR obviates reverse transcription and inter-laboratory standardisation. The principal disadvantage of the method is the initial cost, although this may be able to be amortised over several assays if monitoring is ongoing.

Description: Alfa Interferon, commonly used in chronic phase chronic myeloid leukemia (CML-CP) in the pre-imatinib era, was able to induce a cytogenetic response in a minority of patients (pts). Pegylated interferon (Peg-IFN) is better tolerated than IFN, and increases molecular response rates when used in combination with imatinib (IM) compared with IM monotherapy (Preudhomme NEJM 2010). The phase II Pinnacle (ALLG CML 11) study evaluated the tolerability and molecular response rate of nilotinib (NIL) with Peg-IFN alfa-2B (PegIntron, MSD) in de novo CML-CP. Pts were screened for cardiac / vascular disease and associated risk factors at baseline (EKG, left ventricular ejection fraction, arterial duplex of carotids and lower limbs, blood HbA1c and lipid profiles). Those with uncontrolled vascular risk factors (diabetes, hypertension, dyslipidemia) or a history of vascular events were excluded. Eligible pts received NIL 300mg BID alone for the first 3 months (mths). PegIntron was then added at 30mcg/week in pts without persistent hematological toxicities, increasing to 50mcg/week as tolerated over the following mth. Combination therapy continued until 24 mths, when pts reverted to TKI monotherapy. Switching to IM 400-600mg QD was allowed for pts with persistent grade II or any grade III/IV toxicity from NIL.. Sixty pts were enrolled from 12 Australian centres. Median age was 48.5 years (range 19-72); 45% were female. Sokal risk was low in 43% and high in 18%. Median follow up (FU) was 28 mths (16-51). Data is presented on an intention to treat basis. Figure 1a shows BCR-ABL1 transcript levels over time. The co-primary end points are MMR (BCR-ABL1 ≤0.1% IS) AT 12 mths and MR4.5 (BCR-ABL1 ≤0.0032% IS) at 24 mths. At 12 mths, MMR and MR4.5 rates were 76.7% (95% CI 63.4-87%). and 43.4% (95% CI 30.1-57.3%), respectively. In 40 evaluable pts at 24 mths, MR4.5 was 50% (95% CI 29.9-70.1%). The median time to MMR and MR4.5 was 5.8 mths and 18 mths respectively for pts achieving these responses (Figs 1B & C). Six pts (10%) had BCR-ABL1 ≥10% at 3 mths - 2 of whom had multiple dose interruptions due to toxicity; 3/6 have since achieved MMR, 1 has BCR-ABL 90% of their assigned dose, 13 (22%) received between 50-90% and 25 pts (41%) received

Description: Background: Iron overload in patients receiving red blood cell (RBC) transfusions for treatment of anemia and in patients with non-transfusion-dependent thalassemia (NTDT) can lead to impaired organ function and is associated with significant morbidity and mortality. Regular iron load monitoring is essential and iron chelation therapy (ICT) should be promptly initiated when appropriate. Serum ferritin (SF) 〉1000 ng/mL is commonly used as an indicator of adverse clinical outcome resulting from iron overload. However, this is an indirect measure of tissue iron, whereas MRI allows accurate, reproducible assessment of cardiac and hepatic iron load, and its use may lead to improved patient care. The TIMES study used MRI to assess prevalence and severity of cardiac and hepatic siderosis in a large population of Australian patients with transfusion-dependent anemia or NTDT. Methods: TIMES was an epidemiological study to assess cardiac and hepatic iron load in Australian, adult patients with thalassemia major (TM), NTDT (β thalassemia intermedia, β thalassemia/Hb E, Hb H disease), myelodysplastic syndromes (MDS) or other chronic anemias. Patients with NTDT had SF〉300 ng/mL; others had a lifetime history of ≥20 units RBC transfusions and SF〉500 ng/mL. Past medical history was collected (including anemia, RBC transfusion, ICT and hematologic data). Prospective MRI (FerriScan) was used to determine R2 liver iron concentration (LIC; siderosis 〉5 mg Fe/g dw for NTDT, 〉7 mg Fe/g dw for others) and myocardial T2* (siderosis

Description: Real-time quantitative reverse transcriptase PCR for BCR-ABL (RQ-PCR) is used to monitor treatment response in chronic myeloid leukaemia (CML). BCR-ABL levels continue to decline over several years of imatinib treatment and increasing numbers of patients have BCR-ABL levels at or below the limit of detection. The sensitivity of current RQ-PCR assays limits our ability to identify patients who have a continuing decline in BCR-ABL levels; or to assess the effects of novel therapies to improve outcome in patients who have a good response to ABL kinase inhibitors, but harbour minimal residual disease. Improvements in therapy for these patients will be hard to assess without more sensitive monitoring of BCR-ABL. BCR-ABL levels are usually reported relative to a control gene. The control gene value gives an indication of the sensitivity achieved within each RNA sample; this varies with sample quality and efficiency of reverse transcription (RT). Control gene copy number below a defined value indicates that the analysis may be unreliable. We investigated the use of a random pentadecamer (RP; 15mer) primer in the RT reaction to improve the sensitivity of RQ-PCR. The RP primer is reported to be more efficient than the random hexamer (RH; 6mer) which is commonly used for RQ-PCR. BCR-ABL and BCR control gene transcripts were measured in 30 peripheral blood samples. After Trizol extraction 2μg RNA and 400U Superscript II reverse transcriptase were added to two RT reactions using RP or RH at a final concentration of 25μM. Each RT and quantitative PCR was performed 2–3 times and results compared using the Wilcoxon signed rank test or paired t-test. A more efficient RT is indicated by higher transcript levels. The table shows that BCR-ABL and BCR control gene values were significantly higher with RP primers. There was a proportionate increase in all transcripts so that the change in BCR-ABL/BCR% was not significant. BCR-ABL and control gene values with pentadecamer or hexamer primers No. of Samples Primer Median 25th Percentile 75th Percentile P value (RP v HP) * missing values due to inclusion of samples with undetectable BCR-ABL BCR transcripts 30 RH 438900 241800 1090000 〈 0.001 RP 1203700 836500 2125000 BCR-ABL transcripts 16* RH 280 110 11340 〈 0.001 RP 390 220 32910 BCR-ABL/BCR ratio 16* RH 0.10% 0.06% 5.6% =0.083 RP 0.08% 0.05% 4.2% We tested samples with undetectable BCR-ABL including 10 from CML patients on imatinib, and 10 from BCR-ABL negative control subjects. Each RNA was tested in two independent RTs and Q-PCRs. If the results were discordant a third RQ-PCR was performed and a consensus result determined. Using RP primers 6/10 patient samples were positive; 2/10 were positive with RH. None of the BCR-ABL negative control samples was positive with either primer. Sensitivity was calculated using the lower limit of detection and the standardised baseline value for untreated CML patients.The median calculated sensitivity increased from 4.5-log with RH to 5.0-log with RP. The use of pentadecamer primer made possible the detection of BCR-ABL in a small number of patients who had undetectable BCR-ABL with random hexamer RQ-PCR. This initial analysis suggests that the sensitivity of BCR-ABL detection may be improved 2–3-fold with a simple modification to RT methodology. This also has the potential to reduce the number of samples deemed unsatisfactory due to low control gene levels.

Description: Background: Clinical scoring systems, such as Sokal risk, continue to have prognostic relevance for patients (pts) treated with tyrosine kinase inhibitors (TKI) and may have utility in combination with emerging biomarkers. The BCR-ABL value at 3 and 6 months (mo) of TKI are the strongest predictors of response. However, recent data demonstrate that the rate of BCR-ABL decline from the pre imatinib level adds significant predictive information (Hanfstein, Leukemia 2014; Branford, Blood 2014). Among poor risk pts with 〉10% at 3 mo in our cohort of first line imatinib, those with a slow rate of decline from their pre imatinib value, assessed by calculating the number of days over which BCR-ABL halved (halving time), predicted significantly poorer outcomes. Notwithstanding the importance of the 3 and 6 mo values, a prognostic biomarker obtained at an earlier timepoint may allow opportunities for therapy optimization. We therefore examined the prognostic significance of the rate of BCR-ABL decline at 1 mo in the context of other predictors of response. Aim: To determine whether baseline factors (age, gender, Sokal risk and imatinib starting dose: 400, 600 or 800 mg) and the BCR-ABL halving time at 1 mo of imatinib have predictive significance. Method: 528 first line imatinib treated pts were evaluated (median 45 mo of imatinib). Molecular assessment was performed pre imatinib and at 1 mo for 470/528. 453 of these 470 pts had a Sokal score available and were included in the analysis of outcome. Results: The median BCR-ABL halving time at 1 mo of imatinib was 17 days, quartiles 11, 29. An initial rapid BCR-ABL decline, indicated by halving times in the lowest quartile of ≤11 days (n=115), was associated with significantly superior rates of MMR by 12 mo, MR4.5 and failure-free survival (FFS) by 4 years compared with longer halving times of 〉11 days (n=338), Table. MMR by 12 mo was assessed since it represents an optimal response and is associated with subsequent deep molecular response. By univariate and multivariate regression analysis only the 1 mo halving time and Sokal risk significantly predicted MMR, MR4.5 and FFS. These factors were independent and there was no difference between the median 1 mo halving times among the Sokal risk groups, P = .36. The high Sokal risk pts had significantly poorer outcomes. To improve response prediction, these pts were divided into 2 groups according to their 1 mo halving time; ≤11 days (n = 28) and 〉11 days (n=90). A 1 mo halving time of ≤11 days was associated with significantly improved outcomes for these pts, Table and Figure. The responses equated to those of pts with low Sokal risk: high risk ≤11 days vs low risk: MMR by 12 mo 57% vs 59%, P = .95; MR4.5 by 4 years 36% vs 40%, P = .82; FFS by 4 years 79% vs 84%, P = .39. The high Sokal risk pts with the rapid initial BCR-ABL decline also had a lower probability of BCR-ABL 〉10% at 3 mo (early molecular response [EMR] failure), which is considered a warning or treatment failure; ≤11 days vs 〉11 days: 14% vs 33%, Table. Table 1 Outcome* by Sokal risk and BCR-ABL halving time at 1 mo of imatinib Factor No. of pts EMR % 3 mo MMR % 12 mo MR4.5 % 48 mo FFS % 48 mo Sokal Low 195 90 59 40 84 Intermediate 140 79 50 35 71 High 118 71 43 26 59 P value 29 days) was associated with a significantly lower cumulative incidence of MMR by 12 mo: low Sokal risk ≤29 days (n = 151) vs 〉29 days (n = 44) 65% vs 39%, P = .002; intermediate Sokal risk ≤29 days (n = 103) vs 〉29 days (n=37) 57% vs 31%, P = .004, Figure. Conclusion: Imatinib treated high Sokal risk pts have a higher rate of treatment failure and poorer molecular response. However, our data suggest their prognosis can be refined by taking into account the kinetics of BCR-ABL decline after only 1 mo of treatment. A rapid initial decline defined a subgroup of high Sokal risk pts with outcomes equivalent to those of low Sokal risk pts. Frequent molecular monitoring in the critical first months of treatment could enhance outcome prediction and limit the indication for a change of treatment. Figure 1 Figure 1. Disclosures Branford: Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding. Yeung:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ross:Novartis: Honoraria, Research Funding; BMS: Honoraria. Seymour:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Hughes:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding.

Description: Introduction:An attempt at tyrosine kinase inhibitor (TKI) withdrawal in deep molecular remission leads either to treatment free remission (TFR) or early molecular relapse (MolR) in chronic myeloid leukaemia (CML) patients. We hypothesise that immune responses promote sustained TFR and immunological markers may predict response following TFR attempt. Methodology: We studied 54 CML patients (from ALLG trials CML 8, median follow-up 66 mo, and CML 10, median follow-up 24 mo) at baseline on TKI (minimum 24 mo MR4.5) and 3 mo and 6 mo following TKI discontinuation. MolR was defined as any single sample on follow-up with BCR-ABL1 〉0.1% or two consecutive BCR-ABL1 positive samples at any value. Effector immune responses of CD56dim natural killer (NK) cells and NK cell receptor repertoire were characterised by flow cytometry and cytotoxic T lymphocyte (CTL) responses to leukaemia-associated-antigens (LAAs) WT1, BMI-1, PR3 and PRAME by interferon-gamma ELISPOT. Immune suppressor regulatory T cells (Treg; CD4+CD25brightCD127-FoxP3+), Granulocytic and Monocytic Myeloid-Derived Suppressor Cells (MDSCs; HLA-DR-Lin-CD11b+CD33+CD66b+CD15+ and HLA-DR-Lin-CD11b+CD33+CD66b-CD14+, respectively), Programmed cell death-1 (PD-1) expression on T cells, NK cells, B cells and Monocytes, and major B cell subsets were characterized by flow cytometry. Results: TFR patients displayed increased CD3-CD56dimCD16bright cytolytic NK cells as a proportion of total lymphocytes at baseline (n=23, 27.1% ± 2.9) vs MolR (n=23, 19.1% ± 2.0, p=0.02). TFR patients displayed a more mature CD56dim CD57+ NK cell phenotype at baseline (74.5% ± 2.2 of total NK cells) vs MolR (66.3% ± 2.7, p=0.04). Extensive characterisation of NK cell receptor repertoire revealed NKG2D activating receptor expression was increased in TFR patients (baseline= 56.8% ± 3.8, 3 mo= 61.4% ± 5.0, 6 mo= 49.9% ± 5.8) vs MolR (baseline= 44.2% ± 3.7, 3 mo= 42.2% ± 5.5, 6 mo= 22.0% ± 8.3, all p=0.02). KIR2DL2/DL3/DS2-positive NK cells were increased in MolR patients at 3 and 6 mo vs TFR. (MolR; 3 mo= 44.8% ± 4.6, 6 mo= 48.8% ± 4.9. TFR; 3 mo= 31.5% ± 4.0 p=0.05, 6 mo= 31.1% ± 2.1, p=0.001). No significant differences were observed in CD56brightCD16-/dim immunoregulatory NK cells, C-type lectin receptor expression (CD94/NKG2A/NKG2C, CD161, CD69), Natural cytotoxicity receptors (NKp30, NKp44, NKp46), CD62L (on T cells and NK cells) and KIR2DL5 expression. No difference in NK Cell-mediated K562 degranulation as a surrogate marker of NK cell function was observed between TFR and MolR patients. Functional CTL immune responses were observed in TFR and MolR patients. BMI-1 CTL responses were increased at baseline in TFR (23%) vs MolR (9%). PR3 CTL responses were not detected in TFR at baseline, 3 mo or 6 mo (0%) vs MolR (baseline= 18%, 3 mo= 50%, 6 mo= 50%). No difference was observed in WT1 or PRAME CTLs. Quantification of immune suppressor cell types revealed decreased Monocytic MDSCs in TFR patients at baseline (10.0% ± 2.3) vs MolR (17.7% ± 3.1, p=0.02). There was no difference in granulocytic MDSCs or Treg between TFR and MolR. No difference in PD-1 expression was observed on NK cells, T cells, B cells and Monocytes. Extensive characterisation of B cell subsets revealed no difference in TFR vs MolR (Table 1). Conclusion: In keeping with STIM and EURO-SKI trials, a threshold level of particular NK cell subsets may be important in maintaining TFR. We found additionally that enhanced NK and CTL effector responses and decreased inhibitory NK KIR2DL2/DL3/DS2 expression, in combination with reduced monocytic MDSC may promote sustained TFR. Methods to enhance nett immune effector responses, such as mature CD56dimCD57+ NK cells and BMI-1 CTL responses or targeting inhibitory KIR may increase TFR success rates. Disclosures White: Ariad: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ross:Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria. Hughes:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria. Yong:Celgene: Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Description: Epidermal growth factor receptor (EGFR) expression is elevated in peripheral blood (PB) cells of polycythemia vera (PV) patients (Skov et al, Eur J Haematol 2011;87:54-60) and EGFR inhibitors (AEE788, erlotinib) inhibit erythroid burst-forming units (BFUE) from PV patients but not normal donors. The mechanism underlying the effect of EGFR inhibitors on MPN progenitor growth has not been established but could be due to an off-target effect on JAK2 activity (Li et al, J Biol Chem 2007;282:3428-32; Gaikwad et al, Exp Hematol 2007;35:1647-56). Therefore, we investigated the growth of BFUE in the presence and absence of the EGFR inhibitor gefitinib (10µM), which does not inhibit JAK2, and observed inhibition of growth of both erythropoietin (Epo)-dependent and -independent colonies from PB mononuclear cells (PBMNC) from PV patients but not from normal individuals. These results suggest a potential role for EGFR signalling in supporting growth and/or survival of PV progenitors. Therefore, to evaluate the possibility of somatic genetic abnormalities leading to EGFR hypersensitivity in MPN, we performed targeted exon capture and massively parallel sequencing, and sensitive mass array screening of 155 MPN patient samples. We identified a low-frequency, recurrent somatic variant of EGFR (p. C329R) in 3/155 MPN patients. The human EGFR C329 residue is homologous to the residue C359 of the C. elegans gene let-23, target of a known gain-of-function mutation (Katz et al, Mol Cell Biol 1996;16(2):529-37); it also aligns with the cysteine residue affected in the highly-transforming mutant ErbB2 C334S found in lung cancer (Greulich et al, PNAS 2012; 109:14476–14481); and it lies within the extracellular cysteine-rich region of EGFR that is the target of frequent somatic mutations in glioma. To confirm the hypothesis that the EGFR C329R mutant leads to altered cytokine response, we transduced Ba/F3 cells with empty vector, EGFR wild type (WT) or mutant constructs (BaF3/MIG, BaF3/EGFR and BaF3/EGFRC329R, respectively). Both WT and mutant receptors showed constitutive activation and transforming ability when expressed at high levels. However, BaF3/EGFRC329R cells display increased levels of STAT activation associated with a slight proliferation advantage when compared to BaF3/EGFR. Given that gefitinib inhibited the growth of both BaF3/EGFR and BaF3/EGFRC329R but did not affect BaF3/MIG cells grown in IL-3, we next compared the effect of gefitinib (10µM) on the growth of BFUE from PV patient samples with and without the EGFR C329R mutation. We observed significant inhibition of Epo-independent BFUE from all PV samples but not of Epo-dependent BFUE from normal controls (Figure A). Furthermore, genotyping of JAK2 and EGFR from the individual colonies obtained in BFUE assay (treated or not with gefitinib, 10µM) for a PV patient that is positive for EGFR C329R showed that drug treatment significantly reduced the proportion of JAK2 V617F heterozygous BFUE compared to the vehicle-treated control (chi-squared test = 0.0002, Figure B). This suggests that signalling from EGFR contributes to proliferation and/or survival in JAK2 V617F heterozygous BFUE from this patient. The results presented here are consistent with an EGFR signalling role in supporting growth of PV progenitors, particularly in the context of a heterozygous JAK2V617F mutation. STAT5 signalling is essential for PV (Walz et al, Blood 2012;119:3550-3560; Yan et al, Blood 2012;199:3539-3549) and JAK2-independent activation of STAT5 through EGFR (Quesnelle et al, J. Cell. Biochem. 2007; 102:311–319) via various mechanisms may contribute to the level of STAT5 activation required for the PV phenotype. A recent study demonstrating a role for EGFR in hematopoietic stem cells (Doan et al, Nat Med 2013;19:295-304) also highlights the potential of aberrant EGFR signalling to contribute to altered properties of MPN stem cells. Finally, given that gefitinib is currently in clinical use for treatment of solid tumors, these findings raise the possibility that gefitinib may have clinical utility in the context of MPN. Figure 1 Figure 1. Disclosures Branford: Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding.

Description: Introduction: Anemia is one of the commonest presenting features of MDS and approximately 30-40% of patients require regular RBC-transfusion. RBC-transfusion dependency (RBC-TD) is a poor-prognostic factor independent of revised International Prognostic Scoring System (IPSS-R) (Hiwase et al ASH 2014). Although RBC transfusion increases the risk of alloimmunization, there is limited literature characterizing this risk in MDS patients as compared to other hematological disorders (such as thalassemia). Methods: This retrospective study assessed the alloimmunization rate in 784 MDS and AML (20-30% blasts) patients registered in the South Australian-MDS registry (SA-MDS registry) between 1991 and 2015. RBC-TD was defined as ≥1 unit of RBC transfused every eight weeks for four months according to WHO based Prognostic Scoring System. The cumulative incidence of RBC-alloimmunization was calculated using competing risk analysis (death being the competing risk). Factors associated with increased rate of RBC antibody formation were investigated by Cox regression analysis. Results: The median age of the 784 patients at diagnosis was 75 years with 66% males. The estimated median follow up time was 7.3 years. 70% of patients (549/784) were diagnosed with primary MDS, while the remaining patients were diagnosed with AML (20-30% blasts; n=57), CMML (n=91) or therapy-related myeloid neoplasm (T-MN; n=87). At last follow-up 30% patients were alive, 67% were deceased and 3% were lost to follow-up. During the study period, 658 (84%) patients required ≥1 unit of RBC transfusion and median RBC units transfused were 29 (range 0-708). The WPSS definition of RBC-TD was met in 47% (366/784 patients), while 36% (282/784) patients required intermittent RBC-transfusions (RBC-TI). During follow up, 83 (13%) patients formed 155 RBC-alloantibodies and 50% of these cases (42/83) developed 〉1 RBC-alloantibody. Autoantibodies were also detected in 31 cases, mainly in association with RBC-alloantibodies (n=27; complex alloimmunization) while 4 cases had only autoantibodies. Interestingly, in 19/27 of cases autoantibodies were detected only after alloimmunization. The pathophysiologic mechanism of this remains unclear. The most common alloantibody specificities were Rh (57%) and Kell (21%) (Table 1). The median interval between 1st RBC transfusion and antibody detection was 10 (0.2-225) months. In 9 cases (6 females) alloantibodies were detected prior to the 1st unit of RBC-transfused. The incidence of RBC alloimmunization reached a plateau at 16% by 100 units of RBC (Fig. 1A), however 80% of antibodies were detectable by 30-40 RBC units transfused. It indicates that most "responders" will form antibodies during the first 30-40 units of RBC transfused. Since most chronically transfused MDS patients do not form RBC alloantibodies it is important from a clinical and resource-utilization standpoint to identify who is at greatest risk of RBC alloimmunization. Multivariate analysis using Cox-regression model was performed. The only factor which was associated with significantly higher risk of RBC alloimmunization was RBC-TD (HR 2.52; p=0.0005). Age, sex, IPSS-R category and number of RBC units transfused did not independently predict alloimmunization rate. Using competing risk analysis, the cumulative incidence of RBC-alloimmunization was significantly higher in RBC-TD group compared to RBC-TI group (p=0.0004; Fig. 1B). Conclusion: RBC-alloimmunization is a substantial risk in MDS patients, especially in RBC-transfusion dependent cases. Extended phenotype matching (D,C,c,E,e and Kell) could have prevented alloantibody formation in 79% of alloimmunized MDS patients. Table 1. Specificity of 155 RBC-alloantibodies Table 1. Specificity of 155 RBC-alloantibodies Disclosures Yeung: Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.