Primary Resistant Patients Research Articles

Distinct DNA Methylation and Expression Profiles Underlie CMML Responsiveness to Decitabine and Uncover Novel Mechanism of Resistance

Myelodysplastic syndromes (MDS) and the related disorder chronic myelomonocytic leukemia (CMML) are characterized by abnormal DNA hypermethylation and the DNA methyltransferase inhibitors (DMTis) azacytidine and decitabine (DAC) are frequently used as frontline therapy in these patients. However, DMTis are ineffective for ~50% of the patients who must still undergo treatment for at least 6 months before they can be deemed resistant. Therefore, it is of critical importance to identify baseline molecular differences associated with DMTi sensitivity that can help (i) to improve patient risk-stratification at diagnosis and (ii) identify the underlying mechanisms of resistance to these agents. Previous efforts to identify baseline DNA methylation differences at promoter regions between DMTi responders and non-responders have not been successful, so we hypothesized that any potential differences would be located distally from promoter regions. For this purpose we studied 40 CMML patients at diagnosis, all of whom had been uniformly treated with DAC 20 mg/m2/day x 5 days as frontline therapy. After 6 cycles of therapy patients were classified as responders (n=19, hematological improvement or better), or non-responders (n=21, stable or progressive disease). Mutational analysis showed no significant differences in the frequency of mutations in TET2, ASXL1, DNMT3A, RUNX1, TP53, JAK2, KIT, KRAS, EZH2, IDH1/2 and spliceosome genes. Using Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) we analyzed the baseline methylation status at ~3M CpG sites across the genome of 39/40 CMML patients. We identified 158 statistically significant differentially methylated regions (DMRs) (FDR<0.1 and methylation difference ≥25%) between the two groups. DAC-sensitive patients displayed both regions of higher methylation as well as regions with lower methylation compared to DAC-resistant patients. As predicted, DMRs were depleted at promoters (DMRs 9% vs. Background [BG] 21%, p-value: 3.4×10-5) and CpG islands (DMRs 8% vs. BG 25%, p-value: 1.5×10-8). Further analysis showed that hypermethylated DMRs were enriched at intronic regions (Hyper DMRs 58% vs. BG 33%, p-value: 3.7×10-6) while hypomethylated DMRs were enriched at intergenic regions (Hypo DMRs 49% vs. BG 38%, p-value: 2.6×10-2). Moreover, hypermethylated DMRs were significantly enriched for enhancer regions, and in particular, enhancers located within gene bodies (hyper DMRs 38% vs. BG 18%, p-value: 2.3×10-5). KEGG pathway analysis showed a significant enrichment of DMRs in the MAPK signaling pathway (FDR<0.01). Next, using a support vector machine algorithm with 10-fold cross validation we were able to develop a classifier capable of predicting response to DAC with high level of accuracy (ROC AUC: 0.99) based solely on the DNA methylation status at diagnosis of 17 genomic regions. Three different random splits of the cohort into training and test sets achieved correct predictions for 85.7%, 89.47%, and 100% of cases, respectively, demonstrating the accuracy and potential utility of such a classifier. Finally, RNA-seq analysis identified 53 differentially expressed genes between responders (n=8) and non-responders (n=6) at diagnosis. Genes implicated in cell cycle and DNA replication were overexpressed in responders. By contrast, very few genes were overexpressed at the time of diagnosis in primary resistant patients. Among these were CXCL4 and CXCL7 which, given their reported contributions to cell cycle arrest and chemoresistance, were tested for their functional roles in DAC resistance. Pre-treatment of normal CD34+ cells for 72 h with 10nM DAC significantly reduced colony formation (p<0.05) but the addition of 50ng/mL of CXCL4 and CXCL7 restored colony formation to that of untreated cells. Moreover, treatment of primary CMML cells with 10 nM DAC for 72h significantly reduced viability of these cells, while concomitant treatment with 50ng/mL of CXCL4 and CXCL7 was sufficient to abrogate this effect. Taken together, our findings demonstrate that (i) specific DNA methylation profiles targeting non-promoter regulatory regions are associated with DAC sensitivity, (ii) these differences can be harnessed for the development of clinical biomarkers predictive of response and (iii) we identified a novel mechanism of resistance to DAC mediated through two chemokines that are exclusively overexpressed in non-responders. DisclosuresNo relevant conflicts of interest to declare.

Mutational Analysis of Patients with Primary Resistance to Single-Agent Ibrutinib in Relapsed or Refractory Mantle Cell Lymphoma (MCL)

▪Introduction: Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton’s tyrosine kinase is highly effective in relapsed or refractory mantle cell lymphoma (MCL) patients with an ORR of 68% (Wang M, et al. N Engl J Med. 2013;369:507-516). Similar efficacy results were observed in a recent phase 2 study in MCL patients who progressed after rituximab containing chemotherapy and bortezomib therapy (SPARK study, NCT01599949). However, despite the very promising efficacy data, some patients with MCL do not respond to ibrutinib. Here, we report analyses of potential mechanisms associated with primary resistance to ibrutinib therapy in the SPARK study.Methods: In this phase 2, international, multicenter, single-arm study, patients with MCL received 560 mg/day oral ibrutinib continuously until progressive disease (PD) or unacceptable toxicity. Patients who had PD at the first disease evaluation were considered to have primary resistant disease. DNA was extracted from baseline/pretreatment tumor samples (biopsy or CD19-enriched cells from PBMC) and enriched libraries were constructed with probesets specific for the coding region of 97 genes possibly involved in ibrutinib response and resistance, using the Ovation Target Enrichment system (NUGEN). Deep sequencing (150bp, single end reads) was performed on an Illumina HiSeq instrument . Sequences were aligned to the hg19 reference genome, variants were called using samtools and filters were applied to identify possible somatic mutations (minor allele frequency < 1% in dbSNP, > 5% and < 95% variant allele, > = 10 total reads).Results: Twenty five (22.7%) of the 120 patients enrolled in this study had Independent Review Committee (IRC)-confirmed disease progression. In these primary treatment resistant patients, the median number of prior lines of systemic therapy was 3 (range 1-5 lines); 37% of patients had high risk MIPI score; 64% had bulky disease (longest diameter ≥ 5 cm), 52% had extranodal disease; 32% had bone marrow involvement and 20% had blastoid subtype. None of these baseline clinical parameters were found to be predictive for primary treatment resistance. The median duration of treatment was 1.54 months. Sequence data could be collected from 23 of the 25 patients, with an average of 9 million reads. After data filtering as described above, 27 genes were found with nonsynonymous variants in at least 2 or more patients. The majority of these variants were previously unreported in dbSNP or COSMIC databases. No mutations previously described in chronic lymphocytic leukemia (CLL) patients with acquired resistance to ibrutinib (BTK C481S, PLCg2 R665W) were seen in these patients, although one patient had a different mutation in PLCg2. Genes previously implicated in diffuse large B-cell lymphoma (DLBCL) pathogenesis (Pasqualucci L, et al. Nature Genetics. 2011;43:830-837) such as MLL2 and CREBBP were also found to be mutated in these patients. In addition, mutations in PIM1 and ERBB4 kinase genes were more frequent in patients with PD compared with those non-resistant to therapy. PIM1 kinase has been associated with increased proliferation, survival, and migration of tumor cells, and somatic mutations in this gene have been described in several cancers including hematologic malignancies. Interestingly, several of the mutations detected affect NF-kB signalling inhibition which is thought to contribute to ibrutinib’s mechanism of action. These results will be updated at the time of final presentation.Conclusion: Sequence analyses were conducted on tumor DNA from the fraction of patients with primary resistance to ibrutinib treatment in this study. These studies have revealed a number of known and novel mutations, including genes involved in NF-kB signalling. Among others, the mutational status of PIM1 kinase and ERBB4 kinase genes may be of interest with respect to primary resistance to ibrutinib therapy in MCL. DisclosuresBalasubramanian:Janssen R&D: Employment, Equity Ownership. Schaffer:Janssen: Employment. Deraedt:Johnson & Johnson: Employment, Equity Ownership. Davis:Janssen: Employment. Aquino:Janssen R&D: Employment. Yuan:Johnson & Johnson : Employment, Equity Ownership. Kranenburg:Janssen Biologics: Employment; Johnson & Johnson: Equity Ownership. Dreyling:Janssen, Pfizer (support of IITs (institutional): Research Funding; Janssen, Pfizer (speaker’s honoraria): Honoraria. Rule:Pharmacyclics, J&J: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Wang:Pharmacyclics, Janssen, Celgene, Onyx, OnyPep, : Research Funding; Onyx, Janssen: Honoraria. Zhuang:Johnson & Johnson: Employment, Equity Ownership. Rizo:Janssen R&D: Employment, Equity Ownership. Lenz:Janssen: Membership on an entity’s Board of Directors or advisory committees.

Braf Mutation Analysis in Cell Free Tumoral Dna (Cfdna) of Melanoma Patients: Preliminary Results from the Spanish Melanoma Group Prospective Study Gem1304

ABSTRACT Aim: Our data in BRAFV600 mutant melanoma patients treated with BRAF inhibitors showed a significant difference in median PFS by cfDNA status (3.5 months vs 13.57 months for BRAFV600 positive and negative, respectively p = 0.026)1. The Spanish Melanoma Group initiated a translational study to assess BRAFV600 mutation in cfDNA of stage IV BRAF mutant melanoma patients to determine the prognostic value of BRAFV600 mutation and its role in monitoring disease evolution (GEM1304) (ClinicalTrials.gov Identifier: NCT01960634). Methods: A quantitative 5'-nuclease PCR based assay for determination of BRAFV600 mutation in cfDNA was developed and validated in 92 previously genotyped cancer patients. Planned accrual for GEM1304 study was 58 BRAFV600 mutated melanoma patients. Serum and plasma samples are collected before and during treatment until disease progression. Results: The assay detected 2.5 pg mutated DNA/µL and a ratio of V600E versus wild type allele of 1:20000 with clinical sensitivity of 58% and specificity of 100%. Twenty-six patients, 47 samples, have been included to date. Pretreatment analysis was positive in serum in 16 (64%) and in plasma in 15 (60%) patients. Eleven patients have more than one sample; in four patients no mutation was detected in pretreatment and follow up samples (2 m+. 5 m+. 5 m+. 7 m+). BRAFV600 was detected in pretreatment samples in 6 patients.: in 4 responding patients BRAFV600 mutation was not detectable in subsequent analysis until disease progression (3m + , 6m, 8m + , 9m+). In two primary resistant patients, BRAFV600 persisted in follow up samples and time to death was less than 3 months. Discordant results between clinical response and cfDNA were found in one single patient: radiologic partial response was diagnosed but BRAFV600 mutation cfDNA load increased and patient died after 3 months on treatment. Re-evaluation of the previously performed scan showed new bone metastasis that had been inadvertent. Conclusions: Noninvasive genotyping of ctDNA by PCR assay could allow effective translation to the clinical setting. BRAFV600 mutation in pretreatment samples and persistence of BRAFV600 mutation at first evaluation could be prognostic markers of survival. Updated results will be presented during the meeting. Disclosure: M. Gonzalez-Cao: Work on this study was supported by a grant from Roche Pharmaceuticals. All other authors have declared no conflicts of interest.

The combined treatment with orbital and pretarsal botulinum toxin injections in the management of poorly responsive blepharospasm

Blepharospasm (BS) is a focal dystonia involving involuntary contractions of muscles around the eyes. Botulinum toxin (BoNT) is the most effective treatment for BS and the technique of injection changes depending on the clinical picture. Usually typical BS benefits from the injection in the orbital part of the orbicularis oculi (OOc) muscle (orbital injection), while BoNT injection in the pretarsal part of OOc muscle is helpful especially for the atypical BS (opening eyelid apraxia). The aim of this study was to compare the efficacy of two injection techniques, the orbital versus the combined (injection in both orbital and pretarsal part of OOc) in BS patients with unsatisfactory response to BoNT. Nineteen patients with typical BS not having a satisfactory response from BoNT treatment with the orbital injection (primary and secondary resistant patients) were studied. After 3 months from the last orbital injection patients received the combined injection; they were assessed with the JRS and BSDI scales after 4 weeks from the last orbital and the first combined injection. Statistical analysis showed a significant reduction (p < 0.05) of the mean score of JRS and BSDI scales comparing the combined with orbital injection. This study shows that the treatment of typical BS can have better results when BoNT is injected with the combined technique in primary and secondary resistant patients.

The gefitinib long-term responder (LTR)—A cancer stem-like cell story? Insights from molecular analyses of German long-term responders treated in the IRESSA expanded access program (EAP)

BackgroundIn selected patients with advanced non-small cell lung cancer (NSCLC) the EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor (TKI) gefitinib (IRESSA) shows response rates of ≥70% and a significant prolongation of progression free survival (PFS). However, cogent biomarkers predicting long-term response to EGFR-TKIs are yet lacking. Cancer stem-like cells (CSC) are thought to play a pivotal role in tumor regeneration and appear to be influenced by the EGFR-pathway. This makes them a promising candidate for predicting long-term response to EGFR-TKIs. Materials and methodsWe analyzed pre-therapeutic tissue specimens of a rare and specific subset of previously treated German patients with advanced NSCLC who experienced ≥3 year response to gefitinib within the International IRESSA EAP. 11/20 identified long-term responders (LTRs) had appropriate tissue specimens available. Those were analyzed for EGFR and k-ras (Kirsten rat sarcoma) mutations, EGFR and c-met (met proto-oncogene) amplifications and protein expression of EGFR, E-cadherin/vimentin and the CSC antigens CD133 and BCRP1 (breast cancer resistance protein 1). The results were compared to primary resistant patients (RPs) and intermediate responders (IRs) showing a median response of 8.6 months. ResultsEach group consisted of 6 women and 5 men, with 1 squamous cell carcinoma (SCC) and 10 adenocarcinoma (AC). Along the LTRs, all but the SCC had EGFR mutations, whereas the RPs had no EGFR, but k-ras mutations in 5/11 cases. 8/11 IRs had EGFR and 3/11 k-ras mutations, of which 2 occurred concomitantly. One patient of each group had an EGFR and/or c-met amplification. EGFR and E-cadherin/vimentin expression was not different between the groups, whereas CD133 was expressed only in 4/10 LTRs and BCRP1 predominantly in responders. The LTRs showed a substantially longer mean PFS to previous therapies, a substantially lower number of metastatic sites and almost exclusively pulmonary or pleural metastasis. ConclusionLTRs display established properties of EGFR-TKI responders. Antigens characterizing CSC might identify a fraction of LTRs and matter of interest for further evaluation.

Pretreatment prognostic factors for overall survival in primary resistant acute myeloid leukemia

AimPrimary resistant acute myeloid leukemia has a very poor prognosis. We assessed pretreatment parameters for their significance as prognostic factors in the overall survival (OS) of 53 acute myeloid leukemia (AML) patients who had failed to achieve complete remission (CR) after first-line standard-dose remission-induction therapy. ResultsDuring the period January 2005–December 2009, 53 with acute myeloid leukemia received two cycles of the 3+7 protocol as a first-line standard-dose remission-induction therapy (ARA-C, days 1–7 and daunorubicin, days 1–3). The HiDAC (5 patients), MiDAC (7 patients), and FLAG-IDA protocols (3 patients) were given as salvage therapy. None of these patients achieved CR. There were 27 (51%) males and 26 (49%) females (median age, 55 years, range 28–76). The median white blood cell count was 53 (range 0.9 –350)×109/L, platelets 44 (range 3–856×109/l) and bone marrow blasts 67%. HCT-IC comorbidity scores were 3 in two (3.8%) patients, 2 in 11 (20.8%), 1 in 12 (22.6%) and 0 in 16 (30.2%) patients. Median OS was 3.9 months (range 1 –20 months). The hepatomegaly, white blood cell count, ECOG PS, serum level of lactate dehydrogenase, dysplastic changes, coexpression of CD64, CD15, CD11b, comorbidities and disease cytogenetics influenced survival. ConclusionThis single-center study evaluated the significance of pretreatment factors, and found that patient age, comorbidities, ECOG performance status, leukocytosis, hepatomegaly, LDH, and the disease cytogenetics were factors which influenced the outcomes of primary resistant patients with acute myeloid leukemia. An understanding of these factors may help to predict OS in cases where CR has not been achieved and may help when making further treatment decisions.

Response-Adjusted Transplantation in Primary Resistant and Relapsed Hodgkin Lymphoma

Abstract 336Therapeutic options for patients with primary resistant or relapsed Hodgkin Lymphoma (HL) have remained largely unchanged for the past 20 years, comprising salvage chemotherapy consolidated by autologous transplantation. Newer agents such as brentuximab vedotoxin may increase response rates, and are being evaluated in the maintenance setting after transplantation. For patients with chemosensitive disease, autologous transplantation confers 5yr progression-free-survival rates of 40–50%. Prognosis is worse in primary resistant or early relapsing disease (within 12 months of primary therapy). Functional imaging also provides prognostic information. Patients with metabolic complete response (mCR) prior to autograft have progression-free survival (PFS) rates in excess of 70% at 3–5yrs, whilst in those with chemosensitive disease (CR/PR) but residual metabolically active lesions these figures drop to 25–30%. The feasibility of performing allogeneic hematopoietic stem cell transplants in patients with HL using reduced intensity conditioning is well established. However, reduced non-relapse mortality rates have been associated with increased relapse, which is now the major cause of treatment failure. Registry data suggest the importance of dose intensity in determining relapse risk. Consideration of transplantation earlier in the disease pathway may improve the tolerability of increased dose intensity. We evaluated the feasibility of a response-adjusted transplantation strategy, based on assessment of disease status by FDG-PET-CT following salvage chemotherapy. Between November 2007 and December 2010 we treated 61 patients with primary refractory or relapsed HL at our institution. Patients were restaged following 1 line of salvage. Those in mCR were consolidated with a BEAM autograft, whilst non-progressive patients with <mCR were offered a BEAM-alemtuzumab allograft. Those with progressive or bulky residual disease were given further salvage until they demonstrated stable disease or better, at which point they were eligible for an allograft. Patients were monitored following the allograft for lineage-specific chimerism and restaged by PET-CT, and received dose-escalating donor-lymphocytes from 6 months post-transplant for mixed chimerism or residual disease/progression. 53/61 (87%) patients achieved sufficient response and were fit for high-dose therapy. 28 patients proceeded to autograft and 25 to allograft (8 related donor, 12 HLA-matched unrelated donor, and 5 HLA-mismatched unrelated donor). The majority had received ABVD as first-line therapy (n=48), and ESHAP as first-line salvage (n=48). 21 of those proceeding to allograft received mini-BEAM as additional salvage. The median number of lines of salvage was 2 (1–8) in the allograft cohort. 12/13 ‘late’ relapses, 5/9 ‘early’ relapses and 11/31 primary resistant cases received an autograft. The median age at transplant was 32 in both groups (range 18–66 for autograft vs 16–50 for allograft). Median follow-up is 2.5 vs 2.3 years respectively (range 0.5–3.6yrs in both). Outcomes in the autograft group were understandably good. NRM was 4%, and relapse incidence 11%. 3yr OS and PFS were 92% and 85% respectively. Outcomes following allografting in the higher risk cohort were also encouraging. All engrafted. NRM was 8% and relapse incidence only 16%, with all relapses occurring within 9 months of transplantation. 4 patients received donor-lymphocytes for relapse. All responded and 3 (all CR) are maintained. 3yr OS, PFS and ‘current’ PFS are therefore 88%, 71% and 84% respectively. 4 patients developed grade II and 2 patients grade III acute graft-versus-host disease, and no patients were taking immune suppressants beyond 18 months after transplantation. The combined 3yr outcomes for all 53 transplanted patients are 90% OS, 78% PFS and 84% cPFS. In conclusion, the data demonstrate favorable survival outcomes for the entire cohort of primary resistant and relapsed patients. They illustrate that selected patients with primary resistant and early relapsed disease may do well following autografting, but more strikingly that those predicted to have poor outcomes based on functional imaging may have very favorable outcomes following alemtuzumab-based allografting with aggressive post-transplant immune modulation. The data now form the basis for 2 national UK studies. Disclosures:No relevant conflicts of interest to declare.

Safety and efficacy of HiDAC induction in de novo AML patients age 60 and older.

6587 Background: High dose (>1.5 gm/m2) Ara-C (HiDAC) given during induction is effective and well-tolerated in patients with AML <60. We report safety and efficacy of HiDAC in AML patients ≥60. Methods: Retrospective analysis of newly diagnosed de novo AML ≥ 60 who underwent induction with 6 daily doses of cytarabine 2 gm/m2 and 3 daily doses of daunorubicin 45 mg/m2, which was repeated in patients achieving CR. Subsequent consolidations consisted of either 2 additional courses of 6 daily doses of cytarabine 2 gm/m2 or hematopoietic stem cell transplantation (HSCT). Study end points were toxicities, remission, relapse-free (RFS) and overall survival (OS). Kaplan-Meier curves were used to obtain RFS and OS. Results: 59 patients (30 M/29 F) with a median age 68 (range 60-86) received HiDAC induction between July 1996 and February 2005. 4 (7%) had favorable cytogenetics, 36 (61%) intermediate, 15 (25%) unfavorable, and 4 (7%) unknown. Median WBC was 20,000/mcL (700- 279,000/mcL). Overall, 41/59 (69%) achieved CR. Induction-related toxicities are listed in the table and contributed to the death of 11/59 (19%) by day 60. Eighteen primary resistant patients after 1-2 inductions received alternative therapies and expired. 33/ 41 patients in CR, received additional 1 (N=6), 2 (N=11), or 3 (N=10) courses of HiDAC consolidation of which 6 received HSCT (5 auto and 1 allogeneic matched sibling donor). 8/41 patients in CR received other consolidations (N=5), refused/was unable to receive further therapy (N=1), and returned to referring MD (N=1). Median follow-up for surviving patients was 55 (17-114) months. Median OS was 12(1-114) for the cohort and for those in CR, median RFS was 14 months (1-113) and OS was 27 months (2-114). Conclusions: In a group of 59 unselected de novo AML patients age 60 and older, HiDAC induction was well- tolerated, had comparable early mortality rates to those observed with standard 7+3 induction in this age group, and was associated with high rates (69%) of durable remissions. Induction toxicity No. of patients (%) n = 59 (%) Neutropenic fever 17 (29) Pneumonia/pneumonitis/hypoxia 15 (25) Arrhythmia 10 (17) Acute renal failure 5 (8) Cerebellar 3 (5) Congestive heart failure 2 (3) Hepatic 1 (2) No significant financial relationships to disclose.

KRAS mutation in the primary or acquired resistance of Chinese NSCLC to EGFR tyrosine kinase inhibitors

e19071 Background: Prevalence of KRAS mutation in Asian NSCLC is different to population of white ethnicity. Data about KRAS mutation after development of resistance to EGFR tyrosine kinase inhibitors (TKIs) are rare. This study was to investigate the prevalence of KRAS mutation in Chinese NSCLC after primary or acquired resistance to EGFR TKIs. Methods: 155 EGFR TKI-naive NSCLC patients and corresponding tumor specimens were collected to establish baseline of KRAS mutation. 45 specimens from six patients with primary resistance to TKIs and twenty-nine patients with acquired resistance to TKIs were analyzed. Genomic DNA was prepared after purified cancer cells were isolated by laser microdissection. Mutations in codon 12 and 13 of the KRAS gene in TKI-naive patients were analyzed by direct sequencing. Mutations in TKI-resistant patients were meanwhile detected by DxS KRAS Mutation Test Kit and direct sequencing. Results: KRAS mutation (G12X) was detected in 7.1% (11/155) of the TKI- naive patients by direct sequencing. In 6 primary resistant patients (two paired specimens and four resistant specimens) and in 29 acquired resistant patients (eight paired specimens and twenty-one resistant specimens), absence of KRAS mutation was detected in all pre- treatment and post-treatment tissues by both methods. Conclusions: KRAS mutation exists in TKI-naive NSCLC patients instead of TKI-resistant setting. KRAS mutation couldn’t thoroughly explain the primary resistance to EGFR TKIs and it might not be the crucial consideration in the mechanisms of acquired resistance. No significant financial relationships to disclose.

Gene expression signature of primary imatinib-resistant chronic myeloid leukemia patients

Although the selective tyrosine kinase inhibitor imatinib is successfully used in the treatment of chronic myeloid leukemia (CML), inherent mechanisms confer primary resistance to leukemic patients. In order to search for potentially useful genes in predicting cytogenetic response, a retrospective gene expression study was performed. Leukocyte RNA isolated before imatinib from interferon-alpha-pretreated chronic phase CML patients (n=34) with or without major cytogenetic remission (< or =35% Philadelphia (Ph)+ metaphases) during the first year of treatment was comparatively analyzed using Affymetrix U133A chips. Using support vector machines for gene classification, an outcome-specific gene expression signature consisting of 128 genes was identified. Comparative expression data of specific genes point to changes in apoptosis (e.g. casp9, tumor necrosis factor receptor-associated protein 1, hras), DNA repair (msh3, ddb2), oxidative stress protection (glutathione synthetase, paraoxonase 2, vanin 1) and centrosomes (inhibitor of differentiation-1) within primary resistant patients. Independent statistical approaches and quantitative real-time reverse transcriptase-polymerase chain reaction studies support the clinical relevance of gene profiling. In conclusion, this study establishes a candidate predictor of imatinib resistance in interferon-alpha-pretreated CML patients to be subjected to future investigation in a larger independent patient cohort. The resulting expression signature point to involvement of BCR-ABL-independent mechanisms of resistance.

More Than 4 Cycles of ABVD for the Treatment of Stage I–II Hodgkin's Lymphoma?.

Patients with stage I-II A non bulky Hodgkin's lymphoma (HL) are successfully treated with 2–4 cycles of ABVD plus IF radiotherapy (RT). Although the negative impact in DFS and OS of B symptoms and bulky disease(X) is widely accepted, other risk factors such as number of lymph node regions involved or extensive spleen involvement have also been reported. What is the best treatment for such patients? Is radiotherapy necessary? We report here our experience with protocol HD 98 in 62 patients, with stage I–II HL. Since January 98 we have included 30 individuals without risk factors and 32 with one of the following risk factors: B symptoms (n:11), X (n:9), extensive spleen involvement (n:1) and involvement of more than two lymph node regions (n:11). Other patient characteristics were: age: x 31yrs (15–69), sex: females 31, males :31. Ann Arbor stage IA :11, IB :1, IIA: 36, IIB :14. Histologic subtypes were: LP: 4, NS: 42, MC: 15, lymphocyte rich :1. Patients with favourable features received 4 cycles of ABVD plus IFRT, those with unfavourable factors received 6–8 cycles of ABVD plus RT in areas of bulky or residual disease.Results: 59/61 evaluable patients obtained CR or CRu. Two patients were considered primary resistant. Three patients relapsed at 7, 13 and 30 months after completion of treatment. Autologus bone marrow transplant was performed in four patients. The two primary resistant patients relapsed after the transplant and died with proggressive disease. Those patients transplanted in a chemosensitive relapse are currently in CR. With a median follow up of 46m (13–84) 96.7% of the patients are alive, in CR.Conclusions: In our experience patients with unfavourable stages I–II HL are succesfully treated with 6–8 cycles of ABVD. In such patients RT might only be necessary in areas of bulky or residual disease.

Relationship between elevated levels of the alpha 1 acid glycoprotein in chronic myelogenous leukemia in blast crisis and pharmacological resistance to imatinib (Gleevec ®) in vitro and in vivo

The Abl tyrosine kinase inhibitor imatinb is becoming a standard for the treatment of chronic myelogenous leukemia (CML). However, Bcr-Abl gene mutations have been reported mainly in relapsing or resistant patients. In primary resistant patients, only few mutations have been documented so far, suggesting alternative mechanisms. We aimed to investigate if alpha 1 acid glycoprotein (AGP), an acute phase drug binding protein, could be a biological marker for pharmacological resistance to imatinib in nine patients in acute phase CML. All patients (3/3) with high AGP dosages (2.31±0.17 mg/mL; normal values, 0.5–1.3 mg/mL) were primary resistant to imatinib whereas an early clinical response was observed for the six patients with normal AGP levels (1.13±0.2 mg/mL). No mutation in the adenosine triphosphate domain of Abl were detected before the initiation of imatinib therapy. By using in vitro tests combining various imatinib concentrations (1–10 μM) with purified human AGP (1 and 3 mg/mL), we demonstrate that imatinib-induced apoptosis of K562 or fresh leukemic CML cells is abrogated or reduced. The same effect was observed using sera from donors with high AGP levels (1.9–3.28 mg/mL). In patients with CML in blastic phase, AGP levels could reflect pharmacological resistance to imatinib, suggesting that increased dosage of imatinib or the use of a competitor to drug binding should be recommended to optimize the therapeutic effect of the drug.