Introduction. Overall survival (OS) of patients in chronic phase (CP) chronic myeloid leukemia (CML) dramatically increased in the era of tyrosine kinase inhibitors (TKI). Meanwhile nearly half of patients discontinue 1-st line Imatinib due to resistance or intolerance. Half of them subsequently failure treatment with second line TKI. It seems that 20-25% of CP CML patients need 3-d line therapy (TKI-3l). There are a few reports regarding durable outcome of TKI-3l. Materials and Methods. In our retrospective study 53 patients (20 male, 33 female) with CML CP treated either by Nilotinib 400 mg BID (n=18), Dasatinib 100 mg QD (n=33) or Bosutinib 500 mg QD (n=5) as TKI-3l were included. The median age at the time of diagnosis was 46 years (23-88 years). The main reason for previous TKIs discontinuation was resistance: 48/53 (91%) had failure of one and 42/53 (79%) patients had failure of both previous TKIs treatment. Median CML duration before TKI-3l was 55 months (2-314 months). Before TKI-3l mutation analysis was performed in 35 patients: 18 mutations were revealed in 16 (46%) patients including T315I mutation in 3 cases. At the moment of 3-d line TKIs therapy initiation, all patients were in CP and 43/53 (81%) had at least complete hematologic response (CHR), 8/53 (15%) patients had major cytogenetic response (MCyR) including 1 patient with complete cytogenetic response (CCyR). Results. At the time of analysis, the median duration of TKI-3l therapy was 21 months (1-67 months). No additional patients achieved CHR, but during observational time CHR was maintained nearly in all 40/43 (93%) patients with CHR at baseline. New cases of MCyR and CCyR were observed in 15/45(33%) and 11/52(21%) of patients, respectively. Median time to MCyR and CCyR was 3.4 (3-8) and 5.2 (3-13) months, respectively. Median duration of MCyR and CCyR was 9.3 (1-43) months and 4.5 (3-6) months, respectively. Patients with resistant mutations did not obtain any cytogenetic response. TKI-3l treatment was discontinued in 21 patients. Intolerance was the reason of treatment discontinuation in 5/53(10%) cases. Progression to accelerated or blastic phases during therapy or after discontinuation occurred in 8/53 (15%) patients. Median time to progression was 14.7 months (1-46 months). There were 13 deaths in overall group of 53 patients. Two-year OS in TKI-3l was 67%. All patients with MCyR were alive and preserved CP phase. Concluson: Our results showed that 20% of patients might obtain at least CCyR and benefit with TKIs as third line. Therefore, after two TKI lines patients, who are not eligible for allogeneic transplantation and without resistant mutations should be treated with one more line of TKI therapy. Disclosures Lomaia: Novartis: Consultancy. Zaritskey:University of Heidelberg: Research Funding; Novartis: Consultancy.
Clinical data: Subject: Male, 45 years old, diagnosed in 2001 with Ph+ CMLCP (low Sokalrisk).From 2001 to 2005 he was treated with Hydrea and Interferon with low dose Cytarabine. In 2005 he was switched to imatinib 400 mg/day. Due to cytogenetic resistance Imatinib dose was escalated to 600mg/day in 2006 and 800mg/day in 2007. No cytogenetic response was obtained. In 2009 patient was switched to nilotinib. Nilotinib therapy was discontinued in 2012 due to loss of complete hematologic response. Patient refuses unrelated donor allo HSCT. INFa 3000 U/3 times a day was given with prolonged stable complete hematologic response. Myeloid blasts crisis was diagnosed in Jan 2013. (The time patient was hospitalized to Almazov medical research center). Dasatinib 140mg/day was started without rapid blast reduction.The response lost in a few weeks, followed by 2 couses of FLAG regimen. Patient died in Dec 2013. BCR-ABL KD mutations history: 2007- no mutations found; 2009- no mutations found, February 2013 -L248V mutation; July 2013 mutation analysis returns no result due to the poor sequence quality;T315I mutation revealed in samples obtained from October 2013. BCR-ABL mutation analysis: Bcr-Abl analysis performed as described before (Branford et al., 2002). KD sequence was amplified from cDNA using seminested PCR, PCR product extracted from gel and sequenced in the both directions on ABI 3130 genetic analyzer. For confirmation we used genomic DNA extracted from stored peripheral blood samples. Primers specifically amplifying exon 4 and 5 of ABL used for genomic DNA analysis. Analysis of the sequences was done using UGENE software. Results: We analyzed two sets of probes- one dated March 2011 and another dated October 2013. After performing semi-nested PCR on BCR-ABL KD sequence we found at least two bands in each of the samples. Sequencing of this bands revealed 81 bp deletion translated into absence of 27 aa position at 248-274. Genomic DNA analysis confirmed alternative splicing and appearance of the truncated form. We found SNP resulted in T315I mutation presented either in full or in short isoform of BCR-ABL in samples from October 2013. Also we confirmed L248V mutation presented in all analyzed samples. Conclusion: SNP that cause L248V mutation in BCR-ABL transcript known to activate cryptic promotor in ABL exon4 with subsequent appearance of alternative splicing form described before (Gruber et al., 2006). Truncated form known to be kinase dead and have no influence on disease course (Shebenou et al., 2008). On the other hand it disturbs the Sanger sequence quality, masking the appearance of the additional mutations. Extensive attention requires in obtain proper sequence quality: use genomic DNA for Sanger sequencing or alternative sequencing methods, e.g. single-molecule long-read sequencing for revealing mutations in multiple splice isoforms. References: Branford S, Rudzki Z, Walsh S, Grigg A, Arthur C, Taylor K, Herrmann R, Lynch KP, Hughes TP. High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571)resistance. Blood. 2002 May 1;99(9):3472-5. Gruber FX, Hjorth-Hansen H, Mikkola I, Stenke L, Johansen T. A novel Bcr-Abl splice isoform is associated with the L248V mutation in CML patients with acquired resistance to imatinib. Leukemia. 2006 Nov;20(11):2057-60. Sherbenou DW, Hantschel O, Turaga L, Kaupe I, Willis S, Bumm T, Press RD,Superti-Furga G, Druker BJ, Deininger MW. Characterization of BCR-ABL deletion mutants from patients with chronic myeloid leukemia.Leukemia. 2008, Jun;22(6):1184-90. Disclosures Lomaya: Novartis: Consultancy. Zaritskey:University of Heidelberg: Research Funding; Novartis: Consultancy.
We analyzed CALR mutations in cohort of patients with ET and PMF. Diagnoses were determined according to the WHO criteria. 33 PMF and 45 ET patients that were negative for JAK2V617F and MPL515L/K mutations have been studied for CALR mutations presence as described in original paper (Т.Klampfi , 2013). 29 ET and 25 PMF patients found mutated. In PMF group sex ratio was equal - 12 males/13 females, in ET group females prevailed - 21 males/69 females. Median age in PMF group was 64 (41-78) and in ET group 58 (22-80). Exon 9 was Sanger sequenced and mutation type identified. All the mutated CALR sequences were translated with verified +1 frameshift with alternative C-end. In PMF group we found type 1 52bp deletion p.L367fs*46 (n=11), type 2 TTGCT insertion p.K385fs*47 (n=5), type 3 p.L367fs*48 (n=3), type 7 p.K368fs*45 (n=2), type 19 p.E372fs*48 (n=2) and single cases of p.K368fs*51, p.K385fs*47 , p.Q365fs*50. In ET group we found type1 52bp deletion p.L367fs*46 (n=10), type 2 TTGCT insertion p.K385fs*47 (n=6), and single cases of p.L367fs*52, p.K368fs*51, p.E378fs*45, p.K374fs*49), p.E364fs*49. Also in ET group we identified two cases with alternative insertion c.1154_1155insGTGTC p.E386fs*46. In one ET case we found complex CALR mutation variant c. с.1126_1142delInsACCTTTGCATTTT (p.R376fs*53). In this case alternative C-end, different from what was described before will be formed. CALR mutations were grouped in Type1 like and Type2 like according to protein sequence similarity (A. Tefferi, 2014). In PMF group type1/2 like mutation ratio was 18/7 and in ET group it was 16/13, confirming higher CALR type2 like mutations rate in ET. We analyzed CALR mutations in cohort of patients with ET and PMF. Diagnoses were determined according to the WHO criteria. 33 PMF and 45 ET patients that were negative for JAK2V617F and MPL515L/K mutations have been studied for CALR mutations presence as described in original paper (Т.Klampfi, 2013). 29 ET and 25 PMF patients found mutated. In PMF group sex ratio was equal - 12 males/13 females, in ET group females prevailed - 21 males/69 females. Median age in PMF group was 64 (41-78) and in ET group 58 (22-80). Exon 9 was Sanger sequenced and mutation type identified. All the mutated CALR sequences were translated with verified +1 frameshift with alternative C-end. In PMF group we found type 1 52bp deletion p.L367fs*46 (n=11), type 2 TTGCT insertion p.K385fs*47 (n=5), type 3 p.L367fs*48 (n=3), type 7 p.K368fs*45 (n=2), type 19 p.E372fs*48 (n=2) and single cases of p.K368fs*51, p.K385fs*47 , p.Q365fs*50. In ET group we found type1 52bp deletion p.L367fs*46 (n=10), type 2 TTGCT insertion p.K385fs*47 (n=6), and single cases of p.L367fs*52, p.K368fs*51, p.E378fs*45, p.K374fs*49), p.E364fs*49. Also in ET group we identified two cases with alternative insertion c.1154_1155insGTGTC p.E386fs*46. In one ET case we found complex CALR mutation variant c. с.1126_1142delInsACCTTTGCATTTT (p.R376fs*53). In this case alternative C-end, different from what was described before will be formed. CALR mutations were grouped in Type1 like and Type2 like according to protein sequence similarity (A. Tefferi, 2014). In PMF group type1/2 like mutation ratio was 18/7 and in ET group it was 16/13, confirming higher CALR type2 like mutations rate in ET. Klampfl T. et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013 Dec 19;369(25):2379-90. Tefferi A. et al. The prognostic advantage of calreticulin mutations in myelofibrosis might be confined to type 1 or type 1-like CALR variants. Blood. 2014 Oct 9;124(15):2465-6. Disclosures Zaritskey: Janssen: Consultancy; Novartis: Consultancy.
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