Search

B-cell acute lymphoblastic leukemia (B-ALL) is a diverse group of malignant blood disorders both with regard to the biological properties of the tumor and to therapeutic approaches. Immunophenotyping, molecular genetic techniques, whole-genome sequencing characterize B-ALL as a very diverse group for sensitivity to chemotherapy and prognosis. We present three clinical cases of patients with B-ALL and expected good response to standard therapy, in whom standard protocol treatment failured: refractoriness, persistence of minimal residual disease (MRD), and progression (MRD increase). The remission in these patients was achieved after chemotherapy change to immunological targeted therapy. Nowadays a unified therapeutic approach to all primary patients of the B-ALL is considered generally outdated. Great efforts are carrying out to develop molecular genetic classifications. The molecular dissection of subtypes of B-ALL goes on, and new protocols for selective treatment with targeting are clearly outlined for each subtype of B-ALL.

A wide range of toxic complications of BCR-ABL tyrosine kinase inhibitors therapy due to non-targeted inhibition of other protein kinases. This is of particular importance in the treatment of patients with Ph-positive lymphoblastic leukemia, who receive, in addition to tyrosine kinase inhibitors, cytotoxic drugs, enhancing myelotoxicity and, as a consequence, infections and thrombo-hemorrhagic complications. In addition, complex inter-drug interactions lead to the manifestations of combined toxicity. This article presents a case report of patient with relapsed acute Ph-positive lymphoblastic leukemia, treatment of which was accompanied by numerous complications, especially during treatment of II and III generation tyrosine kinases inhibitors.

CONTEXT:A substantial number of patients with acute lymphoblastic leukemia (ALL) die from progressive disease after relapse that is frequently associated with clonal evolution. Clinical exome sequencing (CES) is a useful tool for mutation investigation, particularly in leukemic subclones during relapse. OBJECTIVE:The objective of this pilot study was to determine the specific genetic abnormalities associated with primary tumor comparing with normal hematopoietic tissue and to define clonal evolution patterns at relapse. DESIGN:CES (TruSightTM One Sequencing Panel) was performed on samples obtained from the patient with B-cell Ph-negative ALL at diagnosis, remission and relapse. SETTING:DNA was extracted from bone marrow samples according to modified salting out method. Libraries were pooled together and underwent two rounds of hybridization and capture, and additional quantification and size assessment. Between 21 and 26 million reads for each sample were obtained with a MiSeq (Illumina, USA), with a coverage of at least 20x for 97% of all sequences. More than 99% of reads passed quality and length control and were used in the analysis. De-multiplexing of indexed reads and the generation of FASTQ files were performed by the MiSeq system on-instrument software. The sequencing data, composed of 150 bp paired-end reads, were processed for MiSeq Reporter (Illumina), which is endowed with Burrows-Wheeler Aligner (BWA) for alignment on a reference sequence of hg19 and Genome Analysis Toolkit (GATK) for variant calling. Then, the Variant Call format (VCF) file was processed for analyzing on VariantStudio (Illumina) and wANNOVAR (Yang H. et al, 2015), which contain annotation databases, such as the ClinVar database, OMIM, the dbSNP database), the Polymorphism Phenotyping v2 database (Polyphen-2), and the Sorting Intolerant from Tolerant database (SIFT) and some others. PATIENTS OR OTHER PARTICIPANTS :A 27-year-old woman, who was admitted to the National Research Center for Hematology (Moscow, Russia) on March, 2017, presented with a three-month history of paleness and ossalgia. Common B-ALL (BII) ALL with complex karyotype and extramedullary lesions in ovaries, kidneys, intra-abdominal and peripheral lymph nodes has been diagnosed. Patient achieved complete remission (CR) after first induction therapy according to ALL-2016 protocol (ClinicalTrials.gov NCT03462095). After 16 months of CR during maintenance therapy (July, 2019) was diagnosed a relapse, which was refractory to multiple chemotherapy approaches and the patient died from the disease (January, 2020). RESULTS:Primary dataset analysis included more than 9000 gene variations for each of the three samples. Only clonal variations (at diagnosis) that are pathogenic either by the type of mutations (insertions (frameshift) and splice site mutations) or by prediction databases (PolyPhen-2, SIFT) were selected. As a result, about 70 variations were identified for each of the samples. Table 1 shows all clonal somatic mutations by which tumor tissue differs from normal hematopoietic tissue. At the time of diagnosis, a clone with monosomy of chromosome 3 was identified, that disappeared at CR and reappeared as a minor clone at relapse. Taking in consideration that the loss of one allele was caused by monosomy, we searched for pathogenic variations in genes localized on the chromosome 3 in order to identify genes inactivated according to Knudson's "Two-Hit" Theory of cancer causation (Knudson A., 1971). Out of 500 genes variations on chromosome 3, mutations in 5 genes according to prediction databases (PolyPhen-2, SIFT) were defined as pathogenic. All mutations were germinal. Moreover, four out of five mutations were homozygous. Table 2 shows the dynamics of pathogenic variations in genes localized on the chromosome 3 at different stages of the disease. The missense mutation ofCOL7A1gene (c.2791C> T), which was found in a small percentage (8%), was localized on the disappeared copy of chromosome 3, and its detection in this case was possible due to the presence of normal cells. CONCLUSIONS:There is an evident change in tumor clones at relapse: a major clone diagnosed inde novoB-cell ALL patient became minor at relapse. The data obtained from this analysis suggest that at the time of diagnosis, several clones are present in the tumor, and that most of the diagnosed pathogenic variations are not associated with oncogenesis and are germinal. Disclosures No relevant conflicts of interest to declare.

Introduction. For more than a decade it's postulated that the addition oftyrosine kinase inhibitors (TKI) to chemotherapy has dramatically improved the long term outcome in Ph-positive adult acute lymphoblastic leukemia (Ph+ ALL). Nevertheless whether do we need chemotherapy at all and if yes - how intensive it should be, is still the matter of debates. The only randomized trial addressing this issue (GRAAL, Blood 2015, 125: 3711-3719) has demonstrated the lack of benefit of more intensive induction at all checkpoints: complete remission (CR) rate, major molecular complete remission (MMolCR), molecular complete remission (MolCR), progression disease (PD) and resistance. We have conducted two consecutive trials in Ph+ ALL aiming to evaluate the efficacy of more and less intensive chemotherapy approach in combination with constant non-stop 600 mg Imatinib application. Aim. Toanalyze of the protocol RALL-2009 with ITK and RALL-Ph+-2012 effectiveness in patients with Ph+ ALL. The primary objective was the major molecular complete remission (MMolCR) rate after induction (70th day), patients being then eligible for allogeneic stem cell transplantation (SCT) if they had a donor, or autologous SCT if in MMolR and no donor. Patients and methods. Since Jan 2010 till July 2016, 120 new cases of ALL were verified in our National Research Center for Hematology with 68 (56,7%) of them being B-cell precursors ALL and 25 diagnosed as Ph-positive (36,8%). Since 2010 till 2012, 10 Ph+ ALL pts (median age 35 years (19-68), m/f (50%)/(50%), CNS disease=1, WBC> 30*109/l=5(50%), bcr/abl transcript p190/p210/p190+210 6(60%)/3(30%)/1(10%)) were treated according to RALL-2009 protocol (ClinicalTrials.gov public site; NCT01193933) with parallel Imatinib. This protocol includes 8 cytostatic drugs and no intervals between treatment phases. Since 2012 till now 15 other pts (median age 40 years (17-61); m/f 7(46,7%)/8(53,3%); CNS disease=1, WBC>30*109/l=5(33,3%), bcr/abl transcript p190/p210/p190+210 9(60%)/5(33%)/1(7%)) were included in RALL- Ph+-2012 protocol, based mainly on 600 mg Imatinib with prednisolone, VNCR, L-asp, followed by 6-MP and MTX. Both protocols suggested the shift to Dasatinib (100-140 mg) after non-achievement of MolCR at day 70 of treatment. MolCR was stated if no bcr/abl chimeric transcript was detected by PCR with 10-4 sensitivity. All patients were considered as candidates for allogeneic HSCT if HLA-identical donor was available. Results. At day 70th disregarding the chemotherapy intensity there was 40% of MolCR on both protocols (RALL-2009 - n=4 and RALL-2012 - n=6). No death within 2 months of induction/consolidation were registered on less intensive protocol in comparison with 2 cases on RALL-2009. Hematological CR was achieved in all pts (except two early deaths on RALL-2009) - 23 of 25 (92%). There was one autologous HSCT in older pts, included in RALL-2012 (n=3, aGVHD and severe infections, at a median +4 months after HSCT and more than 12 months of CR duration). The 3y OS, DFS and relapse probability (RP) for all 25 pts constituted 37,8%, 32,5% and 52,1% (Fig. 1). The long-term outcome on both protocols (RALL-2009 and RALL-2012) was similar: OS - 45% vs 27,7% (p=0,27), DFS - 45% vs 22,1% (p=0,94), RP - 35,7% vs 57% (p=0,29), respectively (Fig.2). Conclusion. De-intensification of the chemotherapy does not affect the effectiveness of the therapy Ph-positive acute lymphocytic leukemia in era of the tyrosine kinase inhibitors. We haven't seen differences in achievement of molecular remission when we deescalated chemotherapy (40% vs. 40%). However, when we reduced toxicity of the chemotherapy in ALL-2012 protocol, we were able to realize more extra allo-BMT and it could improve long-term results of the therapy Ph+ ALL. Figure Overall, disease-free survival and relapse probability in patients with Ph+ ALL on RALL protocols. Figure. Overall, disease-free survival and relapse probability in patients with Ph+ ALL on RALL protocols. Figure Overall, disease-free survival and relapse probability in patients with Ph+ ALL on RALL-2009 and RALL-2012 protocols. Figure. Overall, disease-free survival and relapse probability in patients with Ph+ ALL on RALL-2009 and RALL-2012 protocols. Disclosures No relevant conflicts of interest to declare.