Un profundo estudio de secuenciación de la leucemia mieloide crónica los pacientes en crisis blástica (CB-CML) detecta mutaciones en el 76,9% de los casos

He decidido publicar desde el original este informe, para que junto a los profesionales que nos acompañan en el tratamiento podamos compartirlas. Mi apuesta esta en mantenernos informados, de todos los estudios cientificos que  alienten una esperanza en la mejor calidad de vida ya sea por el control efectivo y tal vez, la cura de la enfermedad.A deep-sequencing study of chronic myeloid leukemia patients in blast crisis (BC-CML) detects mutations in 76.9% of casesBlast crisis (BC) is the terminal phase of chronic myeloid leukemia (CML) and is characterized by a rapid expansion of myeloid or lymphoid differentiation-arrested blast cells leading to short median survival.1, 2 In approximately 70% of cases the blast lineage is myeloid, whereas in 20–30% of cases the blasts are lymphoid.3 It has been suggested that the progression of CML to BC-CML is a two-step process. The initial step for chronic phase is the occurrence of the Philadelphia chromosome and genetic instability caused by the BCR–ABL tyrosine kinase.4 The second step is the acquisition of additional chromosomal aberrations or mutations of transcription factors by failed DNA repair processes.5 However, at present, little is known about the molecular mechanisms underlying disease progression, but, most likely, activation of oncogenic factors and/or mutations leading to loss of function of tumor suppressor genes in hematopoietic stem cells are involved.2Only limited changes occurring during clonal evolution of chronic phase to BC, both resulting in altered gene expression patterns or DNA copy number alterations, have been described. We hypothesized that specific molecular alterations that regulate gene transcription occurring in other myeloid and lymphatic malignancies may be acquired during the malignant disease progression from chronic phase to BC.In this study, in total, 39 BC-CML cases (n=24 myeloid, n=10 lymphoid, n=5 not specified) were analyzed to elucidate the molecular mechanisms underlying disease progression. Between September 2005 and July 2009, cells were collected from the purified fraction of mononuclear cells after Ficoll density centrifugation. With respect to the karyotype, 12/34 cases analyzed (38.2%) harbored a t(9;22) translocation chromosome without additional chromosomal aberrations at BC-CML stage (cytogenetic data not available in five cases), whereas the other 22 cases carried additional alterations such as +8, +Philadelphia, +19, i(17)(q10), −7 and inv(3)(q21q26).In 22/39 cases of this cohort, whole-genome 2.7M cytogenetic array profiling (Affymetrix, Santa Clara, CA, USA) was performed to identify recurrent submicroscopic gains and losses, as well as regions of copy-neutral loss of heterozygosity. In addition to two patients with copy-neutral loss of heterozygosity for chromosome 1p (both ranging to the telomere as being typical for acquired copy-neutral loss of heterozygosity), 28 submicroscopic alterations were observed: n=9 gains, n=16 losses and n=3 copy-neutral loss of heterozygosity regions, for example, gains for AKT2, MLLT4 and ELN, and losses for CBFB, MLLT10 or MYC (Supplementary Spreadsheet 1). Besides microdeletions in the breakpoint regions of BCR (n=2) or ABL (n=2), the only recurrent submicroscopic alteration detected was a deletion confined to a subset of exons from the IKZF1 gene, located on 7p12.2 (n=3/22, 13.6%) (Supplementary Spreadsheet 1). IKZF1 encodes for a transcription factor, which is an important regulator of lymphoid cell differentiation. Subsequently, the complete cohort of 39 BC-CML cases, including the three patients with an IKZF1 deletion already detected by cytogenetic arrays, was investigated or confirmed for IKZF1 deletions by PCR using specific primer pairs for the common intragenic deletions spanning from exon 2 to 7 or from exon 4 to 7, as published by Iacobucci et al.6 In total, in 17.9% (7/39) of all cases intragenic IKZF1 deletions were observed (1/7 exon Δ2–7; 6/7 exon Δ4–7).Further, next-generation deep sequencing (454 Life Sciences, Branford, CT, USA) was applied for a broad molecular screening used to investigate for mutations in all the 39 patients. For this purpose, 11 candidate genes that play important roles in differentiation and self-renewal of hematological stem cells were selected in order to identify novel targets that are important for malignant progression of CML. In detail, hotspot regions were sequenced for CBL (exons 8 and 9), NRAS(exons 2 and 3), KRAS (exons 2 and 3), IDH1 (exon 4), IDH2 (exon 4) and NPM1(exon 12). Complete coding regions were analyzed for RUNX1, TET2, WT1 andTP53. Further, ASXL1 exon 12 aberrations were investigated by Sanger sequencing. To perform this comprehensive study, amplicon-based deep sequencing was applied using the small-volume Titanium chemistry assay (Roche Applied Science, Mannheim, Germany). In median, 472 reads per amplicon (in total 65; primer pair sequences are available online, Supplementary Spreadsheet 2) were obtained, thus yielding sufficient coverage for detection of mutations with high sensitivity. A 500-fold coverage, representing a sensitivity of less than 5%, was aimed at according to the manufacturer's recommendation. The sequencing data were analyzed using Sequence Pilot version 3.4 (JSI Medical Systems, Kippenheim, Germany).After excluding known polymorphisms and silent mutations, 54 abnormalities were identified in 30/39 patients (Table 1): RUNX1: 13/39 patients (33.3%), ASXL1: 8/39 patients (20.5%, excluding 4 additional cases (10.2%) with the controversial 1934dupG variant7), WT1: 6/39 patients (15.4%), NRAS: 2/39 patients (5.1%),KRAS: 2/39 patients (5.1%), TET2: 3/39 patients (7.7%), CBL: 1/39 patients (2.6%), TP53: 1/39 patients (2.6%), IDH1: 3/39 patients (7.7%), IDH2: 0/39 patients and NPM1 0/39 patients. Thus, in summary, in 76.9% of all BC-CML patients, at least one molecular aberration was detected. In median, one affected gene per patient was observed (range 0–4). Of note, few cases harbored multiple aberrations in the same gene. When taking the observed percentage of next-generation sequencing reads carrying the respective abnormalities into account, four patients with more than one mutated gene harbored mutations that presumably occurred in the same or a dominant clone, for example, case T208 harbored a mutation in WT1 (49% of sequencing reads) and concomitantly inRUNX1 (45% of reads). In 9/39 (23.1%) cases, no mutation was detectable. With respect to the karyotype, 4/8 of these patients (n=1, data not available) were harboring chromosomal alterations in addition to t(9;22), and two carried an additional monosomy 7, resulting in a deletion of one IKZF1 allele (Table 1).Regarding recurrent molecular associations among the distinct genes (Figure 1),RUNX1 was observed to be associated with mutations in other genes, that is, 8/13 of cases were harboring additional mutations in combination with RUNX1. Similarly, in 4/8 of patients with ASXL1 mutations, additional molecular aberrations were detected. Further, certain mutations seemed to be highly associated with myeloid or lymphoid phenotype, for example, ASXL1 mutations (n=8) were exclusively observed in patients with myeloid BC, whereas, in contrast, IKZF1 cases were preferentially detected in cases with lymphoid features (n=5 lymphoid, n=1 myeloid and n=1 not specified). Interestingly, besides mutations in IKZF1 (n=5) and RUNX1(n=3), which regulate transcription of genes relevant for both myeloid and lymphoid development,5 there was no other mutated gene occurring in lymphoid BC-CML (Figure 1). Moreover, no aberration was detected in NPM1, and, in contrast to published data,2 in our cohort only one patient harbored a mutation in the tumor suppressor gene TP53 (Tyr205Asn, mutation load 85%).In addition to the above-mentioned mutations, secondary mutations in BCR-ABLwere detected in 33.3% (13/39) of the cases. Patients with mutated BCR-ABL are predisposed to acquire genetic aberrations of transcription factors.5 The BCR-ABL tyrosine kinase is localized in the cytoplasm and is responsible for constitutive activation of several signal transduction pathways.3 After translocating from the cytoplasm to the nucleus, it may cause genetic instability by unfaithful DNA repair, which contributes to structural chromosomal aberrations or mutations of transcription factors in hematopoietic stem cells.1, 5 Ultimately, the accumulation of chromosomal and molecular alterations is responsible for the transformation to BC-CML. Of note, no significant differences were observed with respect to associations between BCR–ABL mutations and additional molecular mutations or chromosomal alterations in addition to t(9;22).In this study, RUNX1, being essential for the self-renewal of hematological stem cells and definitive hematopoiesis, was identified as the most frequently mutated transcription factor (13/39 patients). As only 4/13 of cases with mutated RUNX1were concomitantly mutated in BCR–ABL (Figure 1), we suggest that abnormalities in RUNX1 have an independent role in tyrosine kinase inhibitor resistance and may contribute to treatment failure.1, 8Moreover, for eight patients with mutations in IKZF1 (n=3), RUNX1 (n=3), ASXL1(n=1), WT1 (n=2) and IDH1 (n=2), matched DNA samples from initial diagnosis at chronic phase were available. In none of the chronic phase CML samples were the respective IKZF1 deletions or RUNX1 and ASXL1 mutations detectable, indicating that mutations in IKZF1 and RUNX1 were acquired at the time of transformation to BC-CML, and thus act as driver mutations in these cases. In contrast, WT1 andIDH1 mutations were detected at diagnosis in chronic phase in one case each.With respect to clinical data, associations with survival for RUNX1, ASXL1, IKZF1and WT1 alterations were investigated. No molecular parameter was significantly associated with outcome, which may be due to the short median survival in BC-CML (n=34 patients with survival data available; median overall survival: 9.3 months).In conclusion, the aberrant BCR–ABL kinase causes genomic instability of the CML clone by inefficient DNA repair, resulting in chromosomal alterations and molecular aberrations of transcription factors. This study on 12 genes demonstrated for the first time that in 76.9% of the BC-CML patients, molecular mutations are detectable. The high mutation rate of RUNX1 (33.3%), ASXL1 (20.5%) and IKZF1(17.9%) represented important molecular abnormalities in the progression of CML. In particular, IKZF1 and RUNX1 alterations, both involved in cell differentiation, were identified as important markers of disease progression from chronic phase to BC. Although this is a comprehensive study, further investigations are required to identify additional pathogenetic alterations, as in four cases (10.2%) of our cohort no chromosomal or molecular genetic alterations were observed in addition to t(9;22)(q34;q11).In addition to the above-mentioned mutations, secondary mutations in BCR-ABLwere detected in 33.3% (13/39) of the cases. Patients with mutated BCR-ABL are predisposed to acquire genetic aberrations of transcription factors.5 The BCR-ABL tyrosine kinase is localized in the cytoplasm and is responsible for constitutive activation of several signal transduction pathways.3 After translocating from the cytoplasm to the nucleus, it may cause genetic instability by unfaithful DNA repair, which contributes to structural chromosomal aberrations or mutations of transcription factors in hematopoietic stem cells.1, 5 Ultimately, the accumulation of chromosomal and molecular alterations is responsible for the transformation to BC-CML. Of note, no significant differences were observed with respect to associations between BCR–ABL mutations and additional molecular mutations or chromosomal alterations in addition to t(9;22).In this study, RUNX1, being essential for the self-renewal of hematological stem cells and definitive hematopoiesis, was identified as the most frequently mutated transcription factor (13/39 patients). As only 4/13 of cases with mutated RUNX1were concomitantly mutated in BCR–ABL (Figure 1), we suggest that abnormalities in RUNX1 have an independent role in tyrosine kinase inhibitor resistance and may contribute to treatment failure.1, 8Moreover, for eight patients with mutations in IKZF1 (n=3), RUNX1 (n=3), ASXL1(n=1), WT1 (n=2) and IDH1 (n=2), matched DNA samples from initial diagnosis at chronic phase were available. In none of the chronic phase CML samples were the respective IKZF1 deletions or RUNX1 and ASXL1 mutations detectable, indicating that mutations in IKZF1 and RUNX1 were acquired at the time of transformation to BC-CML, and thus act as driver mutations in these cases. In contrast, WT1 andIDH1 mutations were detected at diagnosis in chronic phase in one case each.With respect to clinical data, associations with survival for RUNX1, ASXL1, IKZF1and WT1 alterations were investigated. No molecular parameter was significantly associated with outcome, which may be due to the short median survival in BC-CML (n=34 patients with survival data available; median overall survival: 9.3 months).In conclusion, the aberrant BCR–ABL kinase causes genomic instability of the CML clone by inefficient DNA repair, resulting in chromosomal alterations and molecular aberrations of transcription factors. This study on 12 genes demonstrated for the first time that in 76.9% of the BC-CML patients, molecular mutations are detectable. The high mutation rate of RUNX1 (33.3%), ASXL1 (20.5%) and IKZF1(17.9%) represented important molecular abnormalities in the progression of CML. In particular, IKZF1 and RUNX1 alterations, both involved in cell differentiation, were identified as important markers of disease progression from chronic phase to BC. Although this is a comprehensive study, further investigations are required to identify additional pathogenetic alterations, as in four cases (10.2%) of our cohort no chromosomal or molecular genetic alterations were observed in addition to t(9;22)(q34;q11).
Leukemia (2011) 25, 557–560; doi:10.1038/leu.2010.298; published online 28 January 2011
V Grossmann¹, A Kohlmann¹, M Zenger¹, S Schindela¹, C Eder¹, S Weissmann¹, S Schnittger¹, W Kern¹, M C Müller², A Hochhaus³, T Haferlach¹ and C Haferlach¹

1. ¹MLL Munich Leukemia Laboratory, Munich, Germany
2. ²III. Medizinische Klinik, Universitätsmedizin Mannheim, Mannheim, Germany
3. ³Klinik für Innere Medizin II, Abteilung Hämatologie und Onkologie, Universitätsklinikum Jena, Jena, Germany
4. Correspondence: V Grossmann, E-mail: vera.grossmann@mll.com