Translocation In Chronic Myeloid Leukemia Research Articles

The first case of six-way complex translocation of t(4;7;9;22;8;14) in a patient with chronic myeloid leukemia.

In chronic myeloid leukemia (CML), patients exhibit the t(9;22)(q34.1;q11.2) translocation, resulting in the formation of a Philadelphia chromosome (Ph). However, a subset of CML patients display variant complex translocations, characterized by three-way, four-way, and five-way translocations, which have been occasionally associated with a poor prognosis. This case report presents the first case of a t(9;22) variant six-way complex translocation in CML. The R banding chromosome karyotyping technique was used to obtain preliminary karyotyping results, and the multi-probe FISH technique was used to assist in the verification of chromosome results. Both FISH and PCR proved the existence of fusion genes. A 45-year-old male patient admitted to our hospital due to elevated WBC and anemia. Bone marrow smears revealed a significant proliferation of mature granulocytes, accompanied by an increase in eosinophils and basophils. Karyotype analysis indicated abnormalities in six chromosomes, including 4, 7, 8, 9, 14, and 22. Further analysis using FISH technology demonstrated the presence of the BCR::ABL1 fusion gene, as well as the mapping of the BCR (22q11), MYC (8q24), IGH (14q32), D4S163 (4q35.1), and D7S486 (7q31) genes to new chromosomes. Ultimately, the karyotype findings were described as t(4;7;9;22;8;14)(q27;q22;q34;q11;q22;q12). PCR showed that BCR::ABL1 was p210. After treatment with imatinib for 4months, the patient achieved complete cytogenetic response (CCyR) and early molecular response (EMR). This is the first report of complex chromosomal karyotype involving six-way translocation in CML; the combination of chromosome analysis and FISH techniques is an effective strategy in determining the karyotype result.

Asciminib - ABL Kinase Inhibitor in the Treatment of Philadelphia Chromosome Positive Chronic Myeloid Leukemia: Chemistry and Pharmacology Perspectives

Abstract: Asciminib, also known as ACP-196, is the FDA-approved low-molecular ABL kinase inhibitor. The ABL kinase is a non-receptor tyrosine kinase that helps in cell growth and survival and is a key player in the development of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL). The BCR-ABL fusion protein, which is formed by chromosomal translocation in CML and Ph+ ALL, results in the constitutive activation of ABL kinase, leading to uncontrolled cell growth and proliferation. To have a high binding affinity for the active site of the enzyme, structural biology and computer-aided drug design (CADD) concepts were applied to the design of asciminib so that it could specifically target the ABL kinase enzyme. The drug was synthesized and characterized in a laboratory. In its pharmacological studies, it has shown that asciminib is a potent and selective inhibitor of ABL kinase. Phase I clinical trials assessed its safety and efficacy, revealing that it is effective against tumors while causing minimal discomfort to patients. In addition to this, it was able to induce apoptosis and a cytogenetic response as well as inhibit the proliferation of CML and Ph+ALL cells in patients with CML. As this trial gave a positive response, phase II and III trials were conducted. In that sense, asciminib has shown to be highly effective, with response rates of over 90% in patients with these diseases. The safety and efficacy of asciminib were also evaluated in combination with other drugs, such as tyrosine kinase inhibitors and immunomodulatory drugs, and the results were promising. Overall, the discovery and development of asciminib showed that by using the concepts of pharmacology and CADD, a drug with a 90% positive rate response can be developed with a high tolerance level and lower side effects

Patient-Derived In Vitro and In Vivo Models of Cancer.

Over the last two decades, cancer researchers have taken the promise offered by the Human Genome Project and have expanded its capacity to use sequencing to identify the genomic alterations that give rise to and sustain individual tumors. This expansion has allowed researchers to identify and target highly recurrent alterations in specific cancer contexts, such as EGFR mutations in non-small cell lung cancer (Lynch et al, N Engl J Med 350:2129-2139, 2004; Sharifnia et al., Proc Natl Acad Sci U S A 111:18661-18666, 2014), BCR-ABL translocations in chronic myeloid leukemia (Deininger, Pharmacol Rev 55:401-423. https://doi.org/10.1124/pr.55.3.4 , 2003; Druker et al, N Engl J Med 344. 1038-1042, 2001; Druker et al, N Engl J Med 344:1031-1037. https://doi.org/10.1056/NEJM200104053441401 , 2001), or HER2 amplifications in breast cancer (Slamon et al, N Engl J Med 344:783-792. https://doi.org/10.1056/NEJM200103153441101 , 2001; Solca et al, Beyond trastuzumab: second-generation targeted therapies for HER-2-positive breast cancer. In: Sibilia M, Zielinski CC, Bartsch R, Grunt TW (eds) Drugs for HER-2-positive breast cancer. Springer, Basel, pp 91-107, 2011). Despite these advances in our capacity to identify the genetic alterations that drive tumor initiation, survival, and proliferation, our ability to target these alterations to provide effective treatment options for patients in need, particularly those with rare or advanced cancers, remains limited (Gould et al, Nat Med 21:431-439. https://doi.org/10.1038/nm.3853 , 2015). Patient-derived models of cancer offer one potential mechanism to overcome this barrier between the bench and bedside. Through the development and testing of patient-derived models of cancer, functional genomics efforts can identify tumor-specific drug sensitivities and thereby provide a connection between tumor genetics and effective therapeutics for patients in need of treatment options.Recognizing that cancer is a multifaceted set of disease states, the development of personalized models of cancer that can be used to compare treatment options, identify tumor-specific vulnerabilities, and guide clinical decision-making has tremendous potential for improving patient outcomes. This chapter will describe a representative set of patient-derived models of cancer, reviewing each of their strengths and weaknesses and highlighting how selecting a model to suit a specific question or context is critical. Each model comes with a unique set of pros and cons, making them more or less appropriate for each specific research or clinical question. As each model can be leveraged to gain new insights into cancer biology, the key to their deployment is to identify the most appropriate model for a specific context, while carefully considering the strengths and limitations of the selected model. When used appropriately, patient-derived models may prove to be the missing link needed to bring the promise of personalized oncology to fruition in the clinic.

Predictive and Prognostic Implications of Variant Philadelphia Translocations in CML: Experience From a Tertiary Oncology Center in Southern India.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by Philadelphia (Ph) chromosome with classical t(9;22)(q34;q11) seen in up to 90% of cases. However 5% to 10% of patients who present with variant Ph translocations (vPh) have been an area of research for their significance in predicting response to various therapies including tyrosine kinase inhibitors as well as prognosticating survival outcomes for many years involving varied patient populations, with conflicting results. We retrospectively analyzed our data from January 2002 to December 2014. Patients with vPh in chronic phase of CML (CML-CP) were analyzed with respect to their demographic parameters, response to imatinib therapy, and survival and their data were compared with data of patients with classical Ph translocation (cPh). Of 615 patients diagnosed with CML-CP, 72 patients (11.7%) showed vPh. Most common chromosomes involved in these translocations were 14 (13.9%), 11 (12.5%), 19 (9.7%), and 7 (8.3%). Rates of complete hematological response, complete cytogenetic response, and major molecular response were not statistically different between the groups. At 5 years, event-free survival, failure-free survival, progression-free survival, and overall survival were 60% versus 67.9%, 62.7% versus 69.7%, 84.7% versus 92.1%, and 87.5% versus 92.4%, respectively, in vPh and cPh. The differences in survival were statistically not significant. To our knowledge, this is the largest series of variant translocations in CML-CP, pertaining to the Indian population. Our data suggest that the presence of vPh in CML has no significant effect in predicting response to imatinib as well as in prognosticating survival.

Additive Effects of Anti-Leukemia Capacity by Gfi-1B- and Bcr-Abl-Specific siRNA In Advanced Chronic Myeloid Leukemic Cells

AbstractAbstract 4466 Background:Growth factor independent-1B (Gfi-1B) is a transcription factor with six C2H2 zinc fingers and one unique N-terminal SNAG (Snail/Gfi-1) transcriptional repressor domain. The Gfi-1B gene was mapped to chromosome 9q34.13 and is so located downstream the bcr-abl translocation in chronic myeloid leukemia (CML) cells. Selective inhibition of the BCR-ABL tyrosine kinase by RNA interference has been demonstrated in leukemic cells. Therefore, we evaluated the specific bcr-abl small inferring RNA (siRNA) and gfi-1B siRNA silencing in K562 cells and in primary CML cells with myeloid blast crises. Method:For in vitro transfection with gfi-1B siRNA, a total of 175 pM gfi-1B siRNA, No. AF 081946 was added to 1×10E5 cells and transfection was performed as previously described by us. Sequences of siRNA directed against the BCR-ABL transcript were published previously by Scherr (Blood 2003) and transfection with 54 pM bcr-abl siRNA were preformed as described for gfi-1B siRNA. As control we used two or more non-silencing siRNA (mismatched or scrambled siRNA) from Qiagen. Result:Using fluorescence marked non-silencing siRNA (Qiagen) we evaluated either the transfection rate in K562 cells and in primary CML cells. At mean (± standard deviation) we found a transfection rate of 69.4 ± 5.7% in K562 cells and 45.8 ± 12.8% in primary CML cells. The bcr-abl siRNA or the combination with both siRNAs revealed the strongest effects in reducing the viability of K562 cells by 28.5 to 5.3 % (mean) compared to controls (controls were set up to 100%). The gfi-1B siRNA reduced the viability of these cells by 49.8 % after the start of transfection. To confirm that cell growth is dependent on the timing of siRNA silencing, we next investigated the time course of the cotransfection of bcr-abl siRNA and gfi-1B siRNA induced effects in K562 cells. A strong reduction of cell growth to 21.9 + 4.5% compared to the rate of spontaneous viable cells in K562 cells was observed after 24 h of siRNA transfection, which was statistically significant (p<0.0001). We further observed a strong inhibition of cell growth to 5.3 + 1.8% (p<0.0001) compared to the cell growth rate after 48 h of cotransfection with these above siRNAs. After 72 h of the cotransfection of bcr-abl siRNA and gfi-1B siRNA no further reduction of cell growth was measured in K562 cells, in fact cell growth increased to 8.6 + 3.2% compared to the spontaneous cell growth rate. The combination of both bcr-abl siRNA and gfi-1B siRNA reduced the bcr-abl mRNA level to 2 fold higher comparing the amount after bcr-abl siRNA alone (p<0.0001). Cotransfection with bcr-abl siRNA and gfi-1B siRNA resulted in an exaggerated decrease of proliferation rate to 9.9 + 1.7% compared to controls. The inhibitory efficiency of bcr-abl siRNA and gfi-1B siRNA has shown a 3-fold increasing result comparing to bcr-abl siRNA alone and a 5-fold increasing effect comparing to gfi-1B siRNA alone. Concordantly, we observed an additive effect on induction of apoptosis by cotransfection of bcr-abl siRNA and gfi-1B siRNA in K562 cells. The rate of induced apoptosis increased from 1 + 0.09 (controls set to 1.0) to 3.3 + 0.2, whereas the use of either bcr-abl siRNA alone or gfi-1B siRNA alone was again less effective. In order to verify the above anti-leukemic results, we assessed bcr-abl siRNA and gfi-1B siRNA transfection in primary cells from patients with blast crisis of CML. We found either a reduction of the bcr-abl mRNA- and of the gfi-1B mRNA amount in these advanced CML patients compared to untreated controls. Furthermore we found in primary CML cells a strongly decreased level of c-myc gene expression after cotransfection with bcr-abl siRNA and gfi-1B siRNA to 65% compared to controls. Conclusion:Our data suggested that silencing by both bcr-abl siRNA and gfi-1B siRNA may be associated with an additive antileukemic activity against K562 cells and in primary CML cells in myeloid blast crises, and might be a suitable target to treated advanced CML. Disclosures:No relevant conflicts of interest to declare.

Genomic Segmental Duplications at the Basis of t(9;22) Rearrangement in Chronic Myeloid Leukemia.

Abstract 3261Poster Board III-1A crucial role of segmental duplications (SDs) of the human genome has been demonstrated in chromosomal rearrangements associated with several genomic disorders. Limited knowledge is yet available on the molecular processes resulting in chromosomal rearrangements in tumors. The t(9;22)(q34;q11) causing the 5'BCR/3'ABL gene formation has been detected in more than 90% of chronic myeloid leukemia (CML) cases. Some years ago, a 76-kb duplicon was reported, closely located to both ABL and BCR genes which are involved in the t(9;22)(q34;q11) translocation associated with CML. However, the exact role of this duplicon in mediating the t(9;22) rearrangement remained mostly speculative. In 10–18% of CML patients genomic deletions were detected on der(9) chromosome next to translocation breakpoints. The molecular mechanism triggering the t(9;22) and deletions on der(9) is still unknown. Our study presents an experimental evidence of the involvement of SDs in the genesis of the t(9;22) translocation in CML and in the occurrence of genomic deletions on der(9) chromosome.We identified 71 (17%) cases with der(9) deletions by FISH screening of 416 CML patients at diagnosis. Fine-mapping of deletions was performed using appropriate bacterial and Phage P1-derived artificial chromosome clones. The deletions sizes were heterogeneous, ranging from 230 Kb to 12.9 Mb on chromosome 22 and from 260 Kb to 41.8 Mb on chromosome 9. The mapping of all breakpoints revealed an evident breakpoints clustering, on both chromosomes 9 and 22, in two regions of about 2 Mb in size. Indeed, these regions contained the breakpoints detected in 54 out of 60 (90%) patients bearing chromosome 9 deletions and in all patients with chromosome 22 sequences loss. Bioinformatic analysis of chromosome 9 and chromosome 22 genomic regions involved in the deletions was performed to search for features that could correlate with the breakpoints clustering. To this aim, the breakpoint regions were subdivided into 250 Kb intervals.The most striking result was the fact that both clusters contain the above reported 76-kb duplicon, shared by chromosome 9 and 22 (SD_9/22). The SD_9/22 is the only duplication located inside the breakpoints clustering region on chromosome 9, whereas the chromosome 22 clustering region harbors several duplications. A remarkable feature of the chromosome 9 clustering region was the high frequency of Alu repeats. The mean Alu frequency overall on chromosome 9 is 10.8%, whereas the average Alu content on this cluster is 31.3%. Accordingly, as expected, the content in LINE sequences of the region was relatively low (average overall on chromosome 9: 21.2%, as opposed to 8.7% on the cluster region). Gene distribution analysis of chromosome 9 and 22 showed that both SD_9/22 map inside gene-poor regions, of about 460 Kb and 250 Kb in size, respectively. To corroborate the observations on the distribution of SDs and Alu/LINE repeats, the chromosome 9 and 22 regions surrounding the SD_9/22 were once more divided into 250 Kb segments, positioning the SD_9 and SD_22 as landmarks. A statistically significant negative association was observed between the number of breaks and the distance from SD_9/22, on both chromosomes 9 (p=0.01) and 22 (p=0.006), respectively. The relationship between the breaks and the interspersed repeats revealed, on chromosome 9, a positive linear regression with Alu repeats (p=0.04), and a negative one with LINEs (p=0.04). Very similar conclusions were obtained by comparing the distance from the SD_9 and the Alu (p= 0.03, positive) and LINE distribution (p=0.02, negative). No statistically significant relationship was observed on chromosome 22.In our study the involvement of SDs was proposed to explain the recurrent t(9;22) translocation in CML and the genomic deletions that could accompany the rearrangement. Although the chromosomes 9 and 22 breakpoints clustering regions are quite large, the strong non-randomness of SD_9/22 location and the genomic features identified in our work suggest that the chromosomal segments near the ABL and BCR genes are brought together by an active process, facilitating recombination. At the light of these findings, the analysis of secondary non-recurrent events could represent a new methodological approach able to identify architectural elements involved in the occurrence of recurrent primary rearrangements in human neoplasia. Disclosures:No relevant conflicts of interest to declare.

Molecular cytogenetic characterization of variant Philadelphia translocations in chronic myeloid leukemia: genesis and deletion of derivative chromosome 9

The mechanisms for the formation of variant Philadelphia (Ph) translocations that occur in 5–10% of patients with chronic myeloid leukemia (CML) are not fully characterized. Studies on the prognosis of these variant translocations have yielded conflicting results, especially regarding imatinib outcome and the status of deletions on the derivative chromosome 9. To shed light on these controversial subjects, we sought to analyze all variant translocation cases presented at diagnosis and identified in our institution between the years 2001 and 2008. Of 336 CML patients who presented at diagnosis and were studied by conventional cytogenetics and fluorescence in situ hybridization (FISH), 25 patients (7.44%) exhibited variant Ph-rearrangements. All chromosomes could be implicated in variant Ph rearrangements, with 32 breakpoints defined. Their distribution was located preferentially in the CG-richest regions of the genome. Deletions on der(9) were observed in 15 of the 25 cases (60%), a greater proportion in typical Ph translocations (12–15%). Both one- and two-step mechanisms were encountered in our series, as well as multiple-step mechanisms, which originate more complex rearrangements. Higher prevalence was observed for the two-step mechanism (56%). Proper assessment of the prognostic significance of variant translocations requires better categorization of these translocations based on their mechanisms of genesis and 9q34 deletion status.

T(3;9;22) 3-Way Chromosome Translocation in Chronic Myeloid Leukemia is Associated with Poor Prognosis

Chronic myelogenous leukemia (CML) is genetically characterized by the reciprocal translocation of chromosome 9 and 22. Around 5–8% of CML develop complex variant Ph translocations involving one or more chromosomal regions besides 9 and 22. Chromosome 3 is not frequently involved in complex translocations in CML. We report in this study a case of CML displaying a t(3;9;22) 3-way translocation. A review of the literature appears to indicate that CML patients with this translocation tend to have an aggressive course and poor outcome. Additional 3-way chromosome translocations associated with CML are also reviewed.

Biological therapy for pediatric malignancy: Current perspectives

Biologicals are defined as agents that are either uniquely or partially tumor-specific. Great expectations were raised by the success in agents that target a specific genetic translocation: all-trans retinoic acid, targeting the chronic myeloid leukemia retinoic acid receptor in acute promyelocytic leukemia and imatinib, a small molecule targeting the BCR-ABL translocation in chronic myeloid leukemia (CML). Thus far, the search for similar "druggable" genetic targets in pediatric cancers has not yet resulted in such dramatic results. The rarity of pediatric cancer as well as ethical considerations necessitate that the agents for testing be carefully and rigorously selected. Biologicals present an additional challenge, as they often do not lend themselves to in vitro testing. Early approaches to specific targeting of solid tumors utilized monoclonal antibodies. The microenvironment provides an interesting new biological approach to treating tumors and alteration of the host immune response provides another avenue. Biological agents are a step forward in supportive care to reduce the hematological toxicity of high-dose chemotherapy and to manage the frequent infectious complications.

Mechanisms of genesis of variant translocation in chronic myeloid leukemia are not correlated with ABL1 or BCR deletion status or response to imatinib therapy

Many published studies have indicated that various mechanisms could be involved in the genesis of variant chronic myelogeneous leukemia (CML) translocations. These are mainly one-step or two-step mechanisms, associated or not with deletions adjacent to the translocation junction on der(9) or der(22) chromosomes (or both). Based on the mechanism of genesis, it has been suggested that the complexity may affect the occurrence of ABL1 and BCR deletions (either or both), or may be associated with the CML disease course, and thus could determine the response to imatinib therapy. Through a retrospective molecular cytogenetic study of 41 CML patients with variant Philadelphia chromosome (Ph), we explored the genesis of these variant rearrangements and analyzed the correlation with deletion status and imatinib efficiency. Our results confirmed that the one-step mechanism is the most frequent, evidenced in 30 of 41 patients (73%); 3 patients demonstrated other more complex multistep events and 8 patients (19.5%) harbored ABL1 or BCR deletions that are not significantly associated with the complexity of translocation genesis. We also found no association between one-step, two-step, or multistep mechanisms and the response to imatinib therapy.

On the genesis and prognosis of variant translocations in chronic myeloid leukemia

Variant translocations involving 9q, 22q, and at least one additional genomic locus occur in 5–10% of patients with chronic myeloid leukemia (CML). The mechanisms for the formation of these variant translocations are not fully characterized. Studies on the prognosis of these variant translocations revealed conflicting results. In addition, deletions in the derivative chromosome 9 are reportedly more frequent among variant translocation cases. We analyzed cytogenetic and FISH data from 22 CML patients with variant translocations tested at our laboratory. Deletions were observed in 6 of the 14 cases with FISH data available (43%), consistent with the literature and higher than in typical translocation cases (12–15%). Sequential changes of 9q deletions are possible and could be acquired as the disease progresses in addition to simultaneous formation of the Philadelphia chromosome with the deletion. Variant translocation CML patients with a deletion showed a worse cytogenetic response 1 year after therapy than those without a deletion ( P < 0.05). Variant translocations may be formed by either a one-step or a two-step mechanism. Proper assessment of the prognostic significance of variant translocations requires better categorization of these translocations based on their mechanisms of genesis and the deletion status.

Breakpoints of variant 9;22 translocations in chronic myeloid leukemia locate preferentially in the CG‐richest regions of the genome

From 5% to 10% of 9;22 translocations in chronic myeloid leukemia (CML) are reported to occur in variant form, that is, with the involvement of other regions of the genome in 3-way or more rearrangements. The literature indicates that the alternative breakpoints are not distributed randomly in the genome but show hotspots. We present data on 289 unpublished cases of CML with variant 9;22 translocations having a total of 342 variant breakpoints, the largest independent series to date. We found that the distribution of breaks was in loose agreement with the literature but that some new hotspots were identified; furthermore, some published hotspots were not fully supported by our data. Moreover, when our 342 variant breakpoints were plotted against profiles of CG heterogeneity in the genome, a significant positive correlation between breakpoint locations and CG composition was observed. In an ancillary study, we compared the frequency of variant t(9;22) with that of variants of t(15;17) associated with acute promyelocytic leukemia (AML M3). We found that the frequency of the former, 9.3%, was significantly higher than that of the latter, 2.6%.

Kinetics of BCR-ABL fusion transcript levels in chronic myeloid leukemia patients treated with STI571 measured by quantitative real-time polymerase chain reaction.

The activated tyrosine kinase, which arises as a result of the balanced t(9,22) translocation in chronic myeloid leukemia (CML), is thought to be essential for the development of the leukemic phenotype. Recently, designer drugs have been introduced which specifically inhibit such specific kinases. Among these, STI571 (Glivec) has entered clinical trials and shown promising activities in chronic phase (CP), accelerated phase (AP) and blast crisis (BC) as evidenced by significant hematological and cytogenetic responses in CML patients. To evaluate the effect of STI571 at the molecular level we have employed quantitative real-time PCR (RQ-PCR) to measure the amount of BCR-ABL fusion transcript in a series of 19 patients treated with STI571, either in CP(11) or in (AP)(8) of the disease for 3--9 months (median 6 months). Employing this method, which is able to detect at least one BCR-ABL+ cell in 500,000, in serial blood and bone marrow specimens we found decreases in transcript levels in 10/11 CP patients, but only in 1/8 of the AP patients. When present such decreases were gradual and became evident only after 3 months of STI571 treatment, and their kinetics in blood closely mirrored those seen in parallel marrow samples. Moreover, decreases were between 10- and 100-fold in 11/13 patients, with only two patients reaching residual disease levels below 10(-2) (a 900-fold decrease). Thus, no patient reached PCR negativity. We conclude that the RQ-PCR method is a highly suitable tool for following the effect of STI571 in CML and that further validation of the method, performed in a prospective manner, will contribute significantly to the elucidation of the proper role of STI571 in CML.

Instability of the Ph Chromosome in Chronic Myelocytic Leukemia: A Case Report

Chronic myelocytic leukemia (CML) is a clonal myeloproliferative disorder characterized cytogenetically by the Philadelphia (Ph) chromosome, which arises from a reciprocal translocation, (9;22)(q34;q11), and is characterized molecularly by the fusion of parts of the C-ABL gene (9q34) with parts of the BCR gene (22q11), generating an abnormal BCR/ABL fusion gene [ 1 Rowley J.D. A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining. Nature. 1973; 243: 290-292 Crossref PubMed Scopus (3339) Google Scholar , 2 de Klein A. Hagemeijer A. Cytogenetic and molecular analysis of the Ph translocation in chronic myeloid leukemia. Cancer Surv. 1984; 3: 515-529 Google Scholar ]. In general, more than 85% of patients diagnosed as having CML are found to have the Ph chromosome [ 3 Sandberg A.A. The Chromosomes in Human Cancer and Leukemia. ed 2. Elsevier Science, New York1990 Google Scholar ]. Variant forms of Ph translocations are seen in 5–10% of CML cases [ 3 Sandberg A.A. The Chromosomes in Human Cancer and Leukemia. ed 2. Elsevier Science, New York1990 Google Scholar ]. When CML progresses, additional chromosome aberrations are noted in 75–80% of the cases, the most common ones being trisomy 8, an additional Ph, an i(17q), trisomy 19, trisomy 21, and loss of a sex chromosome [ 3 Sandberg A.A. The Chromosomes in Human Cancer and Leukemia. ed 2. Elsevier Science, New York1990 Google Scholar ].

Identification of variant translocations in chronic myeloid leukemia by fluorescence in situ hybridization

We studied two cases of chronic myeloid leukemia (CML) having variant complexes translocations detected by trypsin G-banding and fluorescence in situ hybridization (FISH). Application of dual color- (DC-) FISH using abl and bcr cosmid probes permitted us to detect the bcr-abl fusion event on both interphase nuclei and metaphase spread. Furthermore, FISH using combinatorial hybridization (centromeric-library and library-library probes) demonstrated the content and the position of the translocations in CML patients with variant (complex type) Ph-positive rearrangements. FISH analysis appears to be superior than conventional cytogenetic analysis.

Detection of the Mbcr/abl translocation in chronic myeloid leukemia by fluorescence in situ hybridization: Comparison with conventional cytogenetics and implications for minimal residual disease detection

The correlation between the detection of the Philadelphia chromosome by conventional cytogenetics and the identification of Mbcr/ abl translocation by fluorescence in situ hybridization (FISH) in both metaphase and interphase cells is prospectively analyzed in a group of 21 chronic myeloid leukemia (CML) patients. To gain insight into the sensitivity and specificity of the detection of the bcr/abl translocation by FISH, a group of 10 healthy volunteers was also studied. Our results show that for the detection of bcr/abl translocation in CML patients, FISH is more sensitive than conventional cytogenetics because it detects significantly higher proportions of cells carrying the translocation both in metaphase ( P < .0002) and interphase nuclei ( P < .003). Moreover, in the metaphases of the controls analyzed, no bcr/abl+ chromosome was detected that makes the colocalization of bcr and abl signals in the CML patients highly specific. Conversely, in control interphase nuclei, a small proportion of cells (ranging between 0% and 3%, mean value of 1.7% ± 0.9%) displaying colocalization of both signals is usually detected. This limits, at least for the moment, the routine use of FISH for the detection of minimal residual disease in CML patients at levels lower than 10 −1.

BCR-ABL-mediated inhibition of apoptosis with delay of G2/M transition after DNA damage: a mechanism of resistance to multiple anticancer agents

A critical determinant of the efficacy of antineoplastic therapy is the response of malignant cells to DNA damage induced by anticancer agents. The p53 tumor-suppressor gene is a critical component of two distinct cellular responses to DNA damage, the induction of a reversible arrest at the G1/S cell cycle checkpoint, and the activation of apoptosis, a genetic program of autonomous cell death. Expression of the BCR-ABL chimeric gene produced by a balanced translocation in chronic myeloid leukemia, confers resistance to multiple genotoxic anticancer agents. BCR-ABL expression inhibits the apoptotic response to DNA damage without altering either the p53-dependent WAF1/CIP1-mediated G1 arrest or DNA repair. BCR-ABL-mediated inhibition of DNA damage-induced apoptosis is associated with a prolongation of cell cycle arrest at the G2/M restriction point; the delay of G2/M transition may allow time to repair and complete DNA replication and chromosomal segregation, thereby preventing a mitotic catastrophe. The inherent resistance of human cancers to genotoxic agents may result not only by the loss or inactivation of the wild-type p53 gene, but also by genetic alterations such as BCR-ABL that can delay G2/M transition after DNA damage.

BCR-ABL-Mediated Inhibition of Apoptosis With Delay of G2/M Transition After DNA Damage: A Mechanism of Resistance to Multiple Anticancer Agents

A critical determinant of the efficacy of antineoplastic therapy is the response of malignant cells to DNA damage induced by anticancer agents. The p53 tumor-suppressor gene is a critical component of two distinct cellular responses to DNA damage, the induction of a reversible arrest at the G1/S cell cycle checkpoint, and the activation of apoptosis, a genetic program of autonomous cell death. Expression of the BCR-ABL chimeric gene produced by a balanced translocation in chronic myeloid leukemia, confers resistance to multiple genotoxic anticancer agents. BCR-ABL expression inhibits the apoptotic response to DNA damage without altering either the p53-dependent WAF1/CIP1-mediated G1 arrest or DNA repair. BCR-ABL-mediated inhibition of DNA damage-induced apoptosis is associated with a prolongation of cell cycle arrest at the G2/M restriction point; the delay of G2/M transition may allow time to repair and complete DNA replication and chromosomal segregation, thereby preventing a mitotic catastrophe. The inherent resistance of human cancers to genotoxic agents may result not only by the loss or inactivation of the wild-type p53 gene, but also by genetic alterations such as BCR-ABL that can delay G2/M transition after DNA damage.

Molecular analysis of six variant Philadelphia chromosome translocations in chronic myeloid leukemia

In 420 Philadelphia positive (Ph+) chronic myeloid leukemia (CML) patients karyotyped at diagnosis in our laboratory, 26 Ph variants (6.2%) were observed. Twelve of them are reported. Five cases are “simple” variants without detectable involvement of band 9q34, and seven are “complex,” since a third chromosomal band is involved in the Ph formation. Two translocations [t(7;22)(q36;q11) and t(9;22;12)(q34;q11;q11)] are reported for the first time. Six cases were characterized molecularly, and bcr-abl rearrangement was demonstrated, confirming involvement of 9q34 band also in the cases in which chromosomes 9 appear cytogenically normal. Chimeric mRNAs in which M- BCR exon 3 is joined to abl exon 2 (type b3-a2) were detected in four of six cases; one case showed a DNA breakpoint in zone III, which may also give rise to the same transcript. In one case, mRNA junction was b2-a2. The frequency of the b3-a2 junction occurs more frequently in CML patients with a Ph variant than in patients with the standard translocation, suggesting a preferential correlation between this type of transcript and the involvement of other chromosomes in Ph formation.

Variant philadelphia translocations in chronic myeloid leukemia: correlation with cancer breakpoints, fragile sites and oncogenes

Four cases of variant Philadelphia (Ph 1) translocations were found in 72 patients (5.5%) with Ph 1-positive chronic myeloid leukemia (CML). One previously unreported case was a simple variant translocation, namely, 46,XY,t(11; 17) (q13;p13),t(17;22) (q25;q22); 46,XY,t(1;21)(q32;q11),t(11;17)(q13;p13), t(17;22)(q25;q11). Complex variant translocations were observed in three cases, namely, 46,XY,t(5;9;22)(q31;q34;q11),46,XX,t(8;9 ;22)(q22;q34;q11) and 46,XX,t(9;15;22) (q34;q15;q11). The chromosomal breakpoints in the cases of variant Ph 1 translocations were the following: 1q32, 5q31, 8q22, 11q13, 15q15, 17p13, 17q25 and 21q11. Eight of the eight (100%) breakpoints were located in Giemsa-negative bands. Furthermore, seven of the eight (87%) variant Ph 1 breakpoints correspond to the breakpoints present in consistent cancer arrangements. Three of the eight (38%) correspond to fragile sites and four of the eight (50%) correspond to oncogenes.