Ph-positive Clone Research Articles

Bone marrow mesenchymal stromal cell (MSC) gene profiling in chronic myeloid leukemia (CML) patients at diagnosis and in deep molecular response induced by tyrosine kinase inhibitors (TKIs)

Although it has been well-demonstrated that bone marrow mesenchymal stromal cells (MSCs) from CML patients do not belong to the Ph1-positive clone, there is growing evidence that they could play a role in the leukemogenesis process or the protection of leukemic stem cells from the effects of tyrosine kinase inhibitors (TKIs). The aim of the present study was to identify genes differentially expressed in MSCs isolated from CML patients at diagnosis (CML-MSCs) as compared to MSCs from healthy controls. Using a custom gene-profiling assay, we identified six genes over-expressed in CML-MSCs (BMP1, FOXO3, MET, MITF, NANOG, PDPN), with the two highest levels being documented for PDPN (PODOPLANIN) and NANOG. To determine whether this aberrant signature persisted in patients in deep molecular response induced by TKIs, we analyzed MSCs derived from such patients (MR-MSCs). This analysis showed that, despite the deep molecular responses, BMP1, MET, MITF, NANOG, and PDPN mRNA were upregulated in MR-MSCs. Moreover, BMP1, MITF, and NANOG mRNA expressions in MR-MSCs were found to be intermediate between control MSCs and CML-MSCs. These results suggest that CML-MSCs exhibit an abnormal gene expression pattern which might have been established during the leukemogenic process and persist in patients in deep molecular response.

Molecularly Defined Clonal Evolution in Patients with Chronic Myeloid Leukemia Independent of the BCR-ABL Status

Molecularly defined clonal evolution has been identified as a key phenomenon in the biology of acute myeloid leukemia. Molecular aberrations may be markers of individual subclones of the disease, but also responsible for treatment resistance. In most patients with chronic myeloid leukemia (CML), treatment with tyrosine kinase inhibitors (TKI) induces complete cytogenetic remissions (CCyR) characterized by polyclonal hematopoiesis. However, some CML patients show emergence of clonal cytogenetic abnormalities (CCA) in Philadelphia (Ph) negative cells during treatment indicating clonal hematopoiesis. We searched for BCR-ABL independent gene mutations in both Ph-negative and Ph-positive clones in 29 chronic phase CML patients (male, n=16; median age 58 years, range 29-73 years) using targeted deep next-generation sequencing of 25 genes frequently mutated in myeloid disorders: ASXL1, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, LNK, MPL, NPM1, NRAS, RUNX1, SF3B1, SRSF2, TP53, TET2, U2AF1, UTX, WT, and ZRSR2. Ph-negative clones were analyzed in 14 CML patients who developed CCA in Ph-negative cells (trisomy 8, n=10; nonrecurrent reciprocal translocations, n=2; monosomy 7, n=2). At time of analysis, patients were in CCyR (n=7), partial CyR (n=6), or minimal CyR (n=1) after a median of 21 months (range 7-114 months) of TKI treatment with imatinib, and/or nilotinib, and/or dasatinib. After exclusion of known polymorphisms, mutations were detected in 6/14 patients (43%) affecting the genes DNMT3A, EZH2, RUNX1, TET2, TP53, U2AF1, and ZRSR2. These patients were in CCyR (n=2), partial CyR (n=3), or minimal CyR (n=1), respectively, indicating BCR-ABL independent mutations in both Ph-negative and Ph-positive subclones. In two patients, the mutations were also found in corresponding diagnostic samples at higher or lower mutation level. To further investigate BCR-ABL independent gene mutations in Ph-positive clones, 15 randomly selected CML patients at diagnosis were analyzed. Mutations additional to BCR-ABL were found in 5/15 CML patients (33%) affecting ASXL1, DNMT3A, RUNX1, and TET2. None of the mutations were recognized in corresponding constitutional DNA specimens indicating that all mutations had been somatically acquired. Deep-sequencing of subsequent samples obtained in early CCyR after three months of TKI treatment revealed one DNMT3A mutation in Ph-negative cells which was also present in Ph-positive cells at diagnosis. Follow-up investigation showed that the mutation persisted in Ph-negative cells throughout CCyR and deep molecular remission (MR4.5) up to month 36 thereby not significantly changing its mutation level of approximately 15% implying a clonal hematopoiesis before the acquisition of the BCR-ABL rearrangement. In summary, BCR-ABL independent gene mutations were frequently found in Ph-negative and Ph-positive clones of CML patients and may be considered as important cofactors in the evolution of CML. Additional mutations acquired in the Ph-positive clone may impact on response to TKI treatment. Mutations preexisting to the occurrence of the BCR-ABL rearrangement may predispose patients to secondary hematological neoplasms. Our findings provide novel genetic information regarding CML biology and warrant further studies and impact on the design and performance of discontinuation trials. DisclosuresSchnittger:MLL Munich Leukemia Laboratory: Other. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; ARIAD: Honoraria, Research Funding; Pfizer: Consultancy, Research Funding.

Molecular-defined clonal evolution in patients with chronic myeloid leukemia independent of the BCR-ABL status.

To study clonal evolution in chronic myeloid leukemia (CML), we searched for BCR-ABL-independent gene mutations in both Philadelphia chromosome (Ph)-negative and Ph-positive clones in 29 chronic-phase CML patients by targeted deep sequencing of 25 genes frequently mutated in myeloid disorders. Ph-negative clones were analyzed in 14 patients who developed clonal cytogenetic abnormalities in Ph-negative cells during treatment with tyrosine kinase inhibitors (TKI). Mutations were detected in 6/14 patients (43%) affecting the genes DNMT3A, EZH2, RUNX1, TET2, TP53, U2AF1 and ZRSR2. In two patients, the mutations were also found in corresponding Ph-positive diagnostic samples. To further investigate Ph-positive clones, 15 randomly selected CML patients at diagnosis were analyzed. Somatic mutations additional to BCR-ABL were found in 5/15 patients (33%) affecting ASXL1, DNMT3A, RUNX1 and TET2. Analysis of individual hematopoietic colonies at diagnosis revealed that most mutations were part of the Ph-positive clone. In contrast, deep sequencing of subsequent samples during TKI treatment revealed one DNMT3A mutation in Ph-negative cells that was also present in Ph-positive cells at diagnosis, implying that the mutation preceded the BCR-ABL rearrangement. In summary, BCR-ABL-independent gene mutations were frequently found in Ph-negative and Ph-positive clones of CML patients and may be considered as important cofactors in the clonal evolution of CML.

5q- Syndrome in Transformation to the Philadelphia Chromosome Positive Acute Myeloblastic Leukemia

Background: A rare 5q- syndrome subtype (OMIM:153550) of the myelodysplastic syndrome (MDS) evolved to the Philadelphia (Ph) chromosome positive acute myeloblastic leukemia (AML) is presented. The MDS is an acquired clonal stem cell disorder frequently associated with a variety of chromosomal abnormalities. The most common karyotype abnormalities in MDS are del(5q), -7 and +8. The t(9;22)(q34;q11) results in the formation of the Ph chromosome and generates an active chimerical BCR-ABL tyrosine kinase most commonly associated with chronic myelogenous leukemia (CML) and adult precursor B-acute lymphoblastic leukemia (B-ALL). The Ph chromosome positive MDS and AML are uncommon with only 1-2% of newly diagnosed cases. Case report: We present a 72-year old woman with MDS. The HG-banding technique applied on unstimulated bone marrow cells showed 46,XX,del(5)(q13q33)[10]/46,XX. Ten months later, the white blood cell (WBC) count increased to 72×109/l, with 60% of myeloblasts in the formula. The bone marrow aspirate was hypercellular with 50% blasts. The cytogenetic study demonstrated the presence of Ph chromosome together with the initial del(5q) in the same cellular clone. Chemotherapy with hydroxyurea and low-dose cytosine arabinoside was followed by an increase in WBC count to 98×109/l. The dosage of cytosine arabinoside was escalated to 100 mg/m2, for 7 days resulting in a drop in the WBC count to 12×109/l. The follow-up bone marrow aspirations showed a further progression of disease with >70% of myeloblasts in the myelogram. The patient lived 10 months after the initial diagnosis of MDS was made and another 4 months after the diagnosis of secondary AML. Conclusion: The karyotype evolution from the solely 5q- to the 5q- and Ph-positive clone in the MDS in transformation to secondary AML (sAML) is a rare event. To our knowledge, this is a second such case reported in the literature. Based on current literature, we discuss possible links between the 5q- syndrome, BCR/ABL-negative chronic myeloproliferative neoplasms and the BCR/ABL-positive leukemias.

A significant proportion of patients with chronic myeloid leukemia and suboptimal response according to European Leukemia Net criteria have excellent prognosis without treatment change

The Recommendations of the European Leukemia Net (ELN) have become an essential tool in the management and prognosis of patients with chronic myeloid leukemia (CML) treated with tyrosine kinase inhibitors (TKIs). However, the definition of suboptimal response remains under discussion. We used conventional cytogenetics for the detection of clonal changes in Ph-positive and negative clones. RT-PCR and sequencing were carried out on peripheral blood leukocytes to detect the type of BCR-ABL1 transcript. The BCR-ABL1 mutational status was assessed using sequencing of RT-PCR products. High performance capillary electrophoresis for determination of imatinib (IMA) plasma concentration was used. A retrospective study of 110 patients diagnosed with chronic-phase (CP) CML treated with IMA or 2(nd) generation TKIs in the years 2000-2009 focused on analysis of patients with suboptimal response according to ELN criteria. 40 patients were administered IMA as first-line therapy and 70 had been pretreated with interferon-alpha (IFN-α) with or without Ara-C and/or hydroxyurea (HU) for a median 12 months (range, 1-92 months). After adjusting for the ELN criteria, major molecular response (MMR) was achieved after median 34 and 39 months in 66.7% and 41.7% of patients after the first and second-line IMA therapy with suboptimal response defined as lack of achievement of MMR at the 18(th) month of treatment, respectively. In comparison to patients with optimal response, patients with suboptimal response did not show significant differences in overall survival (OS) or progression-free survival (PFS). Cytogenetic assays demonstrated additional chromosome abnormalities (ACAs): chromosome 8 trisomy in a Ph-negative clone during the IMA treatment (in 1 case) and der(9q) in Ph-positive clone (in 2 cases); in patients receiving first-line IMA only chromosome 8 trisomy was observed which was associated with myelodysplastic syndrome - this was the only case where hematopoietic stem cell transplantation (HSCT) was performed. During the treatment with IMA in both subgroups no regulatory mutations in the ABL kinase domain were confirmed. We believe that the category of suboptimal response should be redefined or withdrawn from the ELN 2009 recommendations for management of CML patients treated with TKIs. Patients with suboptimal response who have no additional risks (additional cytogenetic abnormalities or BCR-ABL1 regulatory mutations) may remain on IMA treatment while patients with these risks should be switched to the 2(nd) generation TKIs.

Development of t(8;21) and RUNX1–RUNX1T1 in the Philadelphia-positive clone of a patient with chronic myelogenous leukemia: additional evidence for multiple steps involved in disease progression

A 65-year-old patient with a high hemoglobin and hematocrit was treated for 14 months with therapeutic phlebotomy when cytogenetics of bone marrow revealed 100% cells with the Ph chromosome and 45% of the Ph+ cells contained trisomy 8. Treatment with tyrosine kinase inhibitors did not reduce the BCR-ABL1 fusion positive clone. Instead, the Ph positive cells acquired further the t(8;21)/RUNX1-RUNX1T1, del(4q) and trisomy 15 chromosomal abnormalities which were resistant to further treatment. Literature review revealed eight other patients who either had t(9;22) and t(8;21) simultaneously or developed t(8;21) in the Ph positive clone. We conclude that there are rare patients with CML who either present in blast crisis with coexistence of t(9;22) and t(8;21) with or without +8, or progress to blast crisis with acquiring RUNX1-RUNX1T1 in the BCR-ABL1 clone which may or may not be therapy related and represent a later event in a multistep pathogenesis.

Demonstration of Ph1-negative host haemopoietic progenitor cells in an allogeneic marrow transplant recipient with chronic myelocytic leukaemia using polymerase chain reaction.

In an attempt to characterize host-derived haemopoietic cells in mixed haemopoietic chimeras, we studied bone marrow cells from male patients with chronic myelocytic leukaemia (CML) transplanted with marrow grafts from female donors. Amplification of a Y chromosome-specific sequence (YDNA) in DNA from marrow mononuclear cells using polymerase chain reaction (PCR) revealed persistence of host cells in 2 of 3 patients studied. On the other hand, persistence of Ph1-positive cells was unable to be demonstrated in the marrow cells from these patients using reverse transcription PCR (RT-PCR) for detecting bcr-abl chimeric messenger RNA. RT-PCR sensitivity for detecting minimal Ph1-positive cells in a background of Ph1-negative cells proved better than that of the PCR for detecting male cells among female cells. When bone marrow progenitor cells from one of the documented mixed chimeras were analyzed after an in vitro colony assay using PCR, 2 of 12 erythroid burst-forming units (BFU-E) proved to be YDNA-positive, i.e., of host origin, whereas none of them was shown to be Ph1-positive, although 12 BFU-E analyzed in the marrow cells obtained pretransplant from the same patient were all YDNA- and Ph1-positive. These findings indicate that, in some marrow transplant recipients with CML, a small number of host-derived normal haemopoietic progenitor cells may persist following lethal chemoradiotherapy and allogeneic bone marrow transplantation despite the fact that Ph1-positive clones can be eradicated.

Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Showing Complete Molecular Response Following Imatinib (Glivec®) - combined chemotherapy

The Philadelphia (Ph) chromosome is the result of a reciprocal translocation between chromosomes 9 and 22 [t (9; 22)] and is characterized at the molecular level by expression of the BCR-ABL fusion gene. It is the most frequent genetic aberration in adult acute lymphoblastic leukemia (ALL), being detectable in 30–40%of patients with B-precursor ALL. In our initial work we report two cases of grossly negative (Ph) ALL, that showed a complete molecular response after an imatinib based chemotherapy.In the first case we report a 17-year-old male who was admitted because of bony aches and fever. A diagnosis of acute lymphoblastic leukemia was made on the basis of a 95% infiltration of leukemic cells in his bone marrow. Standard G-banding chromosome analysis of bone marrow cells revealed a normal karyotype. And subsequent fluorescent in situ hybridization (FISH) examination showed a 20% positivity for the Philadelphia chromosome.As for the second case we report a 19 years old male patient who was presented with complaints of recurrent epistaxis and bony aches. He was also found to have acute lymphoblastic leukemia due to bone marrow infiltration with 55% blasts that showed a normal karyotype using the G- banding, and a positive Philadelphia chromosome-using FISH method - in 31 % of the examined cells.Both patients received combination chemotherapy incorporating imatinib mesylate; Glivec® in a dose of 400 mg/day continuously all through the induction, consolidation and subsequent re-enforcement courses. They both achieved complete hematological, cytogenetic as well as molecular remission that was maintained-thus far- for a total period of 20 and 11 months respectively. We postulated that there might have been two clones, both a Ph-positive clone and Ph-negative clone, with subsequent sensitivity of this Ph positive clone to the incorporated targeted treatment, as was evidenced by disappearance of the Ph-positive cells in subsequent FISH analysis and the negativity of the real time quantitative polymerase chain reaction (RT-QPCR) for BCR/ABL gene.Several investigators have studied the role of Hematopoeitic stem cell transplantation (HSCT) in the context of Ph+ ALL patients, which showed superior results when compared to conventional chemotherapy in means of remission induction as well as relapse rates, Dombret et al.2002. The highly successful introduction of the first ABL-kinase inhibitor imatinib into the treatment of Philadelphia chromosome positive chronic myeloid leukemia (CML) has led to clinical testing of imatinib in Ph+ALL. It has recently been approved for Ph+ALL in Europe and Japan. Given the much more aggressive nature of Ph+ALL when compared with chronic or even accelerated phase CML, a variety of imatinib-based treatment strategies have been explored in ALL(Ottman et al.,2007). Yet thus far the exploration of these treatment modalities in the very rare entity of gross normal karyotype and molecular level cytogenetic aberrations remains to be elucidated.Conclusion: Molecular remission on tyrosine kinase inhibitors in patient with ALL Ph +ve may open the door to revising the treatment strategies offered thus far for that subset of patients, as a new stratifying factor. Our work offers a unique protocol for utilizing imatinib as an integral part of the chemotherapy protocols normally offered for these patients, but yet at a lower dose, and raises the question of whether they should be transplanted in accordance with the current guidelines and if so when? More patients experience and controlled randomized trials are needed and eagerly awaited.

Simultaneous occurrence of t(9;22)(q34;q11.2) and t(16;16)(p13;q22) in a patient with chronic myeloid leukemia in blastic phase

Coexistence of two specific chromosomal translocations in the same clone is an infrequent phenomenon and has only rarely been reported in hematological malignancies. We report a combination of t(16;16)(p13;q22), the Philadelphia translocation t(9;22)(q34;q11.2), and deletion of the long arm of chromosome 7 in a patient with chronic myeloid leukemia in blast phase. Monotherapy treatment with imatinib mesylate resulted in the disappearance of the Ph-positive clone, but with persistence of t(16;16) and del(7) in all of the metaphases examined. The case illustrates that, although imatinib mesylate can be an effective treatment in eradication of the BCR–ABL fusion gene cells, the occurrence of additional specific abnormalities in Philadelphia-positive leukemias may pose a significant therapeutic challenge.

In vivo kinetics of kinase domain mutations in CML patients treated with dasatinib after failing imatinib

We sought kinase domain (KD) mutations at the start of treatment with dasatinib in 46 chronic myeloid leukemia (CML) patients resistant to or intolerant of imatinib. We identified BCR-ABL mutant subclones in 12 (26%) cases and used pyrosequencing to estimate subsequent changes in their relative size after starting dasatinib. Four patients lost their mutations, which remained undetectable, 3 patients retained the original mutation or lost it only transiently, 3 lost their original mutations but acquired a new mutation (F317L), and 2 developed another mutation (T315I) in addition to the original mutation within the same subclone. This study shows that expansion of a mutant Ph-positive clone that responds initially to a second generation tyrosine kinase inhibitor may be due either to late acquisition of a second mutation in the originally mutated clone, such as the T315I, or to acquisition of a completely new mutant clone, such as F317L.

Autologous Stem Cell Transplantation in Chronic Myeloid Leukemia

Autografting was first attempted for patients with chronic myeloid leukemia (CML) in transformation in order to restore a second chronic phase (CP). The principal rationale for autografting in CP resides on the reduction of the tumor burden and the number of leukemic cells at risk of developing blastic transformation, and the possibility of eradicating already mutated cells. In a European Group for Blood and Marrow Transplantation (EBMT) Registry survey, patients with CML in CP, undergoing autologous stem cell transplantation (auto-SCT) for the first time, had an overall survival of 65% at 5 years from transplant with more than 50% of all patients remaining in CP. The main point made by this retrospective study was that in patients refractory to interferon-alpha (IFN), 70% achieved a cytogenetic response post autografting, which was complete or major in 31%. Since the advent of imatinib, autografting in CML has experienced a substantial decline. Theoretically, there are several possible ways of using auto-SCT in combination with imatinib: (1) to reverse resistance to imatinib; (2) to eliminate a Ph-positive clone bearing a BCR-ABL kinase domain mutation; and (3) to reduce the level of residual disease after a cytogenetic response to imatinib in patients in whom Ph-negative cells had been harvested. The exact role of auto-SCT in the present management of CML remains unanswered.

Characterization of Ph‐negative abnormal clones emerging during imatinib therapy

Imatinib is a tyrosine kinase-specific inhibitor widely used for the treatment of chronic myeloid leukemia (CML). Studies reported the occurrence of additional cytogenetic abnormalities in the Philadelphia chromosome (Ph)-negative cell population emerging after treatment-induced suppression of the Ph-positive clone. These abnormalities were described in a relatively high proportion of patients treated with imatinib compared with the anecdotal reports of similar cases in patients treated with other drugs. However, the origin of these abnormalities as well as their biological and clinical significance are unknown. The study involved 13 cases of patients diagnosed with CML carrying cytogenetic abnormalities in their Ph-negative cell population after imatinib treatment. The presence of the markers within the CD34+ stem cell compartment and the cell culture growth were analyzed and patients were followed over time. CD34+ cells express the cytogenetic markers present in Ph- cells, suggesting a possible involvement of the stem cell population. Cultured cells showed normal growth in all but 1 patient. No growth advantage was demonstrated for the Ph-negative or the Ph-positive clone after cell culture. After follow-up of up to 49 months, none of the patients had evolved to myelodysplasia or acute leukemia. Hypothesis regarding the biological and clinical significance of these abnormalities are formulated.

Dynamics of cytogenetic aberrations in Philadelphia chromosome positive and negative hematopoiesis during dasatinib therapy of chronic myeloid leukemia patients after imatinib failure

Clonal cytogenetic aberrations of the Philadelphia chromosome (Ph) positive hematopoiesis have been associated with the natural evolution of chronic myeloid leukemia (CML) to advanced disease. Clonal aberrations of Ph negative metaphases have been described after treatment with interferon or imatinib. This study evaluates the effect of dasatinib on Ph positive clones with additional cytogenetic aberrations and the frequency of novel aberrations in Ph positive and negative metaphases. Seventy-one patients treated with dasatinib after imatinib failure for a median of nine months were evaluated. Novel aberrations within Ph positive and negative clones appeared in six and three patients, respectively.

Dynamics of Cytogenetic Aberrations in Philadelphia Chromosome Positive and Negative Hematopoiesis during Dasatinib Therapy of Chronic Myeloid Leukemia Patients after Imatinib Failure.

Clonal cytogenetic aberrations of the Philadelphia chromosome (Ph) positive hematopoiesis have been associated with the natural evolution of CML to advanced disease. Clonal aberrations of Ph negative metaphases have been described after treatment with interferon alpha or imatinib in a minority of patients (pts) with cytogenetic response. Conflicting data suggest selection of preexisting clones vs. induction of aneuploidy by tyrosine kinase inhibitors. The prognostic impact of aberrations in the Ph negative hematopoiesis for the individual pts remains to be determined. Dasatinib is a multitargeted agent inhibiting both ABL and SRC tyrosine kinases. The efficacy and safety of dasatinib has been demonstrated in phase I and II studies in pts with Ph positive CML after failure of imatinib therapy. We sought to evaluate the effect of dasatinib on Ph positive clones with additional cytogenetic aberrations and the frequency of novel aberrations in Ph positive and Ph negative metaphases during dasatinib therapy. 71 pts (40 m, 31 f) with Ph positive CML after failure of imatinib therapy have been evaluated. Median age was 58 years (range, 28–78), median time from diagnosis 73 months (range, 14–231). Pts were in chronic phase (CP, n=50), accelerated phase (AP, n=6), myeloid (n=8) or lymphoid (n=7) blast crisis (BC). Pretreatment consisted of imatinib (n=71), hydroxyurea (n=48), interferon alpha (n=49), cytosine arabinoside (n=11) and nilotinib (n=2). Dasatinib therapy was commenced at a dose of 100–140 mg/day, median duration of dasatinib therapy was 9 months, (range, 1–16). Prior to dasatinib therapy, 22 pts (31%) demonstrated additional chromosomal aberrations in the Ph positive clone indicating BCR-ABL independent mechanisms of resistance. Of these, 8 pts were in CP (16%), 5 in AP (83%), and 9 in BC (60%). In 35 pts (49%), BCR-ABL mutations associated indicating a BCR-ABL dependent mechanism of resistance were observed. 9 pts (13%) showed both clonal evolution and BCR-ABL mutations. Two pts (3%) had trisomy 8 as an aberration of the Ph negative clone at baseline. During dasatinib therapy, 33 pts (46%) achieved major cytogenetic remission, 26 (37%) being complete. From pts with clonal evolution, 3 (14%) achieved a major with 2 (9%) complete cytogenetic remissions. Novel aberrations of the Ph positive clone appeared during dasatinib therapy in 6 pts (8%), aberrations of the Ph negative clone in 3 pts (4%). In total, 5/71 pts (7%) showed clonal aberrations of Ph negative metaphases after consecutive imatinib/dasatinib therapies. None of these pts had morphological indications for secondary neoplastic changes, like myelodysplasia or acute leukemia. In conclusion, dasatinib was efficacious in pts with clonal cytogenetic aberrations as a marker of BCR-ABL independent imatinib resistance. However, cytogenetic response was delayed compared to pts without additional aberrations. Preexisting clonal aberrations of the Ph negative hematopoiesis were uncovered by a simultaneous gradual elimination of the Ph positive clone. In addition, a minority of pts demonstrated novel aberrations in Ph negative cells. The prognostic significance of aneuploidy of the Ph negative hematopoiesis remains to be evaluated by long term observation.

Long Term Follow up on Patients with Ph-Abnormal Clones Emerged during Imatinib Therapy.

Imatinib (Glivec, Novartis) is a tyrosine kinase specific inhibitor used for the treatment of CML. The occurrence of cytogenetic abnormalities in Ph-negative cells emerging after suppression of the Ph-positive clone has been described; however the origin as well as the biological and clinical significance are unknown. We collected data on 32 patients through the GWP in CML registry. Bone marrow cell segregation, cell culture and morphologic features in a subgroup of these patients were studied to acquire insights into the origin of the Ph-negative clone. Patient characteristics and clinical follow up (up to 49 months) are presented together with hypothesis regarding their biological significance. The emergence of a cytogenetic abnormal clone in Ph-negative cells was evidenced in 32 patients after a median of 16.2 months after starting Imatinib. Median age was 51 years, F:M=18:14, median time from CML diagnosis 35 months. All patients but one have started Imatinib while in chronic phase and were in chronic phase at detection of the abnormal Ph-negative clone. Eight patients were treated with Imatinb at onset. At diagnosis no additional abnormalities were evidenced except for one patient which presented with the Ph and a dup(1q)(q11q21). All patients achieved a good response to Glivec with 21 complete, 5 major and 6 minor cytogenetic remissions when additional abnormalities were noticed in Ph-negative cells. The clonal cytogenetic abnormalities included +8 in 14 patients, −Y in 5 patients, three del(20q), two del(5q) and del(7q), one −7, del(13q), t(6;7)(p24;q21), t(2;6)(p25;q23), with one patient presenting with both +8 and +21, and one three clones with +8, double +8, and double +8 and −X. Retrospective analyses of stored pellet using FISH did not evidence abnormalities in previous samples. Patients that lost cytogenetic response showed that the percentage of the Ph+ cells inversely correlated to the abnormal clone. In 7 patients the abnormal clone was not evidenced in subsequent controls, suggesting the possibility that the abnormalities could be temporary. Two patients that lost response to Glivec were treated with a second generation tyrosine kinase inhibitor Dasatinib (Sprycel, Bristol-Myers Squibb) and, at reduction of Ph+ clone, the del (7q) reappeared in one patient, while the +8 clone of the second patient was not further evidenced. FISH analyses on separated CD34+ and CD34-negative cells evidenced that the abnormal clone was present into the CD34+ compartment suggesting the stem cells involvement. Bone marrow biopsies presented with reduced cellularity, normal differential and mild dysplastic signs as documented in patients responding to Imatinib. No increased angiogenesis was evidenced. We performed cell culture on a subgroup of 6 patients demonstrating normal growth in five patients and an abnormal growth pattern in one patient with reduced CFU formation affecting BFU-Es, CFU-GM, and colony size microclusters. While a longer follow up observation and laboratory analyses are required, we remark that after >4 years follow up the Ph-negative abnormal clone did not tend in our patients to evolve in MDS/AML, nor it seems to be associated with CML clonal evolution and disease progression.

Philadelphia chromosome positive acute lymphoblastic leukemia showing normal karyotype in G-banding chromosomal examination before chemotherapy

A 29-year-old male was admitted because of thrombocytopenia. A diagnosis of acute lymphoblastic leukaemia was made on the basis of a 61.6% infiltration of leukemic cells in his bone marrow. Standard G-binding chromosome analysis of bone marrow cells revealed a normal karyotype. He received combination chemotherapy, and achieved hematological complete remission. However, chromosomal analysis of bone marrow cells after 2 courses of consolidation therapy showed the Philadelphia (Ph) chromosome in two cells out of 20 analysed. We retrospectively examined the sample of bone marrow cells before chemotherapy; It showed minor BCR/ABL positivity with FISH and RT-PCR methods. The Ph chromosome disappeared after consolidation chemotherapy and allogeneic bone marrow transplantation, but the Ph chromosome reappeared at relapse. We postulated that there were two clones, both a Ph-positive clone and Ph-negative clone. At the initial diagnosis, Ph chromosome was not detected because the G-banding method analyzed only metaphase cells, which contained few Ph-positive clones. In order to offer effective therapy with molecular targeting agents, in this poor prognostic disease, it is necessary to detect Ph chromosome before the first chemotherapy and BCR/ABL detection with FISH or RT-PCR methods appears more useful than G-banding chromosome analysis.

Extramedullary Blast Crisis Derived from 2 Different Clones in the Central Nervous System and Neck during Complete Cytogenetic Remission of Chronic Myelogenous Leukemia Treated with Imatinib Mesylate

We describe a patient with Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML) who developed an extramedullary blast crisis in the central nervous system (CNS) and then a subcutaneous tumor of the neck during treatment with imatinib mesylate. Administered 400 mg of imatinib mesylate after the diagnosis of chronic-phase CML, the patient achieved a complete cytogenetic remission 4 months later. However, he developed a mixed myeloid/B-cell blast crisis with additional karyotype abnormalities only in the CNS during a complete cytogenetic remission in the bone marrow. Several doses of intrathecal chemotherapy and whole-brain irradiation were effective in treating the blast crisis in the CNS. After 7 months of complete cytogenetic remission, the patient experienced a subcutaneous tumor in the right neck. A biopsy of the tumor revealed a mixed myeloid/T-cell blast crisis. The cytogenetic analysis showed that the blast crisis clone in the neck tumor was different from that of the CNS. An increased dose of imatinib mesylate was ineffective in treating the neck tumor. Irradiation to the right neck was therefore undertaken. This case suggests that the development of a clone resistant to imatinib mesylate is not always detected in the bone marrow and that multiple Ph-positive clones have the potential to become transformed into a blast crisis.

Reciprocal Relationship Between a Ph‐Negative Clone With Trisomy 8 Associated With Severe Myelodysplasia and a Ph‐Positive Clone Following Imatinib Treatment in a Patient With Accelerated‐Phase Chronic Myelogenous Leukemia (CML)

American Journal of HematologyVolume 77, Issue 4 p. 420-420 Letters and CorrespondenceFree Access Reciprocal Relationship Between a Ph-Negative Clone With Trisomy 8 Associated With Severe Myelodysplasia and a Ph-Positive Clone Following Imatinib Treatment in a Patient With Accelerated-Phase Chronic Myelogenous Leukemia (CML) Paulina Patchenko, Paulina Patchenko Institute of Hematology, E. Wolfson Medical Center, Holon, IsraelSearch for more papers by this authorAbraham Klepfish, Abraham Klepfish Institute of Hematology, E. Wolfson Medical Center, Holon, IsraelSearch for more papers by this authorLuba Trakhtenbrot, Luba Trakhtenbrot Institute of Hematology, Sheba Medical Center, Tel-Hashomer, IsraelSearch for more papers by this authorRachel Rothman, Rachel Rothman Institute of Hematology, Sheba Medical Center, Tel-Hashomer, IsraelSearch for more papers by this authorEliezer A Rachmilewitz, Eliezer A Rachmilewitz Institute of Hematology, E. Wolfson Medical Center, Holon, IsraelSearch for more papers by this author Paulina Patchenko, Paulina Patchenko Institute of Hematology, E. Wolfson Medical Center, Holon, IsraelSearch for more papers by this authorAbraham Klepfish, Abraham Klepfish Institute of Hematology, E. Wolfson Medical Center, Holon, IsraelSearch for more papers by this authorLuba Trakhtenbrot, Luba Trakhtenbrot Institute of Hematology, Sheba Medical Center, Tel-Hashomer, IsraelSearch for more papers by this authorRachel Rothman, Rachel Rothman Institute of Hematology, Sheba Medical Center, Tel-Hashomer, IsraelSearch for more papers by this authorEliezer A Rachmilewitz, Eliezer A Rachmilewitz Institute of Hematology, E. Wolfson Medical Center, Holon, IsraelSearch for more papers by this author First published: 22 November 2004 https://doi.org/10.1002/ajh.20226Citations: 6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat REFERENCES 1O'Brien SG, Guilhot F, Larson RA, et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2003; 348: 994– 1004. 2Braziel RM, Launder TM, Druker BJ, et al. Hematopathologic and cytogenetic findings in imatinib mesylate-treated chronic myelogenous leukemia patients; 14 months' experience. Blood 2002; 100: 435– 441. 3Bumm T, Müller C, Al-Ali HK, et al. Emergence of clonal cytogenetic abnormalities in Ph− cells in some CML patients in cytogenetic remission to imatinib restoration of polyclonal hematopoiesis in the majority. Blood 2003; 101: 1941– 1949. 4Royer-Pokora B, Hildebrandt B, Redmann A, et al. Simultaneous occurrence of a t(9;22) (Ph) with a t(2;11) in a patient with CML and emergence of a new clone with the t(2;11) alone after imatinib mesylate treatment. Leukemia 2003; 17: 807– 810. 5Andersen MK. Clonal Ph-negative hematopoiesis in CML after therapy with imatinib mesylate is frequently characterized by trisomy 8. Spotlight correspondence. Leukemia 2002; 16: 1390– 1395. Citing Literature Volume77, Issue4December 2004Pages 420-420 ReferencesRelatedInformation

Results of Conventional Cytogenetics and Interphase FISH (I-FISH) Analyses in Patients with a Clinical and Morphologic Diagnosis of CML: Analysis of 52 Cases.

The presence of the Philadelphia chromosome (Ph), derived from a balanced translocation between chromosomes 9q and 22q, serves as a hallmark for the diagnosis of CML or Ph-positive ALL. In recent years, I-FISH has become the method of choice to detect Ph-positive clones at diagnosis and during treatment. In addition, I-FISH analysis is also able to identify variant translocations and loss of DNA sequences, some of which are known to be predictors of poor clinical outcome. To determine the contribution of I-FISH in detecting genetic alterations at diagnosis and during therapy, we summarized our experience in a series of patients with CML and Ph+ ALL studied in a single institution between 2000–2004. A total of 52 patients had both cytogenetic and I-FISH analyses available for review. The 52 patients included 30 patients in chronic phase, 10 patients in accelerated phase, 9 patients in blastic phase, and 3 patients with acute lymphoblastic leukemia (ALL). Sixteen of the 52 patients had newly diagnosed CML, and the remaining 36 patients had studies performed while on imatinib. Conventional cytogenetics detected the Ph-chromosome in 44 patients (85%), whereas I-FISH detected Ph chromosome positivity in all 52 patients (100%). In three of the 16 patients with newly diagnosed disease, I-FISH identified variant translocations in the setting of a normal karyotype. Two of these 3 patients were found to be RT-PCR negative for bcr/abl transcripts. Nineteen of 52 patients (37%) had additional abnormalities detected by karyotype. Nine of these 19 patients (47%) had more than one additional cytogenetic abnormality. The most common abnormalities in the Ph+ clone included: 6 patients with 2 copies of the Ph chromosome, 4 patients with trisomy 21, 2 patients with deletion (20q), 2 patients with isochromosome 17, and 2 patients with trisomy 8. Independent clones with trisomy 8 were noted in an additional 2 patients on imatinib therapy. A total of 12 of the 52 patients (23%) had variant signal patterns by I-FISH; loss of DNA sequences on chromosomes 9 and 22 occurred in 4 and 3 cases respectively and a variant signal pattern involving additional chromosomes was seen in 5 patients. While in general we found a good correlation between the proportion of Ph-positive cells detected by conventional cytogenetics and I-FISH, differences between the two types of analyses were observed in 8 patients. Six patients had a normal karyotype (20 cells analyzed) with positive I-FISH analysis (range 3.2–99%). Two of the 8 patients had a small percentage of Ph-positive metaphases with negative I-FISH. These results underscore the significance of utilizing both conventional cytogenetics and I-FISH in CML patients at diagnosis as well as during therapy. The biologic and clinical implications of the disparity between conventional cytogenetics, I-FISH, and in some cases RT-PCR results are not yet fully realized.

Interphase FISH for Y Chromosome, VNTR Polymorphisms, and RT-PCR for BCR-ABL in the Monitoring of HLA-Matched and Mismatched Transplants

Thirty-six sex-mismatched transplants were studied using fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) methods. Molecular cytogenetics was performed using interphase FISH with a centromeric probe for chromosome Y, and PCR amplification was performed with a set of VNTR microsatellite loci. In addition, reverse transcriptase-PCR (RT-PCR) for BCR-ABL fusion was used to investigate cases of Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). Our integrated approach of post-transplant monitoring was helpful in documenting successful transplants and in controlling the size of Ph-positive clones in CML. A striking overlap was found between results from FISH analysis and PCR for polymorphic loci.