P210 Fusion Protein Research Articles

Achievement of Undetectable BCR::ABL1 (uBCR::ABL1) Is Predictive of Improved Survival in Philadelphia Chromosome Positive (Ph+ve) Acute Lymphoblastic Leukemia (ALL) Patients Not Receiving Allogeneic Stem Cell Transplantation

Background With the advent of tyrosine kinase inhibitor (TKI)-based therapy the outcome of Philadelphia chromosome-positive (Ph +) acute lymphoblastic leukemia (ALL) has significantly improved. Moreover, patients (pts) who achieved undetectable (u) BCR::ABL1 at 3 months (mo) from the time of initiating therapy, tend to have improved outcomes (Short et al. Blood 2016; 128 [4];504-507). We conducted a retrospective multicenter study to analyze the predictors of achieving uBCR::ABL1 in adult (> 18 years) Ph + ALL pts. Methods A total of 458 adult Ph + ALL pts from 10 US academic institutions, who were diagnosed between May 2003 and April 2023, were evaluated to assess the rates of uBCR::ABL1 at 3 mo after initiating therapy and factors that predict the achievement of molecular response. Continuous variables were summarized as median (range), while categorical variables were reported as frequency (percentage). Unadjusted comparisons of patient/ treatment related characteristics and molecular response were made using a non-parametric test (continuous variables) or Fisher's exact test (categorical variables). Survival curves were estimated using the Kaplan-Meier method and compared between molecular responses via the log-rank test. Results Baseline characteristics The median age of the pts was 53 years (range [R] 19-85), median white blood cell count (WBC) was 17.6 (10 9/L) (R, 1.2-571), 249 (55%) pts had additional cytogenetic (CG) abnormalities, 98 (22%) pts had p210 fusion protein, and 35 (8%) pts had CNS disease at diagnosis. A higher proportion of pts received TKI in combination with intensive chemotherapy (IC) (69%), than TKI + steroids (15%), TKI + low intensity chemotherapy (LC; combination of vincristine, steroids +/- rituximab) (9%), TKI + blinatumomab (3%) and IC without TKI (4%) during induction. During induction, the highest proportion of pts received 2 nd generation TKI combinations (67%; majority dasatinib 98%), compared to imatinib (19%) or ponatinib (10%): 4% of pts did not receive TKI with induction. The complete remission (CR) rate was 94%; measurable residual disease (MRD) negative by flow cytometry 51% (181/352 evaluable pts) and 58.5% (209/354 evaluable pts) of pts had uBCR::ABL1 by PCR at 3 mo from induction. A total of 212 (46%) pts received allogeneic stem cell transplantation (allo-HCT) in CR1, among them 61% of pts received TKI maintenance post allo-HCT. Predictors of uBCR::ABL1 at 3 months Predictors for uBCR::ABL1 at 3 mo are summarized in Table 1. The proportion of pts with WBC > 100 (45% vs 60%, p= 0.04), p210 fusion protein (35% vs 85%, p= <0.001), additional CG abnormality with monosomy 7 (40% vs 61%, p= 0.03), additional somatic myeloid co-mutation (39% vs 61%, p= 0.09; 94 pts evaluable), received induction with steroid plus TKI (41% vs 63%, p= 0.002) or received imatinib based induction (47% vs 61%,p= 0.07) had inferior uBCR::ABL1 rates at 3 mo. Conversely pts who received ponatinib based induction had significantly better uBCR::ABL1 rates (79% vs 57%, p= 0.009). Survival The median relapse free survival (RFS) was 72.3 mo (95% CI; 50.1-94.4), RFS was numerically higher among pts who achieved uBCR::ABL1 at 3 mo but not statistically significant (84.2 mo vs 62.9 mo, p= 0.20). The median overall survival (OS) was 129.2 mo (95% CI; 81.2-177.2), OS was not significantly different among pts who achieved uBCR::ABL1 at 3 mo (129.2) versus no uBCR::ABL1 (not reached; 50% alive at 94 mo), p= 0.47. In sub-set analysis, among non-transplanted pts, RFS was not statistically significant between those who achieved uBCR::ABL1 vs those who did not (38.9 vs 22.0 mo, respectively p= 0.24). However, the median OS was significantly higher in pts who achieved uBCR::ABL1 at 3 mo compared to pts who did not achieve uBCR::ABL1 at 3 mo: 149.3 vs 77.5 mo, p= 0.04 ( Figure 1). Conclusion In this large multicenter study, we observed significantly inferior uBCR::ABL1 rates among pts with WBC > 100, p210 fusion protein, monosomy 7 CG abnormality, pts receiving induction with steroid plus TKI or imatinib based induction. Conversely, uBCR::ABL1 rates were significantly better in pts receiving ponatinib based induction therapy. Overall, we did not see significant impact of uBCR::ABL1 at 3 mo, on RFS or OS. It is likely subsequent therapies and allo-HCT abrogates the negative impact of not achieving uBCR::ABL1 at 3 mo. However, among non-transplanted pts achievement of uBCR::ABL1 at 3 mo showed a significantly improved OS.

HLA-B8 and HLA-A3 Coexpressed With HLA-B8 Are Associated With a Reduced Risk of the Development of Chronic Myeloid Leukemia

AbstractChronic myeloid leukemia (CML) is characterized by the chromosomal translocation t(9;22) resulting in the chimeric bcr-abl oncogene that encodes the P210 fusion protein, which contains a unique amino acid sequence. If peptides derived from the leukemia-specific part of P210 are expressed in HLA molecules on the cell membrane of leukemic cells, an immunological response may occur. Recent studies using synthetic peptides identical to the bcr-abl fusion region showed that some peptides are capable of binding to HLA-A3, -A11, and -B8 molecules. Cytotoxic T-cell responses have been induced against bcr-abl–derived synthetic peptides bound to HLA-A3 and -B8. We hypothesized that if antigen processing of the P210 fusion protein leads to presentation of peptides from the fusion region by major histocompatibility complex (MHC) molecules in vivo, this may be reflected in a diminished incidence of CML in individuals expressing HLA-A3, -A11, or -B8. Consequently, lower frequencies of these antigens would be expected in patients with CML compared with unaffected individuals. A case-control study and a meta-analysis were performed to test this hypothesis. The multicenter case-control study compared patients with CML from the data base of the European Group for Blood and Marrow Transplantation (EBMT) with unaffected individuals from the registry of Bone Marrow Donors Worldwide. Patients and controls were matched per country. The meta-analysis consisted of five studies reported in the literature. The multicenter case-control study consisting of 1,899 patients and 512,363 bone marrow donors as controls yielded odds ratios (ORs) of 0.90 (95% confidence interval [CI], 0.80 to 1.00) for HLA-A3, 1.16 (95% CI, 1.02 to 1.33) for HLA-A11, and an OR of 0.73 (95% CI, 0.65 to 0.82) for HLA-B8. Coexpression of HLA-A3 and HLA-B8 gave an OR of 0.51 (95% CI, 0.40 to 0.67). This can be translated in a protective effect of 27% for HLA-B8, 10% for HLA-A3, and 49% protection for the combination of HLA-A3 and HLA-B8. The meta-analysis comprising 463 CML patients and 4,912 controls showed a 29% risk reduction for individuals expressing HLA-B8 (OR of 0.71; 95% CI, 0.52 to 0.97), but an OR of 1.19 (95% CI, 0.90 to 1.56) for HLA-A3 and an OR of 1.09 (95% CI, 0.80 to 1.50) for HLA-A11. In conclusion, these results indicate that HLA-B8 expression, in particular when HLA-A3 is coexpressed, is associated with a diminished incidence of CML. A biological mechanism may be that presentation of bcr-abl breakpoint peptides in these HLA molecules can induce a protective immune response.

BCR-ABL fusion genes and laboratory findings in patients with chronic myeloid leukemia in northeast Iran.

Background:A specific chromosomal abnormality, the Philadelphia chromosome (BCR-ABL fusion), is present in all patients with chronic myeloid leukemia (CML). The b2a2 and b3a2 fusion mRNAs encode p210 fusion protein p210 and e1a2 encode p190. The aim of this study was to evaluate the frequency of BCR-ABL fusion transcript variants in Northeast of Iranian CML patients and to compare the laboratory results of our patients.Methods:This study was conducted in 85 peripheral blood and bone marrow samples of CML patients. Ribonucleic acid (RNA) was extracted by a commercial kit, RT- PCR for identifying BCR-ABL fusions was carried out by using designed primers and the PCR products were electrophoresed in agarose gels. Finally, statistical analysis was performed for variant frequency identification and their comparison was performed.Results: All patients examined were positive for BCR/ABL rearrangement. Fusion of b3a2 was detected in 53 (62.35%) patients, b2a2 in 25 (29.41), e1a2 in 1 (1.17%) and coexpression of b3a2 and e1a2 in 6 (7.05%) patients. There were significant differences between the mean age in patients with b3a2 positive ( 44.07 years) and in b3a2 negative group (50.35 years) however, no significant differences were seen between sex and b2a2 (P=0.61), b3a2 (P=0.79) and e1a2 (P=0.20).Conclusions:This study showed higher frequency b3a2 than b2a2 and e1a2 transcripts in CML patients in Northeast Iran and there was no association between e1a2 transcripts frequencies and monocytosis in peripheral blood.

BCR-ABL-specific CD4+ T-helper cells promote the priming of antigen-specific cytotoxic T cells via dendritic cells.

The advent of tyrosine kinase inhibitor (TKI) therapy markedly improved the outcome of patients with chronic-phase chronic myeloid leukemia (CML). However, the poor prognosis of patients with advanced-phase CML and the lifelong dependency on TKIs are remaining challenges; therefore, an effective therapeutic has been sought. The BCR-ABL p210 fusion protein's junction region represents a leukemia-specific neoantigen and is thus an attractive target for antigen-specific T-cell immunotherapy. BCR-ABL p210 fusion-region-specific CD4+ T-helper (Th) cells possess antileukemic potential, but their function remains unclear. In this study, we established a BCR-ABL p210 b3a2 fusion-region-specific CD4+ Th-cell clone (b3a2-specific Th clone) and examined its dendritic cell (DC)-mediated antileukemic potential. The b3a2-specific Th clone recognized the b3a2 peptide in the context of HLA-DRB1*09:01 and exhibited a Th1 profile. Activation of this clone through T-cell antigen receptor stimulation triggered DC maturation, as indicated by upregulated production of CD86 and IL-12p70 by DCs, which depended on CD40 ligation by CD40L expressed on b3a2-specific Th cells. Moreover, in the presence of HLA-A*24:02-restricted Wilms tumor 1 (WT1)235-243 peptide, DCs conditioned by b3a2-specific Th cells efficiently stimulated the primary expansion of WTI-specific cytotoxic T lymphocytes (CTLs). The expanded CTLs were cytotoxic toward WT1235-243-peptide-loaded HLA-A*24:02-positive cell lines and exerted a potent antileukemic effect in vivo. However, the b3a2-specific Th-clone-mediated antileukemic CTL responses were strongly inhibited by both TKIs and interferon-α. Our findings indicate a crucial role of b3a2-specific Th cells in leukemia antigen-specific CTL-mediated immunity and provide an experimental basis for establishing novel CML immunotherapies.

Nilotinib for the Frontline Treatment of Chronic Myeloid Leukemia Carrying the p230 Transcript: Dream or Reality?

OPINION article Front. Oncol., 03 February 2014Sec. Hematologic Malignancies https://doi.org/10.3389/fonc.2014.00017

BCR/ABL-specific CD8+ T cells can be detected from CML patients, but are only expanded from healthy donors.

The BCR/ABL p210 fusion protein has long been considered an ideal target antigen for the development of immunotherapeutic strategies in chronic myeloid leukaemia (CML) due to its central role in malignant transformation and to its unique novel amino acid sequence solely expressed in leukaemia cells. However, the feasibility to expand BCR-ABL-specific T cells remains still controversial. Using BCR/ABL peptide/MHC tetramers, significantly higher frequencies of tetramer positive cells were detected in the peripheral blood of HLA-A*0301 (mean 0.38%) and HLA-B*0801 (mean 0.28%) CML patients than in healthy donors (P = 0.0025 and 0.0026, respectively). However, following stimulation with autologous peptide-pulsed DCs, BCR/ABL-specific T cells were only expanded from some healthy donors, suggesting that CML patients may have a specific immune deficit with respect to the BCR/ABL antigen.

Basophilic leukemia in recurrent chronic myelogenous leukemia blast phase

A 31-year-old man with a four-year history of chronic myelogenous leukemia (CML) presents in his second blast phase. The patient’s initial diagnosis was made after routine analysis revealed a white blood cell count (WBC) of 353 10/L. Initial bone marrow biopsy results and cytogenetics were not available for review. The patient responded initially to hydroxyurea followed by imatinib mesylate, at 400 mg daily. A subsequent bone marrow was consistent with morphologic remission; results of cytogenetics and molecular studies were not available. After *3-years, a repeat bone marrow biopsy revealed myeloid blasts comprising 80% of cells consistent with blast phase of CML. Retrospective review of peripheral blood and bone marrow showed only rare (less than 1%) blasts containing granules resembling immature basophils. RT-PCR was positive for BCR-ABL transcript (p210 and p190), and chromosome analysis showed 47, XY, t(2;3)(p23;q29), +8, t(9,22)(q34; q11.2) in 20 of 20 metaphase spreads. Flow cytometric analysis revealed a CD45 positive blast population with moderate side light scatter that strongly expressed CD34, CD117, and CD7 as well as variably expressed CD13, CD33, CD11c, and CD2. Intracellular staining revealed that 1/3 of these cells were myeloperoxidase positive. The imatinib mesylate dose was increased to 800 mg daily but was not tolerated due to nausea. The imatinib mesylate was discontinued, and a second generation ABL kinase inhibitor was initiated. The patient responded to this drug for several months until severe abdominal pain lead to its discontinuation. Less than one week after discontinuation the patient was found to have a WBC of 17 10/L with 30% blasts in peripheral blood, and the tyrosine kinase inhibitor was restarted. Circulating blasts persisted, and the patient underwent induction chemotherapy with an eventual goal of allogeneic transplant. Following this treatment, blasts were no longer detected in peripheral blood, and the patient was thought to be in hematologic remission. After *3-months our patient presented with a WBC of 47 10/L and 76% blasts. These results are consistent with recurrent CML blast phase, though morphologic remission was not established by bone marrow biopsy and persistence of earlier blast crisis remains a possibility. Multiple blast populations were detected morphologically in peripheral blood (Images 1–3), and presence of metachromatic granules after toluidine blue stain indicated a subset (9% of blasts) of basophilic origin (Image 4); maturing basophils were also present (4% of WBC). The patient has since entered a research protocol and had response to a different second generation ABL kinase inhibitor. After 3-weeks of therapy, his WBC has decreased to 1.9 10/L, although occasional blasts (6%) persist. CML is a myeloproliferative disease of bone marrow derived stem cells that contain fusion of BCR on chromosome 22 with c-ABL on chromosome 9 (i.e. Philadelphia chromosome) and which characteristically produces the p210 fusion protein. The initial indolent chronic phase of CML is unstable, and the disease ultimately progresses to accelerated and blast phases. Since the Philadelphia chromosome can be detected in all hematopoietic lineages the blast phase may include neutrophilic, eosinophilic, monocytic, erythroid, megakaryocytic, and basophilic blasts in predominance or in any combination. In about 20–30% of patients the blast phase is due to proliferation of lymphoblasts, and rare patients may have both myeloid and lymphoid blast populations. Acute basophilic leukemia represents <1% of all cases of acute myelogenous leukemia (AML) and must be differentiated from the blast phase of CML, AML with maturation associated with 12p abnormalities or t(6;9), acute eosinophilic leukemia, and acute lymphoblastic leukemia with prominent coarse granules. The persistence of t(9,22), as in the described case, is strong evidence for a predominant basophilic blast population arising from abnormal CML stem cells.

Apoptotic Death of Bcr-Abl-Expressing Myeloid Progenitors in Response to the m-Tor Inhibitor RAD001 (Everolimus) Is Promoted by the Nuclear Import of c-Abl.

Nuclear accumulation of c-Abl protein in response to genotoxic damage addresses apoptotic cell death. The process, driven by the disruption of its binding to 14-3-3 scaffolding proteins, is independent from c-Abl catalytic state and conditional upon 14-3-3 phosphorylation by the c-jun N-terminal kinase (JNK) (Yoshida et al Nat Cell Biol 7,278,2005). Our work moved from the observation that the constitutive activation of Abl tyrosine kinase (TK) by juxtaposed Bcr sequences in p210 fusion protein Bcr-Abl precludes c-Abl nuclear import in response to gamma irradiation, advancing a role for residual c-Abl protein “loss of function” in the apoptosis-resistant phenotype of Chronic Myeloid Leukemia (CML) progenitors. In 32D cell clones transducing a temperature-conditional Bcr-Abl mutant, active p210 TK is associated with the overexpression of 14-3-3sigma mostly driven by transcriptional events involving histone H4 acetylation (but not methylation) status of the gene promoter. P210 TK inhibition in response to 24 hour exposure to imatinib mesylate (IM) is associated with an early downregulation of 14-3-3sigma followed by c-Abl nuclear import and cell commitment to apoptotic death. The two last events are further enhanced by complementary inhibition of the 14-3-3 binding site by R18 peptide, supporting that the negative impact of p210 TK on pro-apoptotic function of residual normal c-Abl arises from its effects on 14-3-3sigma. Previous studies proved that the activation of p38 MAP kinase contributes to IM effects in CML (Parmar et al, J Biol Chem 279,25345, 2004). Interestingly, p38 MAP kinase is also involved in tuberous sclerosis 2 gene protein (TSC2, also known as tuberin) phosphorylation and enhanced binding to 14-3-3 possibly promoting the activation of IM-compensatory signals, including m-Tor (Li et al, J Biol Chem 278,13663, 2003). Here we show that the m-Tor inhibitor RAD001 (everolimus; kindly provided by Novartis Pharma AG) at 100 nM concentration induces c-Abl nuclear import and apoptotic cell death in 32D cell clones expressing active p210 TK. Both events are further and significantly enhanced by combination with IM. C-Abl nuclear shuttling in response to the RAD001 and IM combination procedes from Akt and p38 MAP kinase inactivating dephosphorylation and 14-3-3 phosphorylation at serine residues critical for client protein binding including the apoptotic death effectors Bad, Bax and Ask1. In conclusion, our work supports the advantage of TK and m-Tor inhibitor association for CML treatment.

Effect of a p210 multipeptide vaccine associated with imatinib or interferon in patients with chronic myeloid leukaemia and persistent residual disease: a multicentre observational trial

Background Although imatinib is the standard treatment for chronic myeloid leukaemia, not all patients reach complete cytogenetic remission (CCR) and most maintain detectable disease at the molecular level. We investigated whether a vaccine targeting the BCR-ABL-derived p210 fusion protein was an active and specific immunotherapy. Methods We recruited 16 patients who had chronic myeloid leukaemia (with the b3a2 fusion point of p210), stable residual disease, a minimum treatment of 12 months of imatinib or 24 months of interferon alfa, and no further reduction of residual disease for at least 6 months preceding enrolment. They were given six vaccinations with a peptide vaccine derived from the sequence p210-b3a2 plus molgramostim and QS-21 as adjuvants (CMLVAX100) before assessment of immunological and disease response, which included detecting amounts of b3a2 transcripts by standardised quantitative real-time reverse-transcriptase PCR. Results Of ten patients on imatinib, nine started CMLVAX100 having had a median of 10 months' stable cytogenetic disease (median 10% Philadelphia-chromosome-positive metaphases), whereas one started in stable CCR. All patients' cytogenetic responses improved after six vaccinations, with five reaching CCR. Notably, three of these five patients also had undetectable amounts of b3a2 transcript (BCR-ABL:β2 microglobulin ratio <0·00001). Six patients on interferon alfa treatment with a median of 17 months' stable residual disease (median 13% Philadelphia-chromosome-positive cells) were also vaccinated. All but one had improved cytogenetic responses, and two reached CCR. Overall, we recorded peptide-specific delayed-type hypersensitivity (in 11 of 16 patients), CD4 cell proliferation (13 of 14 assessed), and interferon γ production (five of five assessed). Interpretation Addition of CMLVAX100 to conventional treatment in patients with chronic myeloid leukaemia might favour further reduction of residual disease and increase the number of patients reaching a molecular response. Although imatinib is the standard treatment for chronic myeloid leukaemia, not all patients reach complete cytogenetic remission (CCR) and most maintain detectable disease at the molecular level. We investigated whether a vaccine targeting the BCR-ABL-derived p210 fusion protein was an active and specific immunotherapy. We recruited 16 patients who had chronic myeloid leukaemia (with the b3a2 fusion point of p210), stable residual disease, a minimum treatment of 12 months of imatinib or 24 months of interferon alfa, and no further reduction of residual disease for at least 6 months preceding enrolment. They were given six vaccinations with a peptide vaccine derived from the sequence p210-b3a2 plus molgramostim and QS-21 as adjuvants (CMLVAX100) before assessment of immunological and disease response, which included detecting amounts of b3a2 transcripts by standardised quantitative real-time reverse-transcriptase PCR. Of ten patients on imatinib, nine started CMLVAX100 having had a median of 10 months' stable cytogenetic disease (median 10% Philadelphia-chromosome-positive metaphases), whereas one started in stable CCR. All patients' cytogenetic responses improved after six vaccinations, with five reaching CCR. Notably, three of these five patients also had undetectable amounts of b3a2 transcript (BCR-ABL:β2 microglobulin ratio <0·00001). Six patients on interferon alfa treatment with a median of 17 months' stable residual disease (median 13% Philadelphia-chromosome-positive cells) were also vaccinated. All but one had improved cytogenetic responses, and two reached CCR. Overall, we recorded peptide-specific delayed-type hypersensitivity (in 11 of 16 patients), CD4 cell proliferation (13 of 14 assessed), and interferon γ production (five of five assessed). Addition of CMLVAX100 to conventional treatment in patients with chronic myeloid leukaemia might favour further reduction of residual disease and increase the number of patients reaching a molecular response. Vaccine development for chronic myelogenous leukaemiaThose familiar with the history of the development of tumour vaccines for chronic myelogenous leukaemia (CML), a myeloproliferative disease that accounts for 10–20% of chronic leukaemias, are often reminded of the labours of Sisyphus, who was condemned by the gods to perpetually roll a heavy boulder up a steep hill, only to helplessly watch it roll back before reaching the summit, and start over. In theory, CML should represent an ideal target for immunotherapy, and, indeed, in the arena of haemopoietic stem-cell transplantation, CML has long been recognised as one of the more responsive malignancies to immunotherapy. Full-Text PDF

Effect of a p210 multipeptide vaccine associated with imatinib or interferon in patients with chronic myeloid leukaemia and persistent residual disease: a multicentre observational trial

Although imatinib is the standard treatment for chronic myeloid leukaemia, not all patients reach complete cytogenetic remission (CCR) and most maintain detectable disease at the molecular level. We investigated whether a vaccine targeting the BCR-ABL-derived p210 fusion protein was an active and specific immunotherapy. We recruited 16 patients who had chronic myeloid leukaemia (with the b3a2 fusion point of p210), stable residual disease, a minimum treatment of 12 months of imatinib or 24 months of interferon alfa, and no further reduction of residual disease for at least 6 months preceding enrollment. They were given six vaccinations with a peptide vaccine derived from the sequence p210-b3a2 plus molgramostim and QS-21 as adjuvants (CMLVAX100) before assessment of immunological and disease response, which included detecting amounts of b3a2 transcripts by standardised quantitative real-time reverse-transcriptase PCR. Of ten patients on imatinib, nine started CMLVAX100 having had a median of 10 months' stable cytogenetic disease (median 10% Philadelphia-chromosome-positive metaphases), whereas one started in stable CCR. All patients' cytogenetic responses improved after six vaccinations, with five reaching CCR. Notably, three of these five patients also had undetectable amounts of b3a2 transcript (BCR-ABL:beta2 microglobulin ratio <0.00001). Six patients on interferon alfa treatment with a median of 17 months' stable residual disease (median 13% Philadelphia-chromosome-positive cells) were also vaccinated. All but one had improved cytogenetic responses, and two reached CCR. Overall, we recorded peptide-specific delayed-type hypersensitivity (in 11 of 16 patients), CD4 cell proliferation (13 of 14 assessed), and interferon gamma production (five of five assessed). Addition of CMLVAX100 to conventional treatment in patients with chronic myeloid leukaemia might favour further reduction of residual disease and increase the number of patients reaching a molecular response.

Influence of BCR/ABL fusion proteins on the course of Ph leukemias.

The hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL) is the presence of the Philadelphia chromosome as a result of the t(9;22) translocation. This gene rearrangement results in the production of a novel oncoprotein, BCR/ABL, a constitutively active tyrosine kinase. There is compelling evidence that the malignant transformation by BCR/ABL is critically dependent on its Abl tyrosine kinase activity. Also the bcr part of the hybrid gene takes part in realization of the malignant phenotype. We supposed that additional mutations accumulate in this region of the BCR/ABL oncogene during the development of the malignant blast crisis in CML patients. In ALL patients having p210 fusion protein the mutations were supposed to be preexisting. Sequencing of PCR product of the BCR/ABL gene (Dbl, PH region) showed that along with single-nucleotide substitutions other mutations, mostly deletions, had occurred. In an ALL patient a deletion of the 5th exon was detected. The size of the deletions varied from 36 to 220 amino acids. For one case of blast crisis of CML changes in the character of actin organization were observed. Taking into account the functional role of these domains in the cell an etiological role of such mutations on the disease phenotype and leukemia progression is plausible.

Influence of BCR/ABL fusion proteins on the course of Ph leukemias.

The hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL) is the presence of the Philadelphia chromosome as a result of the t(9;22) translocation. This gene rearrangement results in the production of a novel oncoprotein, BCR/ABL, a constitutively active tyrosine kinase. There is compelling evidence that the malignant transformation by BCR/ABL is critically dependent on its Abl tyrosine kinase activity. Also the bcr part of the hybrid gene takes part in realization of the malignant phenotype. We supposed that additional mutations accumulate in this region of the BCR/ABL oncogene during the development of the malignant blast crisis in CML patients. In ALL patients having p210 fusion protein the mutations were supposed to be preexisting. Sequencing of PCR product of the BCR/ABL gene (Dbl, PH region) showed that along with single-nucleotide substitutions other mutations, mostly deletions, had occurred. In an ALL patient a deletion of the 5th exon was detected. The size of the deletions varied from 36 to 220 amino acids. For one case of blast crisis of CML changes in the character of actin organization were observed. Taking into account the functional role of these domains in the cell an etiological role of such mutations on the disease phenotype and leukemia progression is plausible.

Allogeneic T-Cell Clones Able to Selectively Destroy Philadelphia Chromosome-Bearing (Ph1+) Human Leukemia Lines Can Also Recognize Ph1- Cells From the Same Patient

Immunocompetent cells in bone marrow allografts have been associated with a graft-versus-leukemia (GVL) effect. To further characterize effector mechanisms that may be involved in this GVL phenomenon, we have previously established an in vitro model to identify allogeneic T-cell clones that selectively mediate cytotoxicity against a patient's leukemic cells, but not against nonleukemic lymphocytes from the same patient. We have modified this in vitro model to test whether the Ph1 chromosome and the P210 fusion protein it controls have a detectable role in leukemia-specific recognition by allogeneic T-cell clones. In this report, T-cell lines reactive with allogeneic Ph1 chromosome-bearing (Ph1+) chronic myeloid leukemia (CML) cell lines were derived and selected to be minimally reactive with Ph1 negative (Ph1-) lymphoid lines from the same patient. However, after prolonged culture, these same T-cell lines also mediated significant destruction of the Ph1- target cells from the same patients. These T-cell lines specifically recognized cells from the allogeneic CML patient to which they were sensitized, and were not contaminated by an outgrowth of natural killer cells. Furthermore, subclones could be derived from these T-cell lines, and some of these subclones again showed selective killing of the allogeneic Ph1+ leukemia cell lines, and not of the Ph1- cell line from the same patient. Analyses of T-cell receptor (TCR) genes showed the alloreactive T-cell lines and the Ph1+ selective subclones derived from them to be of the same clonal origin. This suggests that the same T cells reacting with antigens expressed on the nonleukemic Ph1- targets can at times selectively and preferentially kill the allogeneic Ph1+ cells. As the same TCR that recognizes Ph1+ cells also can recognize the Ph1- targets, it appears that the Ph1+ chromosome does not play a detectable role in recognition by these allogeneic T-cell clones. This in vitro observation may provide a model for evaluating the relationship between GVL and graft-versus-host disease effects.

Allogeneic T-cell clones able to selectively destroy Philadelphia chromosome-bearing (Ph1+) human leukemia lines can also recognize Ph1- cells from the same patient

Immunocompetent cells in bone marrow allografts have been associated with a graft-versus-leukemia (GVL) effect. To further characterize effector mechanisms that may be involved in this GVL phenomenon, we have previously established an in vitro model to identify allogeneic T- cell clones that selectively mediate cytotoxicity against a patient's leukemic cells, but not against nonleukemic lymphocytes from the same patient. We have modified this in vitro model to test whether the Ph1 chromosome and the P210 fusion protein it controls have a detectable role in leukemia-specific recognition by allogeneic T-cell clones. In this report, T-cell lines reactive with allogeneic Ph1 chromosome- bearing (Ph1+) chronic myeloid leukemia (CML) cell lines were derived and selected to be minimally reactive with Ph1 negative (Ph1-) lymphoid lines from the same patient. However, after prolonged culture, these same T-cell lines also mediated significant destruction of the Ph1- target cells from the same patients. These T-cell lines specifically recognized cells from the allogeneic CML patient to which they were sensitized, and were not contaminated by an outgrowth of natural killer cells. Furthermore, subclones could be derived from these T-cell lines, and some of these subclones again showed selective killing of the allogeneic Ph1+ leukemia cell lines, and not of the Ph1- cell line from the same patient. Analyses of T-cell receptor (TCR) genes showed the alloreactive T-cell lines and the Ph1+ selective subclones derived from them to be of the same clonal origin. This suggests that the same T cells reacting with antigens expressed on the nonleukemic Ph1- targets can at times selectively and preferentially kill the allogeneic Ph1+ cells. As the same TCR that recognizes Ph1+ cells also can recognize the Ph1- targets, it appears that the Ph1+ chromosome does not play a detectable role in recognition by these allogeneic T-cell clones. This in vitro observation may provide a model for evaluating the relationship between GVL and graft-versus-host disease effects.