ALBERT

Description: We prove that the SH2-containing tyrosine phosphatase 1 (SHP-1) plays a prominent role as resistance determinant of imatinib (IMA) treatment response in chronic myelogenous leukemia cell lines (sensitive/KCL22-S and resistant/KCL22-R). Indeed, SHP-1 expression is significantly lower in resistant than in sensitive cell line, in which coimmunoprecipitation analysis shows the interaction between SHP-1 and a second tyrosine phosphatase SHP-2, a positive regulator of RAS/MAPK pathway. In KCL22-R SHP-1 ectopic expression restores both SHP-1/SHP-2 interaction and IMA responsiveness; it also decreases SHP-2 activity after IMA treatment. Consistently, SHP-2 knocking-down in KCL22-R reduces either STAT3 activation or cell viability after IMA exposure. Therefore, our data suggest that SHP-1 plays an important role in BCR-ABL–independent IMA resistance modulating the activation signals that SHP-2 receives from both BCR/ABL and membrane receptor tyrosine kinases. The role of SHP-1 as a determinant of IMA sensitivity has been further confirmed in 60 consecutive untreated patients with chronic myelogenous leukemia, whose SHP-1 mRNA levels were significantly lower in case of IMA treatment failure (P 〈 .0001). In conclusion, we suggest that SHP-1 could be a new biologic indicator at baseline of IMA sensitivity in patients with chronic myelogenous leukemia.

Description: Abstract 2212 Poster Board II-189 Treatment of Chronic Myeloid Leukemia (CML) has shown an outstanding progress while new understanding of the disease has increased significantly. Nevertheless in the era of targeted therapy, Sokal index remains a dominant prognostic determinant of newly diagnosed CML patients. Our study has aimed to identify novel prognostic indicators to improve both an initial assessment and subsequent monitoring of CML patients. Initially, we have found that the protein-tyrosine phosphatase SHP1, that has a tumour suppressor activity, may play an important role in the resistance to imatinib treatment. We applied gene profiling and proteomic bidimensional electrophoresis to compare the differential pattern of gene and protein expression between KCL22s (imatinib-sensitive) and KCL22r (imatinib-resistant) cell lines. We found SHP1 to be one of the most differentially expressed genes. By ESI-TRAP MS technique, we found that one of the main interactors of SHP1 is SHP2, a protein phosphatase well known as positive regulator of oncogenic pathways, including the Ras/MAPK pathway. Gain-of-function mutations in SHP2 gene, have been described in various haematopoietic neoplasias and myeloproliferative disorders including Juvenile Chronic Myelomonocytic Leukemia. This protein is regulated throw phosphorylation on 542- and 580-Tyr, and unlike SHP1, acts as a positive regulator of the same oncogenic pathways. We found that KCL22r cell line, that has low SHP1 levels, showed complete phosphorylation of both SHP2 tyrosine residues, while these residues are not phosphorylated in the KCL22s line, which could explain an important mechanism for imatinib sensitivity. Consistently with this hypothesis, knock-down of SHP2 phosphatase in KCL22r by a specific shRNA resulted in 60% inhibition of KCL22r proliferation. Furthermore, the KCL22rSHP2- cells showed significant reduction in STAT3 (60%) and ERK1/2 (70%) phosphorylation. Our initial results from CML patients (Esposito et al , ASH 2008 Abs 1106) have suggested a differential expression of SHP1 in patients with different response to imatinib treatment. To further explore the role of SHP1 as a determinant of imatinib sensitivity we evaluated the expression of SHP1 in 93 newly-diagnosed CML patients enrolled into the TOPS trial investigating 400mg versus 800mg imatinib (Cortes et al, EHA 2008). The results of this study indicate that the mRNA levels of SHP1, as assessed by QPCR in peripheral blood of patients at the time of enrolment, are significantly different between patients who do or don't achieve Major Molecular Response (MMR) by 12 months (7.9±4.0 vs. 5.9±3.4; p=0.01). Logistic regression was used to estimate regression coefficients and corresponding odds ratio using MMR by 12 months as outcome variable in our model. Since the 25th and 75th percentiles of SHP1 were 4.3 and 8.4, respectively (resulting in an interquartile range of 4.1), statistical analysis shown that a value of 4.1 or more in SHP1 is associated with almost 2-fold odds of achieving MMR by 12 months (OR=1.92; 95% CI=1.12, 3.29; p=0.018). Moreover, in a contingency table, chi-square analysis has been shown a high risk of not achieving MMR by 12 month in those patients with either low SHP1 expression and high Sokal score, when compared with patients with high-intermediate SHP1 expression and low-intermediate Sokal score (p=0.0068). In conclusion, these results suggest that, measuring expression levels of SHP1 could be of value in assessing newly diagnosed CP-CML patients and estimating treatment response, which could help optimizing Gleevec treatment, or recommending patients to more potent TKIs. Supported by Novartis Oncology, Clinical Development, TOPS Clinical Correlative Studies Network Disclosures: Saglio: Novartis: Honoraria; Celgene: Honoraria. Pane:Novartis: Research Funding; Ministero dell'Università/PRIN: Research Funding; Regione Campania: Research Funding; Ministero della Salute/Progetto integrato Oncologia: Research Funding.

Description: Imatinib (Ima) has dramatically improved the outcome of patients affected by chronic myeloid leukemia (CML) and became the standard care for patients with newly diagnosed CML in chronic phase (CP). In spite of treatment progresses and novel biological findings, Sokal index is still a dominant prognostic determinant of newly diagnosed CML patients also in the era of targeted therapies. In this study we investigated the predictive role of the levels of expression of two SHP-constitutive non receptor protein tyrosine phosphatase, the SHP-1 and SHP-2, in leukemia cells obtained from 48 newly diagnosed CML patients enrolled into the TOPS (Tyrosine kinase inhibitor Optimization and Selectivity) trial. TOPS is a prospective, open-label, randomized (2:1) Phase III trial that compared Ima 800mg/d to 400mg/d in CP-CML. The findings end point of the trial is the rate of major molecular response (MMR) indicated by several reports as a parameter that predict a benefit for progression free survival (PFS). Results indicate that the mRNA levels of both SHP1 and SHP2 assayed by QPCR in peripheral blood of newly diagnosed the patients and expressed as ratio to ABL, are significantly different between those patients who do and do not achieved MMR by 12 months (7.4 ± 3.8 vs 6.0 ± 3.2, p = 0.017 for SHP1/Abl % and 0.19 ± 0.15 vs 0.10 ± 0.12, p = 0.017 for SHP2/ABL%). There is not statistical evidence that patients who achieved MMR earlier than 12 months i.e. at 6 and 9 months, have different baseline levels of SHP1 or SHP2, however the data are suggestive of a difference which might become statistically significant with a larger sample size. Complete cytogenetic response, CCyR, was a secondary end point of the TOPS study, overall, 65% have achieved CCyR by 6 months, and 85% by 12 months, and although not statistically different, results indicate that both SHP1 and SHP2 levels tended to be higher in patients who obtained CCyR, and the our further study with a larger sample size will show if the differences might become significant. .SHP1/ABL % and SHP2/ABL % are weakly correlated each other in the patients, therefore each independently acts as predictor of MMR at 12 months and logistic regression indicated that the combination increases their prognostic value for predicting MMR in the first 12 months and using logistic regression, Sokal score does not add any discriminating power to either of those markers (either alone or in combination). In conclusion, our results indicate, that the levels of expression of SHP1 and SHP2 are useful predictors of MMR in newly diagnosed CP-CML patients. These data confirm our prior findings from “in vitro” studies, which investigated the role of SHP1 and SHP2 phosphatases in mechanisms which regulate Imatinib sensitivity.