Stephanie G. Willis

A half-log increase in BCR-ABL RNA predicts a higher risk of relapse in patients with chronic myeloid leukemia with an imatinib-induced complete cytogenetic response.

Purpose: Imatinib induces a complete cytogenetic response (CCR) in most chronic myeloid leukemia patients in chronic phase. Although CCR is usually durable, a minority of patients relapse. Biomarkers capable of predicting those CCR patients with a higher risk of relapse would improve therapeutic management. Experimental Design: To assess whether changes in BCR-ABL RNA levels are a prognostic biomarker predictive of relapse, we regularly monitored transcript levels [every 3 months (median)] in 90 patients with CCR during 49 months (median) of imatinib therapy. Results: Throughout follow-up, the 20 patients with eventual relapse had higher transcript levels than the durable responders. Major molecular response (MMR; >3-log reduction of BCR-ABL RNA) was attained by 76 patients (12 with subsequent relapse) and was a significant predictor of prolonged relapse-free survival (P = 0.0008). A minimal 0.5-log increase in transcripts (before relapse; experienced by 42 patients, 16 with subsequent relapse) conveyed a significantly shorter relapse-free survival (P = 0.0017). Loss of MMR (transcript increase to <2.5-log reduction, before relapse; experienced by 33 patients, 11 with subsequent relapse) was also predictive of shortened relapse-free survival (P = 0.0003). A complete molecular response (undetectable transcripts by nested PCR) was attained by 28 MMR patients (one with subsequent relapse) and conveyed a significantly prolonged relapse-free survival (P = 0.0052). Conclusions: In chronic myeloid leukemia patients with an imatinib-induced CCR, a minimal half-log increase in BCR-ABL RNA (including loss of MMR) is a significant risk factor for future relapse. The achievement of a complete molecular response imparts longer progression-free survival than the achievement of an MMR.

A gene expression signature of CD34+ cells to predict major cytogenetic response in chronic-phase chronic myeloid leukemia patients treated with imatinib

In chronic-phase chronic myeloid leukemia (CML) patients, the lack of a major cytogenetic response (< 36% Ph(+) metaphases) to imatinib within 12 months indicates failure and mandates a change of therapy. To identify biomarkers predictive of imatinib failure, we performed gene expression array profiling of CD34(+) cells from 2 independent cohorts of imatinib-naive chronic-phase CML patients. The learning set consisted of retrospectively selected patients with a complete cytogenetic response or more than 65% Ph(+) metaphases within 12 months of imatinib therapy. Based on analysis of variance P less than .1 and fold difference 1.5 or more, we identified 885 probe sets with differential expression between responders and nonresponders, from which we extracted a 75-probe set minimal signature (classifier) that separated the 2 groups. On application to a prospectively accrued validation set, the classifier correctly predicted 88% of responders and 83% of nonresponders. Bioinformatics analysis and comparison with published studies revealed overlap of classifier genes with CML progression signatures and implicated beta-catenin in their regulation, suggesting that chronic-phase CML patients destined to fail imatinib have more advanced disease than evident by morphologic criteria. Our classifier may allow directing more aggressive therapy upfront to the patients most likely to benefit while sparing good-risk patients from unnecessary toxicity.

RNAi screen for rapid therapeutic target identification in leukemia patients

Targeted therapy has vastly improved outcomes in certain types of cancer. Extension of this paradigm across a broad spectrum of malignancies will require an efficient method to determine the molecular vulnerabilities of cancerous cells. Improvements in sequencing technology will soon enable high-throughput sequencing of entire genomes of cancer patients; however, determining the relevance of identified sequence variants will require complementary functional analyses. Here, we report an RNAi-assisted protein target identification (RAPID) technology that individually assesses targeting of each member of the tyrosine kinase gene family. We demonstrate that RAPID screening of primary leukemia cells from 30 patients identifies targets that are critical to survival of the malignant cells from 10 of these individuals. We identify known, activating mutations in JAK2 and K-RAS, as well as patient-specific sensitivity to down-regulation of FLT1, CSF1R, PDGFR, ROR1, EPHA4/5, JAK1/3, LMTK3, LYN, FYN, PTK2B, and N-RAS. We also describe a previously undescribed, somatic, activating mutation in the thrombopoietin receptor that is sensitive to down-stream pharmacologic inhibition. Hence, the RAPID technique can quickly identify molecular vulnerabilities in malignant cells. Combination of this technique with whole-genome sequencing will represent an ideal tool for oncogenic target identification such that specific therapies can be matched with individual patients.

Detection of ABL kinase domain mutations with denaturing high-performance liquid chromatography

Mutations of the ABL kinase domain (KD) are common in patients with chronic myelogenous leukemia (CML) who develop resistance to imatinib. We developed an RT-PCR-based denaturing high-performance liquid chromatography (D-HPLC) assay to detect mutations of the ABL KD. Validation experiments using mixtures of wild type and mutant amplicons showed that the D-HPLC assay could detect mutant transcripts when they represented at least 15% of the total, and was thus twice as sensitive as automated sequencing. When D-HPLC was applied to 30 cDNAs from patients with imatinib resistance that had previously been characterized for KD mutations by direct sequencing of BCR-ABL RT-PCR products, there was concordance in 97% of samples. Resequencing confirmed the original mutations in all cases. In addition, sequencing of individual clones detected a mutation in one sample that had been mutation-positive by D-HPLC but wild type by conventional sequencing. In serial samples from the same individuals, D-HPLC detected mutations as early as 260 days before hematological relapse. D-HPLC is suitable for routine clinical monitoring of CML patients for emergence of KD mutations and may be useful for optimizing therapy. Early detection of emerging mutant clones may aid in guiding decisions regarding alternative treatment options.

Determining the rise in BCR-ABL RNA that optimally predicts a kinase domain mutation in patients with chronic myeloid leukemia on imatinib

In imatinib-treated chronic myeloid leukemia (CML), secondary drug resistance is often caused by mutations in the BCR-ABL kinase domain (KD). As alternative therapies are available for imatinib resistance, early identification of mutations may prevent disease progression. Because most patients are routinely monitored by BCR-ABL quantitative polymerase chain reaction (PCR), it is important to define the optimal increase in BCR-ABL that should trigger mutation testing. Expert panels have provisionally recommended a 10-fold BCR-ABL increase as the trigger for mutation screening, acknowledging the lack of consensus. To address this question, we monitored 150 CML patients by quantitative PCR and DNA sequencing. Thirty-five different mutations were identified in 53 patients, and, during 22.5 months (median) of follow-up after sequencing, mutations were significantly predictive of shorter progression-free survival. An unbiased receiver operating characteristic analysis identified a 2.6-fold increase in BCR-ABL RNA as the optimal cutoff for predicting a concomitant KD mutation, with a sensitivity of 77% (94% if including subsequent samples). The 2.6-fold threshold approximated the analytic precision limit of our PCR assay. In contrast, transcript rise cutoffs of 5-fold or greater had poor diagnostic sensitivity and no significant association with mutations. We conclude that the currently recommended 10-fold threshold to trigger mutation screening is insensitive and not universally applicable.

Absolute quantitative detection of ABL tyrosine kinase domain point mutations in chronic myeloid leukemia using a novel nanofluidic platform and mutation-specific PCR

Absolute quantitative detection of ABL tyrosine kinase domain point mutations in chronic myeloid leukemia using a novel nanofluidic platform and mutation-specific PCR

BCR-ABL SH3-SH2 domain mutations in chronic myeloid leukemia patients on imatinib

Point mutations in the kinase domain of BCR-ABL are the most common mechanism of drug resistance in chronic myeloid leukemia (CML) patients treated with ABL kinase inhibitors, including imatinib. It has also been shown in vitro that mutations outside the kinase domain in the neighboring linker, SH2, SH3, and Cap domains can confer imatinib resistance. In the context of ABL, these domains have an autoinhibitory effect on kinase activity, and mutations in this region can activate the enzyme. To determine the frequency and relevance to resistance of regulatory domain mutations in CML patients on imatinib, we screened for such mutations in a cohort of consecutive CML patients with various levels of response. Regulatory domain mutations were detected in 7 of 98 patients, whereas kinase domain mutations were detected in 29. One mutation (T212R) conferred in vitro tyrosine kinase inhibitor resistance and was associated with relapse, whereas most other mutations did not affect drug sensitivity. Mechanistic studies showed that T212R increased the activity of ABL and BCR-ABL and that T212R-induced resistance may be partially the result of stabilization of an active kinase conformation. Regulatory domain mutations are uncommon but may explain resistance in some patients without mutations in the kinase domain.

Absence of SKP2 expression attenuates BCR-ABL–induced myeloproliferative disease

BCR-ABL is proposed to impair cell-cycle control by disabling p27, a tumor suppressor that inhibits cyclin-dependent kinases. We show that in cell lines p27 expression is inversely correlated with expression of SKP2, the F-box protein of SCF(SKP2) (SKP1/Cul1/F-box), the E3 ubiquitin ligase that promotes proteasomal degradation of p27. Inhibition of BCR-ABL kinase causes G(1) arrest, down-regulation of SKP2, and accumulation of p27. Ectopic expression of wild-type SKP2, but not a mutant unable to recognize p27, partially rescues cell-cycle progression. A similar regulation pattern is seen in cell lines transformed by FLT3-ITD, JAK2(V617F), and TEL-PDGFRbeta, suggesting that the SKP2/p27 conduit may be a universal target for leukemogenic tyrosine kinases. Mice that received transplants of BCR-ABL-infected SKP2(-/-) marrow developed a myeloproliferative syndrome but survival was significantly prolonged compared with recipients of BCR-ABL-expressing SKP2(+/+) marrow. SKP2(-/-) leukemic cells demonstrated higher levels of nuclear p27 than SKP2(+/+) counterparts, suggesting that the attenuation of leukemogenesis depends on increased p27 expression. Our data identify SKP2 as a crucial mediator of BCR-ABL-induced leukemogenesis and provide the first in vivo evidence that SKP2 promotes oncogenesis. Hence, stabilization of p27 by inhibiting its recognition by SCF(SKP2) may be therapeutically useful.

Characterization of BCR-ABL deletion mutants from patients with chronic myeloid leukemia

The BCR-ABL oncogenic tyrosine kinase causes chronic myeloid leukemia and is the target for imatinib therapy. During imatinib treatment, cells are selected in some patients with BCR-ABL kinase domain mutations that render decreased drug sensitivity. In addition, some patients express deletion mutants of BCR-ABL, apparently due to missplicing. Most commonly these deletion mutants lack a significant portion of the kinase domain that includes the P-loop. We describe a screen for such mutations in patients with CML and demonstrate that they are not oncogenic and are catalytically inactive. We hypothesized that coexpressing BCR-ABL deletion mutants has a dominant-negative effect on the native form through heterocomplex formation. However, upon coexpression of native and deletion mutant BCR-ABL in Ba/F3 cells, growth factor independence is maintained and signaling is activated normally. Despite this, these cells have increased imatinib sensitivity compared to cells expressing only native BCR-ABL. Thus, it will be important to investigate the prognostic impact of coexpression of deletion mutants in CML patients during imatinib treatment.

BCR-ABL kinase domain mutations in chronic myeloid leukemia: Not quite enough to cause resistance to imatinib therapy?

Patients with chronic myeloid leukemia (CML) treated with imatinib in early chronic phase tend to have durable remissions, but there is a high rate of relapse in patients with advanced disease. Mutations in the kinase domain of BCR-ABL that impair drug binding have been identified as the major mechanism of resistance. It is not known when exactly these mutations arise, but in some patients retrospective analysis of pretherapeutic samples demonstrated identical mutations, suggesting selection in the presence of drug. In the present study we have used a highly sensitive PCR assay to screen for kinase domain mutations in pretherapeutic samples from CML patients, irrespective of their subsequent response to imatinib. We find that kinase domain mutations are demonstrable in approximately 1/3 of patients with accelerated phase or blast crisis and that the presence of 2 copies of the Philadelphia chromosome is strongly correlated with mutation detection. Unexpectedly, kinase domain mutant clones were not invariably selected in the presence of drug, suggesting that additional mechanisms must contribute to a fully drug resistant leukemia.