Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia.

Through sequencing analysis of blood or bone marrow samples from patients with chronic myeloid leukemia, we identified BCR-ABL kinase domain mutations in 29 of 32 patients whose disease relapsed after an initial response to the tyrosine kinase inhibitor imatinib. Fifteen different amino acid substitutions affecting 13 residues in the kinase domain were found. Mutations fell into two groups-those that alter amino acids that directly contact imatinib and those postulated to prevent BCR-ABL from achieving the inactive conformational state required for imatinib binding. Distinct mutations conferred varying degrees of imatinib resistance. Mutations detected in a subset of patients with stable chronic phase disease correlated with subsequent disease progression. Multiple independent mutant clones were detected in a subset of relapsed cases. Our data support a clonal selection model of preexisting BCR-ABL mutations that confer imatinib resistance.

Molecular classification of melanomas and nevi using gene expression microarray signatures and formalin-fixed and paraffin-embedded tissue.

Melanoma may be difficult to identify histologically and relatively high rates of misdiagnosis leads to many malpractice claims. Currently separation of melanomas from nevi is based primarily on light microscopic interpretation of hematoxylin and eosin stained sections with limited assistance from immunohistology. To increase the accuracy of discrimination of benign and malignant melanocytic lesions we identified DNA microarray-derived gene expression profiles of different melanocytic lesions and evaluated the performance of these gene signatures as molecular diagnostic tools in the molecular classification and separation of melanomas and nevi. Melanocyte-derived cells were isolated by laser capture microdissection from 165 formalin-fixed and paraffin-embedded melanocytic nevi and melanoma tissue sections. RNA was isolated, amplified, labeled, and hybridized to a custom DNA microarray. In all 120 samples were used to identify differentially expressed genes and generate a gene expression classifier capable of distinguishing between melanomas and nevi. These classifiers were tested by the leave-one-out method and in a blinded study. RT-PCR verified the results. Unsupervised hierarchical clustering identified two distinct lesional groups that closely correlated with the histopathologically identified melanomas and nevi. Analysis of gene expression levels identified 36 significant differentially expressed genes. In comparison with nevi, melanomas expressed higher levels of genes promoting signal transduction, transcription, and cell growth. In contrast, expression of L1CAM (homolog) was reduced in melanomas relative to nevi. Genes differentially expressed in melanomas and nevi, on the basis of molecular signal, sub classified a group of unknown melanocytic lesions as melanomas or nevi and had high concordance rates with histopathology. Gene signatures established using DNA microarray gene expression profiling can distinguish melanomas from nevi, indicating the feasibility of using molecular classification as a supplement to standard histology. Our successful use of a standard formalin-fixed and paraffin-embedded tissue further supports the practicability of combining molecular diagnostic testing with histopathology in evaluation of difficult melanocytic lesions.

Clinical validation of an array CGH test for HER2 status in breast cancer reveals that polysomy 17 is a rare event.

The HER2 gene is an important prognostic and therapeutic marker in newly diagnosed breast cancer. Currently, HER2 status is most frequently determined by immunohistochemical detection of HER2 protein expression on the cellular membrane surface or by fluorescence in situ hybridization analysis of HER2 gene copy number in fixed tissue using locus-specific probes for the HER2 gene and chromosome 17 centromere. However, these methods are problematic because of issues with intra- and inter-laboratory reproducibility and preanalytic variables, such as fixation time. In addition, the commonly used HER2/chromosome 17 ratio presumes that chromosome 17 polysomy is present when the centromere is amplified, even though analysis of the rest of the chromosome is not included in the assay. In this study, 97 frozen samples of invasive lobular and invasive ductal carcinoma, with known immunohistochemistry and fluorescence in situ hybridization results for HER2, were analyzed by comparative genomic hybridization to a commercially available bacterial artificial chromosome whole-genome array containing 99 probes targeted to chromosome 17 and the HER2/TOP2 amplicon. Results were 97% concordant for HER2 status, meeting the College of American Pathologists/American Society of Clinical Oncology's validation requirements for HER2 testing. Surprisingly, not a single case of complete polysomy 17 was detected even though multiple breast cancer cases showed clear polysomies of other chromosomes. We conclude that array comparative genomic hybridization is an accurate and objective DNA-based alternative for clinical evaluation of HER2 gene copy number, and that polysomy 17 is a rare event in breast cancer.

Whole-genome scanning by array comparative genomic hybridization as a clinical tool for risk assessment in chronic lymphocytic leukemia.

Array-based comparative genomic hybridization (array CGH) provides a powerful method for simultaneous genome-wide scanning and prognostic marker assessment in chronic lymphocytic leukemia (CLL). In the current study, commercially available bacterial artificial chromosome and oligonucleotide array CGH platforms were used to identify chromosomal alterations of prognostic significance in 174 CLL cases. Tumor genomes were initially analyzed by bacterial artificial chromosome array CGH followed by confirmation and breakpoint mapping using oligonucleotide arrays. Genomic changes involving loci currently interrogated by fluorescence in situ hybridization (FISH) panels were detected in 155 cases (89%) at expected frequencies: 13q14 loss (47%), trisomy 12 (13%), 11q loss (11%), 6q loss (7.5%), and 17p loss (4.6%). Genomic instability was the second most commonly identified alteration of prognostic significance with three or more alterations involving loci not interrogated by FISH panels identified in 37 CLL cases (21%). A subset of 48 CLL cases analyzed by six-probe FISH panels (288 total hybridizations) was concordant with array CGH results for 275 hybridizations (95.5%); 13 hybridizations (4.5%) were discordant because of clonal populations that comprised less than 30% of the sample. Array CGH is a powerful, cost-effective tool for genome-wide risk assessment in the clinical evaluation of CLL.

A novel pyridopyrimidine inhibitor of abl kinase is a picomolar inhibitor of Bcr-abl-driven K562 cells and is effective against STI571-resistant Bcr-abl mutants.

Inhibition of the constitutively active Bcr-abl tyrosine kinase(TK) by STI571 has proven to be a highly effective treatment for chronic myelogenous leukemia (CML). However, STI571 is only transiently effective in blast crisis, and drug resistance emerges by amplification of or development of mutational changes in Bcr-abl. We have screened a family of TK inhibitors of the pyrido [2,3-d]pyrimidine class, unrelated to STI571, and describe here a compound with substantial activity against STI-resistant mutant Bcr-abl proteins. This compound, PD166326, is a dual specificity TK inhibitor and inhibits src and abl in vitro with IC(50)s of 6 and 8 nM respectively. PD166326 inhibits the growth of K562 cells with IC(50) of 300 pM, leading to apoptotic G(1) arrest, whereas non-Bcr-abl cell types require >1000 times higher concentrations. We tested the effects of PD166326 on two of the clinically observed STI571-resistant Bcr-abl mutants. PD166326 potently inhibits the E255K mutant Bcr-abl protein and the growth of Bcr-ablE255K-driven cells. The T315I mutant Bcr-abl protein, which is mutated within the ATP-binding pocket, is resistant to PD166326; however, the growth of Bcr-ablT315I-driven cells is partially sensitive to this compound, likely through the inhibition of Bcr-abl effector pathways. These findings show that TK drug resistance is a structure-specific phenomenon and can be overcome by TK inhibitors of other structural classes, suggesting new approaches for future anticancer drug development. PD166326 is a prototype of a new generation of anti-Bcr-abl compounds with picomolar potency and substantial activity against STI571-resistant mutants.

Array CGH analysis of chronic lymphocytic leukemia reveals frequent cryptic monoallelic and biallelic deletions of chromosome 22q11 that include the PRAME gene.

We used BAC array-based CGH to detect genomic imbalances in 187 CLL cases. Submicroscopic deletions of chromosome 22q11 were observed in 28 cases (15%), and the frequency of these deletions was second only to loss of the 13q14 region, the most common genomic aberration in CLL. Oligonucleotide-based array CGH analysis showed that the 22q11 deletions ranged in size from 0.34 Mb up to approximately 1 Mb. The minimally deleted region included the ZNF280A, ZNF280B, GGTLC2, and PRAME genes. Quantitative real-time PCR revealed that ZNF280A, ZNF280B, and PRAME mRNA expression was significantly lower in the 22q11 deletion cases compared to non-deleted cases.

Phosphorylation of the ATP-binding loop directs oncogenicity of drug-resistant BCR-ABL mutants.

The success of targeting kinases in cancer with small molecule inhibitors has been tempered by the emergence of drug-resistant kinase domain mutations. In patients with chronic myeloid leukemia treated with ABL inhibitors, BCR-ABL kinase domain mutations are the principal mechanism of relapse. Certain mutations are occasionally detected before treatment, suggesting increased fitness relative to wild-type p210 BCR-ABL. We evaluated the oncogenicity of eight kinase inhibitor-resistant BCR-ABL mutants and found a spectrum of potencies greater or less than p210. Although most fitness alterations correlate with changes in kinase activity, this is not the case with the T315I BCR-ABL mutation that confers clinical resistance to all currently approved ABL kinase inhibitors. Through global phosphoproteome analysis, we identified a unique phosphosubstrate signature associated with each drug-resistant allele, including a shift in phosphorylation of two tyrosines (Tyr253 and Tyr257) in the ATP binding loop (P-loop) of BCR-ABL when Thr315 is Ile or Ala. Mutational analysis of these tyrosines in the context of Thr315 mutations demonstrates that the identity of the gatekeeper residue impacts oncogenicity by altered P-loop phosphorylation. Therefore, mutations that confer clinical resistance to kinase inhibitors can substantially alter kinase function and confer novel biological properties that may impact disease progression.

Molecular mechanisms of resistance to STI571 in chronic myeloid leukemia.

Therapeutic use of the recently FDA-approved drug STI571 has been successful in the treatment of Philadelphia chromosome-positive leukemias. STI571 is a small molecule inhibitor with activity against BCR-ABL, the deregulated tyrosine kinase responsible for initiation and maintenance of the disease in the chronic phase of chronic myeloid leukemia (CML). Clinical trials demonstrated the ability of STI571 to induce remissions in patients with chronic phase CML with only rare relapses after 18 months of follow-up. However, in patients with more advanced stages of disease, responses to STI571 were less common and often transient. Studies investigating the molecular mechanisms of resistance to this novel compound have progressed rapidly and point to the continued importance of BCR-ABL in disease maintenance even at its latest stages. Here the authors review recent work aimed at elucidating the nature of STI51 resistance.

BCR-ABL point mutants isolated from patients with imatinib mesylate-resistant chronic myeloid leukemia remain sensitive to inhibitors of the BCR-ABL chaperone heat shock protein 90.

Clinical resistance to imatinib mesylate is commonly observed in patients with advanced Philadelphia chromosome- positive (Ph(+)) leukemias. Acquired resistance is typically associated with reactivation of BCR-ABL due to kinase domain mutations or gene amplification, indicating that BCR-ABL remains a viable target for inhibition in these patients. Strategies for overcoming resistance can be envisioned through exploitation of other molecular features of the BCR-ABL protein, such as its dependence on the molecular chaperone heat shock protein 90 (Hsp90). To determine whether inhibition of Hsp90 could induce degradation of imatinib mesylate-resistant, mutant BCR-ABL proteins, hematopoietic cells expressing 2 mutant BCR-ABL proteins found in imatinib mesylate-resistant patients (T315I and E255K) were examined for sensitivity to geldanamycin and 17-allylaminogeldanamycin (17-AAG). Both compounds induced the degradation of wild-type and mutant BCR-ABL and inhibited cell growth, with a trend indicating more potent activity against mutant BCR-ABL proteins. These data support clinical investigations of 17-AAG in imatinib mesylate-resistant Ph(+) leukemias.

Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop.

The Abl tyrosine kinase inhibitor STI-571 is effective therapy for stable phase chronic myeloid leukemia (CML) patients, but the majority of CML blast-crisis patients that respond to STI-571 relapse because of reactivation of Bcr-Abl signaling. Mutations of Thr-315 in the Abl kinase domain to Ile (T315I) were previously described in STI-571-resistant patients and likely cause resistance from steric interference with drug binding. Here we identify mutations of Tyr-253 in the nucleotide-binding (P) loop of the Abl kinase domain to Phe or His in patients with advanced CML and acquired STI-571 resistance. Bcr-Abl Y253F demonstrated intermediate resistance to STI-571 in vitro and in vivo when compared with Bcr-Abl T315I. The response of Abl proteins to STI-571 was influenced by the regulatory state of the kinase and by tyrosine phosphorylation. The sensitivity of purified c-Abl to STI-571 was increased by a dysregulating mutation (P112L) in the Src homology 3 domain of Abl but decreased by phosphorylation at the regulatory Tyr-393. In contrast, the Y253F mutation dysregulated c-Abl and conferred intrinsic but not absolute resistance to STI-571 that was independent of Tyr-393 phosphorylation. The Abl P-loop is a second target for mutations that confer resistance to STI-571 in advanced CML, and the Y253F mutation may impair the induced-fit interaction of STI-571 with the Abl catalytic domain rather than sterically blocking binding of the drug. Because clinical resistance induced by the Y253F mutation might be overcome by dose escalation of STI-571, molecular genotyping of STI-571-resistant patients may provide information useful for rational therapeutic management.