ALBERT

Description: Contemporary treatment of pediatric acute lymphoblastic leukemia (ALL) requires the assignment of patients to specific risk groups. We have recently demonstrated that expression profiling of leukemic blasts can accurately identify the known prognostic subtypes of ALL, including T-cell lineage ALL (T-ALL), E2A-PBX1, TEL-AML1, MLL rearrangements, BCR-ABL, and hyperdiploid karyotypes with more than 50 chromosomes. As the next step toward developing this methodology into a frontline diagnostic tool, we have now analyzed leukemic blasts from 132 diagnostic samples using higher density oligonucleotide arrays that allow the interrogation of most of the identified genes in the human genome. Nearly 60% of the newly identified subtype discriminating genes are novel markers not identified in our previous study, and thus should provide new insights into the altered biology underlying these leukemias. Moreover, a proportion of the newly selected genes are highly ranked as class discriminators, and when incorporated into class-predicting algorithms resulted in an overall diagnostic accuracy of 97%. The performance of an array containing the identified discriminating genes should now be assessed in frontline clinical trials in order to determine the accuracy, practicality, and cost effectiveness of this methodology in the clinical setting.

Description: Failure of initial remission-induction therapy and disease recurrence remains a major problem in the management of acute leukemia, however the nature of the biologic factors promoting relapse are incompletely understood. To identify genomic abnormalities associated with relapse, we performed high-resolution, genome-wide analysis of DNA copy number abnormalities and loss-of heterozygosity using Affymetrix single nucleotide polymorphism (SNP) microarrays in 33 cases of relapsed acute leukemia. Sixteen ALL (2 ETV6-RUNX1, 2 MLL rearranged, 6 pseudodiploid or cytogenetically normal B-progenitor ALL, and 6 T-lineage) and 17 AML (two RUNX1-RUNX1T1 [AML1-ETO], two MLL-rearranged, one M7, and 12 with normal karyotype or miscellaneous cytogenetic abnormalities) were studied. Samples with less than 80% blasts were flow sorted to at least 90% purity prior to DNA extraction. Diagnostic samples were available for all cases, and germline samples for 22. DNA copy number and LOH analysis was performed using Affymetrix 250k Nsp and Sty arrays was performed for all samples. Data were analysed using a karyotype-guided normalization algorithm, dChipSNP, and circular binary segmentation. In a detailed comparative analysis of paired diagnostic and relapse samples, changes in DNA copy number abnormalities were identified in the relapse sample in 14 of 16 (87.5%) ALL cases. A striking finding was loss of copy number lesions present at diagnosis in 8/16 ALL relapse samples, and the acquisition of new copy number lesions in 4 of these 8 samples. In each case, the pattern of deletions at antigen receptor loci was comparable between relapse and diagnosis, suggesting the emergence of a related leukemic clone, rather than the development of a distinct second leukemia. An additional 8 ALL relapse samples retained the copy number lesions present at diagnosis, but 6 of these acquired additional copy number abnormalities at relapse, a finding further suggestive of significant clonal evolution. Of the newly acquired copy number abnormalities in the relapse samples, deletions (62.5% of cases) were more common than gains (12.5%). In constrast to ALL a more restricted range of copy number abnormalities was seen in AML, with new abnormalities at relapse seen in 5/17 (29.4%) cases, and deletions (29.4%) outnumbering gains (17.6%). The loss of lesions present at diagnosis was only observed in two AML relapse samples. Examining the entire cohort, the CDKN2A/B locus was most commonly involved (N=5), gains of 1q were noted in two cases, otherwise all observed copy number changes were noted in single cases only, and included focal deletions of ERG and RUNX1. Copy neutral loss of heterozygosity was uncommon, with the exception of three AML cases with UPD of the entire chromosome 13. These observations indicate that relapse is frequently the result of the emergence of a leukemic clone that shows significant genetic differences from the diagnostic clone. Whether these represent rare clones present at the time of diagnosis or are the emergence of new clones as the result of ongoing genomic abnormalities can now be determined using genomic probes specific for the newly acquired deletions.

Description: Expression of BCR-ABL1 is the hallmark of chronic myelogenous leukemia (CML) and a subset of de novo acute lymphoblastic leukemia (ALL), but the factors determining disease lineage, and progression of CML to myeloid or lymphoid blast crisis, are incompletely understood. We recently reported deletion of IKZF1 (encoding the lymphoid transcription factor Ikaros) in 85% of de novo pediatric and adult BCR-ABL1 ALL, and in lymphoid blast crisis in a small cohort of CML cases (Nature2008;453:110), suggesting that IKZF1 deletion is important in the pathogenesis of BCR-ABL1 lymphoid leukemia. To identify genetic determinants of disease stage and blast crisis lineage in CML, we have now performed high-resolution, genome wide analysis of DNA copy number abnormalities (CNA) and loss-of heterozygosity (LOH) and candidate gene resequencing in a cohort of 90 CML patients that included 64 samples obtained at chronic phase (CP), 15 samples at accelerated phase (AP), 9 lymphoid blast crisis (LBC) and 22 myeloid blast crisis (MBC) samples. Importantly, 25 patients had sequential samples (CP and/or AP, as well as blast crisis samples) enabling analysis of lesions acquired at progression to blast crisis. All blast crisis samples were flow sorted to at least 90% purity prior to DNA extraction. Germline samples for 28 cases obtained at remission or by flow sorting of blast crisis samples were also examined. Affymetrix SNP 6.0 arrays, interrogating over 1.87 million genomic loci, were used for 85 samples, and 500K arrays for the remainder. Identification of tumor-specific (somatic) copy number analysis was performed by directly comparing CML samples to matched germline samples were available, or by filtering results against databases of inherited copy number variants for samples lacking germline material. Genomic resequencing of IKZF1, PAX5 and TP53 was performed for all AP, LBC and MBC samples. There were few CNAs in CP-CML (mean 0.27 deletions and 0.07 gains per case), with no recurring lesions identified apart from deletions or gains at the chromosomal breakpoints of BCR and ABL1 (3 cases each). Notably, the size of these translocation associated deletions was highly variable, ranging from 6kb (one ABL1 deletion) and 15 kb (one BCR deletion) to deletions extending to the telomeres of chromosomes 9 and 22. No significant increase in lesion frequency was identified in AP cases (0.14 deletions and 0.9 gains per case), however the number and cumulative extent of genomic aberrations was significantly higher in both lymphoid and myeloid blast crisis samples. LBC cases had a mean of 8.1 deletions/case (P

Description: During the years of 2005 to 2008, the MILE (Microarray Innovations in LEukemia) study research program was performed in 11 laboratories across three continents: 7 from the European Leukemia Network (ELN, WP13), 3 from the US and 1 in Singapore. The first stage was designed as biomarker discovery phase to generate whole-genome gene expression profiles (GEP) from recognized categories of clinically relevant leukemias and myelodysplastic syndromes (MDS). These were C1: mature B-ALL with t(8;14), C2: pro-B-ALL with t(11q23)/MLL, C3: c-ALL/pre-B-ALL with t(9;22), C4: T-ALL, C5: ALL with t(12;21), C6: ALL with t(1;19), C7: ALL with hyperdiploid karyotype, C8: c-ALL/pre-B-ALL without specific genetic abnormalities, C9: AML with t(8;21), C10: AML with t(15;17), C11: AML with inv(16)/t(16;16), C12: AML with t(11q23)/MLL, C13: AML with normal karyotype or other abnormalities, C14: AML with complex aberrant karyotype, C15: CLL, C16: CML, C17: MDS, and C18: non-leukemic and healthy bone marrow samples as controls and were compared to conventional diagnostic assays (“Gold Standard”). Data from the completed MILE Stage I included 2143 retrospectively collected adult and pediatric samples tested with HG-U133 Plus 2.0 microarrays (Affymetrix). In total only 47 analyses (2.1%) failed technical quality criteria. Cross-validation accuracy (average of three 30-fold cross-validations) of the final 2096 MILE Stage I samples was 92.1% concordant with the center-specific “Gold Standard” diagnosis (average call rate 99.4%). In nine classes the sensitivity was ≥94.3%: C2, C3, C4, C5, C9, C10, C11, C15, and C16. Lower sensitivities were observed for C7, C8, C14, and C17; which can largely be explained by the biological heterogeneity and non-standardized “Gold Standard” definitions for these entities. Yet, it is notable that all these classes showed specificities above 98.1%. In order to assess the clinical utility of microarray-based diagnostics a prospective Stage II was subsequently performed using a customized microarray representing 1480 probe sets. Overall, 1156 high quality GEP have been generated in MILE Stage II and represent an independent and blinded test set for the algorithms developed. A focused classification scheme aimed at accurately addressing only acute leukemias resulted in a 95.5% median sensitivity and a 99.5% median specificity for the 14 classes included in the classifier (C1 – C14, n=696). Lower accuracies were observed for the interface of C7–C8 in ALL, as well as C12 and C14 in AML. Interestingly, during the process of discrepant results analyses, it was observed that for 7.5% (n=52) of acute leukemias microarray results were correctly diagnosing samples as compared to the initial “Gold Standard” diagnoses entered into the study database, either because of erroneous entries into case report forms (24%) or subsequent re-testing of left-over material following the suggested diagnosis from the microarray (76%). In addition, predicted accuracies for CLL, CML and MDS in Stage II were 99.2%, 95.2%, and 81.5%, respectively. In conclusion, the MILE research study confirms in a final cohort of 3252 patients that microarrays accurately classify acute and chronic leukemia samples into known diagnostic and prognostic sub-categories. This final report underlines that the standardized method of gene expression profiling with low technical failure rate and simplified standard operating procedures may improve current “Gold Standards” as an adjunct to conventional diagnostic algorithms and potentially offers a reliable diagnostic/prognostic tool for many patients who don’t have access to a state-of-the-art “Gold Standard” workup. Our gene expression database, intended to be submitted to the public domain, will further contribute to research that aims to elucidate the molecular understanding of leukemias.

Description: Previous genomic analyses identified a high frequency of genetic alterations targeting B-lymphoid development (〉40%) and cell cycle/tumor suppression (CDKN2A/B, 35%) in pediatric B-progenitor acute lymphoblastic leukemia (B-ALL). The frequency and nature of copy number alterations (CNAs) differs significantly across ALL subtype with less than one CNA/case in MLL ALL and over 8 lesions/case in BCR-ABL1 and ETV6-RUNX1 ALL; and the near obligate presence of IKZF1 (Ikaros) deletion in BCR-ABL1 ALL, but never in ETV6-RUNX1 ALL. Although important insights have been obtained from these genome-wide CNAs, it is important to realize that the SNP array platforms that have been used are limited by uneven probe spacing and poor coverage of some genes (including IKZF1 and CDKN2A/B). Moreover, additional CNAs are detected as array resolution increases (e.g. 6.5 lesions per BCR-ABL1 case using 315,000 markers, and 8.8 for 615,000 marker data). Thus, the true frequency of CNAs in ALL remains unknown. To address this question, we now report ultra-high resolution CNA analysis using 2.17 million feature oligonucleotide arrays (Roche Nimblegen) of 20 MLL, 20 BCR-ABL1, and 4 miscellaneous karyotype B-ALL cases. All had previously been examined using Affymetrix 500K SNP arrays. We identified a mean of 6.2 deletions (range 0–38) and 1.4 gains (0–13) per case. There were more lesions in BCR-ABL1 than MLL ALL cases (mean 11.2 v 1.15 deletions, P