Summary Three major types of rearrangements are involved in acute myeloid leukemias (AML): t(8;21)(q22;q22), inv(16)(p13q22), and 11q23/ MLL abnormalities. Their precise identification becomes essential for diagnosis, prognosis, and therapeutic choices. Resulting fusion transcripts (FT) are also powerful markers for monitoring the efficacy of treatment, the minimal residual disease (MRD) and could become therapeutic targets. Today, the challenge is to propose an individual follow‐up for each patient even for those with a rare fusion event. In this study, we propose a biochip‐based assay integrated in a global strategy for identification of rare FT in AML, after fluorescence in situ hybridization detection, as described by the World Health Organization classification. Using cell lines, we developed and validated a biochip‐based assay called the AML Fusion Chip that identifies every FT of AML1‐ETO , CBFβ‐MYH11 as well as MLL‐AF9 , MLL‐ENL , MLL‐AF6 , and MLL‐AF10 . The original design of our AML Fusion Chip. v01 enables the identification of these FT wherever the breakpoint on the partner gene may be. In case of biochip negative result, our 3′RACE amplification strategy enables to clone and then sequence the new translocation partner. This AML Fusion Chip strategy fits into the concept of personalized medicine for the largest number of patients.

e22082 Background: Currently, up to 20% of HER2 tests in breast cancer may be inaccurate. To address this situation, the 2007 ASCO/CAP guideline for HER2 testing recommended using standardized IHC (for HER2 protein expression) and FISH (for HER2 DNA amplification) in a sequential testing algorithm. However, test standardization and equivocal results still remain an issue potentially leading to inappropriate treatment decisions. A new genomic HER2 (gHER2) assay was designed to address these issues. Methods: Frozen samples of 152 primary breast cancers with a positive or negative IHC result (126 IHC 0+ and 26 IHC 3+ determined by tissue microarray) were profiled using Affymetrix U133 Plus 2.0 gene chips. Recursive feature elimination algorithm by support vector machines (RFE-SVM) was used to select probesets correlated to IHC status; to compute a genomic HER2 (gHER2) expression index; and to set a threshold discriminating between IHC 0+ from IHC 3+ samples. This gHER2 assay was validated in 4 independent datasets (n=309) and tested for its ability to reclassify HER2 equivocal cases (IHC 2+, n=21). The assay was standardized using Ipsogen MapQuant Dx. Results: RFE-SVM identified 6 known and 1 unknown genes correlating with the IHC status, all located within the HER2 DNA amplicon. The gHER2 assay gave unequivoqual HER2 status in 96.2 % of samples. The genomic HER2 assay was concordant with IHC status in 94.5% of cases. 95 % of samples with equivocal IHC status (IHC 2+) could be re-classified in accordance with FISH status in 95 % of cases. Conclusions: We could identify a genomic signature of both HER2 DNA amplification and HER2 protein expression. A new standardized genomic HER2 assay was derived that matches the ASCO/CAP guideline requirements of 95 % concordance with validated techniques. It may contribute to reduce the number of equivocal results. [Table: see text]

Abstract Relapse remains the major cause of treatment failure in AML; however, RQ-PCR assays to detect leukemic fusion transcripts have been shown to identify reliably those patients at highest risk of relapse, allowing development of a more individualized treatment approach. In cases lacking a leukemia-specific MRD marker, quantification of genes over-expressed in AML e.g. Wilms’ Tumor gene (WT1) could provide more a precise measurement of disease response and quality of remission, potentially enhancing risk scores such as the one developed by the MRC used to identify those patients most and least likely to benefit from allogeneic transplant in first CR. WT1 is over-expressed in at least 75% of AML cases. Within the European LeukemiaNet we systematically evaluated 9 published and “in house” WT1 assays. Assays were excluded due to demonstrated lack of RNA-specificity or location within the 3′ region of the gene which has been shown to be subject to deletion or mutation in AML. An assay located within the 5′ region associated with superior sensitivity was ultimately selected following parallel testing in 11 labs and evaluated in 238 diagnostic peripheral blood (PB) and 386 bone marrow (BM) samples. WT1 was over-expressed in the majority, with comparable levels in PB and BM (PB - median 4637 WT1 copies/104ABL copies, range 0–1132709; BM - median 7212, 0–750571), as compared to normal BM (median 19.8, 0–213), PB (median 0.01, 0.01–47.6) and PBSCs (median 6.1, 0–39). In cases over-expressing WT1, kinetics of transcript reduction were evaluated following induction. A greater response was associated with a significantly reduced risk of relapse (hazard ratio 0.65 per log reduction (95% CI 0.43–0.96), p=0.03), although this failed to remain significant when adjusted for age, presenting WBC and cytogenetic risk group, which are key variables in the MRC risk index. Indeed, there was a highly significant correlation between larger log reduction in normalized WT1 transcript level and better risk score (p=0.0001). Sequential analysis of PB and BM samples from 15 AML cases with low WT1 expression (<250 copies) showed no significant modulation in transcript level on regeneration after chemotherapy, indicating that in WT1+ AML, transcript levels detected in follow-up samples reliably reflect disease status. This study provides evidence that recognized pre-treatment risk factors for relapse correlate closely with kinetics of response to induction therapy and lend support to the evaluation of early assessment of MRD to develop more robust risk scores, to enhance risk stratification and identify those patients most suited to proceed rapidly to allogeneic transplant.

La maladie veino-occlusive pulmonaire (MVO) est une forme rare d'hypertension pulmonaire (HTP) se caractérisant par une atteinte veinulaire pulmonaire et capillaire prédominante. On distingue au sein des MVO les formes idiopathiques, héritables (mutations bialléliques du gène EIF2AK4), secondaires à une exposition à un toxique (chimiothérapies, solvants organiques) et les MVO associées à une connectivite, en particulier la sclérodermie. La présentation clinique de la MVO est proche de celle de l'hypertension artérielle pulmonaire (HTAP). Les biopsies pulmonaires sont contre-indiquées dans l'HTP et le diagnostic repose donc sur un faisceau d'argument. Le diagnostic de MVO est généralement évoqué devant la présence d'anomalies scannographiques (lignes septales, nodules flous, adénopathies médiastinales), d'une DLCO effondrée associée à une hypoxémie profonde. Le pronostic des patients atteints de MVO est sombre. Les traitements spécifiques de l'HTAP n'ont pas prouvé leur efficacité dans la MVO et peuvent entraîner des œdèmes pulmonaires sévères. La transplantation pulmonaire constitue aujourd'hui le seul traitement curatif de cette maladie.Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension (PH) characterized by preferential remodelling of pulmonary venules and angioproliferation. PVOD term includes idiopathic, heritable (biallelic mutations of EIF2AK4 gene), drugs and toxins induced (alkylating agents, organic solvents) and connectivite-associated forms (especially systemic-sclerosis associated form). PVOD and pulmonary arterial hypertension (PAH) share a similar clinical presentation. Lung biopsy is contraindicated in PVOD due to high risk of life-threatening bleeding. A noninvasive diagnostic approach, including oxygen parameters, low diffusing capacity for carbon monoxide and characteristic signs on high-resolution computed tomography of the chest, is used to support a diagnosis of PVOD. PVOD prognosis is worse than other forms of PAH. There is no evidence-based medical therapy for PVOD and life-threatening pulmonary edema may occur following PAH targeted therapy in PVOD. Lung transplantation remains the preferred definitive therapy for eligible patients.

Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemia. In acute myeloid leukemia (AML), the reliability of real-time quantitative PCR (RQ-PCR) and its potential clinical value for MRD studies using fusion gene (FG) transcripts as PCR targets such as PML-RARa, AML1-ETO and CBFb- MYH11 has been demonstrated, but these markers are present only in a minority of cases. In order to overcome this problem several groups looked for alternative markers and growing evidence has suggested that the high expression of WT1 in a significant proportion of acute leukemia cases provides a suitable target for therapy as well as for monitoring of MRD. However, heterogeneity of molecular approaches resulted in a lack of comparability between different MRD studies. This has been solved using RQ-PCR in a network of 9 laboratories within the European LeukemiaNet. Overall 8 primer/probe sets were evaluated including published and “in-house” sets. The assays analyzed differed significantly in terms of efficiency and sensitivity. Three assays with superior performance were identified, achieving sensitivities of at least 1 in 10,000 in serial dilutions of HL60 cells (WT1 positive). Subsequent analysis of two of the primer/probe sets, which amplify ex. 6/7 and 7/8 of WT1 respectively, revealed the potential for false negative results, following documentation of deletions of the WT1 gene in this region in primary AML samples. In two of these cases, different deletions of sequences corresponding to part of WT1 ex.8 were documented by WT1 RNA sequencing. Therefore, a primer/probe set amplifying ex. 1/2 of WT1 has been subject to further analysis in a QC round involving 9 labs. The analysis of 33 normal BM, 32 normal PB and 12 CD34 enriched PBMNCs gave the following results: BM, mean 70,32 WT1 copies/104 ABL copies (range 8,99–209,82); PB, mean 3,30 WT1 copies/104 ABL copies (range 0–13,55); CD34 enriched PBMNCs, mean 8,16 WT1 copies/104 ABL copies (range 1,55–26,16). Overall, these analyses underline the importance of standardization in the development of RQ-PCR assays for MRD detection in leukemia. There is increasing interest in identification of genes that are over-expressed in leukemia as potential MRD targets. However, it is clear that incorporation of such MRD targets into risk-directed treatment protocols is critically dependent upon establishing thresholds of expression in normal blood and marrow on regeneration following myeloablative therapy.

Reciprocal rearrangements of the MLL gene are among the most common chromosomal abnormalities in both Acute Lymphoblastic and Myeloid Leukemia. The MLL gene, located on the 11q23 chromosomal band, is involved in more than 40 recurrent translocations. In the present study, we describe the development and validation of a biochip-based assay designed to provide a comprehensive molecular analysis of MLL rearrangements when used in a standard clinical pathology laboratory. A retrospective blind study was run with cell lines (n=5), and MLL positive and negative patient samples (n=31), to evaluate assay performance. The limits of detection determined on cell line data were 10−1, and the precision studies yielded 100% repeatability and 98% reproducibility. The study shows that the device can detect frequent (AF4, AF6, AF10, ELL or ENL) as well as rare partner genes (AF17, MSF). The identified fusion transcripts can then be used as molecular phenotypic markers of disease for the precise evaluation of minimal residual disease by RQ-PCR. This biochip-based molecular diagnostic tool allows, in a single experiment, rapid and accurate identification of MLL gene rearrangements among 32 different fusion gene (FG) partners, precise breakpoint positioning and comprehensive screening of all currently characterized MLL FGs.

9658 Background: Microarray technology has significantly contributed to enhance understanding of the diverse molecular mechanisms driving tumorigenesis. However, results from gene expression profiling (GEP) research have yet to be translated to the clinical setting. In this study, we describe the development of the Breast Cancer ProfileChip (BCPC), a device based on GEP for molecular characterization and management of breast cancer. Methods: 220 patients who received anthracyclin based adjuvant chemotherapy were selected from the Institut Paoli-Calmettes. All tissues were analyzed on nylon cDNA microarrays (DiscoveryChip) containing 9,000 genes by isotopic labeling based on RT of total RNA. Signatures for phenotypic markers (ER, PR, BCL2, EGFR, VEGFA, MIB1, ERBB2) and prognosis (metastasis, lymph node status) were calculated by t-test with moderate correction for multiple variable testing. The number of discriminatory genes required to optimize specificity and sensitivity for each defined gene signature were calculated by ProfileSoftware Corporate, and validated on an independent series of patients from Centre Léon Bérard (n=110). All gene signatures were subsequently cross-validated by correlative immunohistochemistry and/or fluorescence in-situ hybridization. Results: Identified signatures comprised of 30 to 150 genes were transferred to the BCPC, a biochip containing 1200 cDNA's. An amplification method based on linear PCR, colorimetric detection of hybridized complexes, and quantification on a flat bed scanner was developed to ensure clinical feasibility of the BCPC. Expression profiles are performed with 500ng total RNA with high reproducibility (mean CV =5%), and automatically quantified and analyzed with ProfileSoftware Cancer. All derived gene signatures are quantitative, sensitive, and highly reproducible (mean CV = 5%). Conclusions: The BCPC is the first analytical tool based on microarray technology engineered to fit the needs of a hospital-based platform; providing a viable method to assess a series of clinical parameters that may contribute to improved breast cancer management. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Ipsogen Ipsogen; Ipsogen SAS

9577 Background: 11q23/MLL abnormalities are generally associated with unfavorable prognosis in acute myeloid leukemia (AML). More than 50 partners have been described for MLL, and 35 of these partners have been cloned and analyzed at the molecular level to date. The functional role of each specific fusion transcript on the progression and outcome of disease remains to be elucidated. We present here the results of an international multi-center study aimed at assessing the clinical performance of a new biochip based molecular device (MLL FusionChip™ kit) designed to confirm the 11q23 abnormality and identify the MLL fusion gene partner. Methods: Nine laboratories in 7 countries ran 4 sets of 5 assays. Each run included a control sample (positive cell line), 2 negative samples, and 2 positive samples. Sample inclusion criteria were made by current standard diagnostic procedures: MLL status of all samples were characterized by cytogenetics, FISH, and/or RQ-PCR, and RNA sample quality assessed prior to analysis on the MLL FusionChip. Results: Nine different partners were accurately identified, including rare cases such as AF17, AF10, MSF, and P300. Overall agreement between routine diagnostic analysis and the MLL FusionChip was >90%. Conclusions: A previous study showed the analytical performance of this oncodiagnostic device is consistent with its claimed use. Taken together, these results collectively demonstrate the MLL FusionChip provides reliable data that may enrich minimal residual disease (MRD) follow-up in the clinical setting. Further studies are necessary to evaluate the clinical utility of the molecular classification of acute leukemia, and whether additional molecular information acquired with this device will contribute to refinement of the 2001 WHO classification. Expected consequences may be enhanced patient stratification, expansion of personalized therapeutic approaches, and optimized use of molecular targeted-based therapeutics. No significant financial relationships to disclose.