FAT1 Expression and Mutation Status In Adult Acute Lymphoblastic Leukemia

FAT1 belongs to the FAT protocadherin family, a drosophila homologous gene involved in development processes. Recently, FAT1 gained large interest as it is mutated in various cancers. Besides the known function of cell-cell interaction and polarity, FAT1 loss of function mutations have been linked to dysregulation of the WNT pathway in solid tumors. In acute lymphoblastic leukemia (ALL), aberrantly high expression of FAT1 was claimed to be associated with inferior outcome in pediatric B-lineage ALL. Herein, we investigated the yet unknown frequency and relevance of FAT1 expression and mutation in a large, homogenously treated cohort of adult ALL patients.

We investigated FAT1 expression in diagnostic bone marrow (BM) samples of 112 T-ALL, 122 B-lineage ALL, and additional 63 early T-cell precursor (ETP) ALL patients by real time (RT)-PCR. Patients were enrolled in trials of the German Multicenter Study Group for Adult ALL (GMALL) and outcome was investigated for patients into GMALL trials 06/99 and 07/03. Using the T-cell line BE13 as reference, we defined patients with FAT1 expression higher than BE13 as FAT1pos (0.01-38.5) and patients with a lower expression as FAT1neg (<0.01). We additionally examined peripheral blood (PB), BM, CD34+-, CD3+-cells from healthy donors. FAT1mutation status was investigated in 68 T-ALL patients. For mutation analyses customized biotinylated RNA oligo pools (SureSelect, Agilent) were used to hybridize the targeted regions, followed by 76-bp paired-end sequencing on an Illumina Genome Analyzer IIx platform.

Normal hematopoietic cells including unselected BM cells, CD34+-progenitors, or CD3+ T-cells from healthy donors lacked FAT1 expression (<0.01) with the one exception of a CD34+-sample (0.03). In contrast, ALL samples aberrantly expressed FAT1: 32% of B-lineage ALL (0.01-38.5) and 54% of T-ALL cases (0.01-31.2) showed elevated FAT1 expression, with a lower frequency in the immature ETP-ALL subgroup (17%; 0.01-15.2). FAT1 expression was associated with a more mature immunophenotype (T-ALL: thymic 74%, mature 45%, early 4%, p<0.001; B-lineage ALL: pre B-ALL 57%, common ALL 26%, pro B-ALL 9%, p=0.04). No significant differences between FAT1pos and FAT1neg patients were observed regarding age and sex. FAT1pos T-ALL patients more frequently showed a white blood cell count (WBC) >30.000/µL at diagnosis compared to FAT1neg T-ALL patients (78% vs. 42%, p<0.01). Whereas no negative prognostic impact was observed for FAT1 expression in B-lineage ALL or T-ALL with respect to overall survival or remission duration, lack of FAT1 expression was associated with primary resistance to induction therapy in T-ALL (T-ALL: FAT1neg 12%, FAT1pos 0%, p=0.04). In addition, we found an unexpected high rate of FAT1 mutations (exclusively missense mutations) with 8 of 68 T-ALL patients (12%). The mutation spectrum, mainly located in the cadherin domains, was similar to the distribution of FAT1 mutations in solid tumors. No difference were observed between FAT1 mutated (FAT1mut) and FAT1 wild-type patients (FAT1wt) with regard to sex, age, WBC, and presentation of antigens associated with an early differentiation stage. FAT1 mutations were more frequent in early T-ALL (3/12, 25%) and in thymic T-ALL (5/41, 12%) than in T-ALL patients with a mature immunophenotype (0/15, 0%). FAT1 expression was more common in FAT1wt T-ALL compared to FAT1mut T-ALL patients (50% vs. 25%).

This first comprehensive analysis on FAT1 in a large cohort of adult patients with ALL shows a high frequency of FAT1 expression. Higher FAT1 expression occurred in ALL patients with more mature immunophenotype linking FAT1 to cell-cell adhesion and polarity, thymic homing and interaction with the BM niche. This yet unreported high mutation rate of 12 % in adult T-ALL makes FAT1 to one of the most frequently mutated genes in T-ALL. The link of inactivating FAT1 mutations to aberrant activation of the WNT pathway, as reported in solid tumors, might allow the development of refined treatment options. In summary, these data make FAT1 a promising candidate for disease monitoring, risk stratification and development of targeted therapies.

Krebs:Illumina: Honoraria. Greif:Illumina: Honoraria.