Comprehensive Analysis of Immunophenotype and Transcriptome in Indian T-Acute Lymphoblastic Leukemia - a Prospective Study

T-cell acute lymphoblastic leukemias (T-ALLs) are aggressive hematologic tumors that result from the malignant transformation of T-cell progenitors. T-ALLs are classified immunologically into immature (pro- and pre-T), cortical and mature T-ALL. ETP-ALL is a recently described subtype of immature T-ALL. T-ALL is characterized by overexpression of oncogenic transcription factors that drive the malignancy: TAL1, TAL2, LYL1,LMO1, LMO2; TLX1, TLX3, NKX2.1, NKX2.2, NKX2.5, MEF2C and HOX genes. This study was conducted to understand the landscape of protein-coding genes using RNAseq in immature, cortical and mature T-ALL patients in India.

This was a prospective study done on 134 T-ALL cases. The T-ALL patients were divided into 2 cohorts: discovery (n=35; immature 13, cortical 17, mature 5) and validation cohort (n=99; immature 45, cortical 44, mature 10). Total RNA seq was done in the discovery cohort. Clinical and prognostic relevance of lesions identified in the discovery cohort were studied in validation cohort.

On RNAseq, a total of 2,318 genes were most differentially expressed between the 3 subtypes. In immature T-ALL cases, transcription factors controlling early hematopoiesis (MEF2C, TP63, HHEX, RUNX2, HOXA10, HOXA9, RUNX1T1 and ZBTB16) were highly expressed. LYN, a tyrosine kinase gene was also highly expressed. BAALC and MN1 genes, previously reported in acute myeloid leukemia, but not emphasized in reported genomics studies in the West, were overexpressed. Interestingly, LMO2 was overexpressed in the absence of significant overexpression of LYL1, a gene reported to be critical for oncogenic functions of Lmo2. In cortical T-ALL, the cortical thymocyte-defining CD1A gene was overexpressed. Homoebox domain genes NKX2-1, TLX1, TLX3, involved in T-cell development were overexpressed. In addition, FAT1, FAT3, EREG, CD1C, AKAP-2, IL-4, PRTG, TCL-6, ZP1 and TRAV genes were overexpressed. On examining CD1a+/sCD3+ and CD1a+/sCD3- groups, we found that in contrast to reported literature, TLX1 was highly expressed in the former group and TLX3 in the latter. In mature T-ALL, APC2, BCL3, CCR4, CDKN2A, EML4, HIST1H4G, HIST2H2B, NCOR2, ST20 and TRAV22 genes were overexpressed. Conventional drivers of T-ALL leukemogenesis - LYL1, TAL1 and LMO1 were not differentially expressed in our patients suggesting a difference between Indian and Western T-ALL patients.

The clinical significance of expression of MEF2C, BAALC, HHEX, LYL1, TAL1 and FAT1 genes was studied in validation cohort by real-time PCR. High expression of MEF2C and BAALC gene was associated with immature immunophenotype (IPT) as compared to cortical and mature IPT (p=0.009; 0.008, respectively). Patients with ETP-ALL had higher expression of MEF2C and BAALC gene (p=0.018; 0.014, respectively). High MEF2C and BAALC expression were associated with CD34 positivity (p=0.004; 0.032, respectively). High BAALC expression was also associated with aberrant expression of the myeloid markers (p=0.021). Although not statistically significant, high HHEX expression was associated with immature IPT (p=0.072) and ETP-ALL IPT (p=0.06). Although LYL1 expression did not correlate with immature IPT, ETP-ALL was associated with higher LYL1 expression (p=0.039). Higher expression of FAT1 gene was associated with cortical IPT (p=0.026). Low XIST expression was more frequently associated with non-ETP-ALL IPT (p=0.017). TAL1 expression did not correlate with IPT.

On survival analysis, only MEF2C gene expression was associated with EFS and OS. Compared to low MEF2C expressors, patients with high MEF2C had a lower event free survival (EFS) (73.58% vs 51.38%, p=0.01). High expression was found to be a significant predictor of overall survival (OS) (81.11% vs 96.43%, p=0.012). MEF2C emerged as driving oncogene in our T-ALL patients which can be used as predictor of EFS and OS. Expression of BAALC, HHEX, LYL1, TAL1, FAT1 and XIST genes did not correlate with EFS and OS.

In this first study correlating T-ALL IPT with transcriptome profile in Indian T-ALL patients, we noted interesting differences from the West, potential subjects of further study. MEF2C gene expression emerged as the predictor of poor EFS and OS in T-ALL patients. Lastly, increased expression of LYN gene in our patients suggests that our immature T-ALL patients may benefit from tyrosine kinase inhibitors targeting Lyn.