Significance and implications of FHIT gene expression and promoter hypermethylation in acute lymphoblastic leukemia (ALL)

Blood samples from ALL patients were used for DNA extraction through phenol/chloroform method and/or DNA extraction kit (Qaigen, Germany). DNA concentration was estimated for purity at the absorbance of 260/280 nm and verified on 0.8–1% agarose gel.

Around 1.5–2 µg of DNA extracted was modified with bisulfite modification treatment (EZ-DNA Methylation Kit, Zymo Research Corporation, USA). A methylation specific-PCR (MSP-PCR) was done as per the protocol published by Golam Reza et al. [24]. The gel pictures were analyzed for bands by gel documentation system Flourchem HD2 (Cell Bioscience, USA).

From fresh blood samples using TRIZOL reagent (Ambion, Thermo Fisher, USA), RNA was extracted as per the manufacturer’s protocol. The concentration of RNA was examined using a UV–Vis Spectrophotometer (Thermo Scientific,Nano Drop 2000) with a wavelength of 260 nm. Integrity of RNA was checked using 1% Agarose gel (Additional file 1: Figure S1). The DNase, RNase- free (Thermo Scientific, USA) reagent kit was used to remove the traces of genomic DNA from RNA preparation and the Thermo Scientific^™ RevertAid^™ First Strand cDNA Synthesis Kit (Thermo Scientific, USA) was used for cDNA synthesis as per the protocol using primers as shown in Additional file Table S1. For qRT-PCR of FHIT mRNA expression, the PCR amplification was performed on Rotor-Gene Q accessories (Qiagen, Germany) using Maxima SyBR Green /ROX qPCR Master Mix (2x) with an internal control gene GAPDH as per the protocol (primers: Additional file Table S1). The cycle threshold (Ct) was analyzed and fold changes of gene of interest relative to GAPDH were done by the formula of 2^−ΔΔCt. The results were finally presented as an average log2 fold change which represented a decrease or increase in the mRNA expression of the respective gene.

Statistical analysis for the results of the study was done using IBM Statistics SPSS software, version-23 (IBM Corp, NY, USA). For clinico-pathological characteristics, categorical variables (gender and age) in case and controls were matched using chi-squared test. Expression analysis FHIT gene was expressed as cycle threshold values. Comparative analysis was performed Livak Method (^ΔΔCt) where data interpretation was exhibited in terms of relative fold expression.

The association of FHIT gene promoter hypermethylation and its subsequent expression were performed on previously obtained samples. The relative expression analysis of all the cases of ALL is shown in Fig. 2A. Of the 30 samples with decreased FHIT gene expression, 90% (27/30) were hypermethylated, while increased expression exhibited 20% (02/10) as methylated sequences (Table 3 and Fig. 2B). The results showed that FHIT mRNA expression was significantly lower in ALL cases with hypermethylation (p = 0.0008). The association of FHIT hypermethylation and its expression in different clinical parameters of ALL patients are shown in (Table 3).

Among immunophenotypes of ALL, B-ALL showed concordant pattern between hypermethylation and mRNA expression. Decreased FHIT expression was observed in 74.2% (26/35) wherein methylated DNA sequences accounted for 68.5% (24/35). Cases with increased mRNA expression of FHIT gene was observed in 25.7% (9/35) wherein 2.8% (1/35) showed hypermethylation. In comparison to T-ALL, a highly significant association was observed between the pattern of methylation and its mRNA expression with B-ALL cases (p = 0.000: Fig. 3A).

Among pediatric age group (< 18 years), 86% (18/21) showed decreased FHIT mRNA expression and accounted for 81% (21/26) of the methylated cases. Increased FHIT expression was detected in 19% (05/26) of the cases with 20% (01/5) methylated cases (Additional file Figure S3).

In low range WBC (< 10 × 109/L) group, FHIT expression was decreased in 74% (20/27) of the cases where 90% (18/20) showed hypermethylated sequences. When compared to rest of the cases in low range WBC group, a significant association was observed between increased expression (26%) of the cases and methylated (4%)/unmethylated group (86%) (p = 0.0006: Table 3 and Fig. 3B). FHIT mRNA expression level for both lower platelet and higher group of ALL patients were significantly decreased in methylated cases, as compared to increased expression observed in unmethylated cases. In lower platelet groups, highly significant association was observed between the methylation status and its mRNA expression among the ALL cases (p = 0.0002). Fusion transcripts were detected in 32.5% (13/40) of the cases and the frequency of these transcripts were as BCR-ABL (n = 4), ETV-RUNX1 (n = 6), MLL-AF4 (n = 2) and TCF-PBX1 (n = 1). When positive fusion transcripts were correlated with FHIT gene hypermethylation and its subsequent expression, 85% (11/13) cases showed decreased expression amongst which 77%(10/13) belong to methylated cases. However, association between the status of fusion transcript cases to expression and methylation did not reveal any significance (Table 3). A Comparison of fold change expression of FHIT gene with clinical parameters is depicted in Additional file 1: Figure S3

Recent advances in the molecular studies have led to deep understanding of ALL and a number of studies have provided evidence for the constitutive activation of FHIT gene and its association with ALL [16, 17]. Altered expression of FHIT is one of the potential mechanisms that promote tumor progression and its decreased expression is associated with signaling pathways in various cancers [30].

In the present study, the prognostic significance of FHIT expression in ALL patients was evaluated for FHIT hypermethylation. The mRNA expression of FHIT analyzed in ALL cases associated with a reduced pattern of expression. Significantly decreased expression was observed in majority of ALL cases (75%) than healthy controls (p = 0.01). The findings of the current study with FHIT mRNA down regulation clearly demonstrated the plausible involvement of FHIT gene expression to play an important role in the pathogenesis of ALL development. In a study by Hallas et al. [22] that confirms and agree with our report, loss of Fhit protein expression was detected in a majority of primary ALL cases and leukemia cell lines. Our results were in accordance with Chen et al. [31], where the expression of FHIT mRNA or protein was reported to be altered in 70% of cases. Further, Malak et al. [32] also agreed with our results wherein significantly less expression of FHIT mRNA was demonstrated for childhood ALL. In yet another report, Iwai et al. [21] demonstrated the RNA expression of the FHIT gene in cases of leukemia (ALL n = 11) and acute myeloid leukemia (n = 40). These results were consistent with our study and showed FHIT gene expression was totally lost or significantly reduced in 46% of the ALL cases and even 55% AML cases [33]. This also signifies the diverse role of FHIT in different types of leukemia. Reduced or loss of FHIT expression has been confirmed in solid tumors like head and neck, GIT, renal cell, breast, cervical cancers and others [34‐36]. It has been substantiated by Siprashvili et al. [37] that transfection of FHIT into tumorigenic cell lines reduces tumor process in nude mice; pointing to the fact that FHIT acts as a tumor suppresser gene. Studies have observed that dysregulation or loss of FHIT mRNA expression occurs frequently in various cancers [38‐40] but loss of the Fhit protein has been seen in various hematological malignancies as well [40]. Substantial evidence from a few studies suggested that the alteration of normal FHIT function could be an important event in the pathogenesis of many human hematological malignancies including ALL and the altered FHIT gene expression is specifically identified factor and highly persistent event in leukemia [21, 41]. The frequent alteration in particular reduction in FHIT expression as found in this study in ALL and other quoted studies gives an evidence that inactivating changes at the FHIT locus could supplement for the development to leukemia’s [22]. When the results of the studies are taken together with our report where clinical sample of ALL were directly examined for FHIT expression, the results demonstrate that dysregulation of FHIT expression has a plausible link in the pathogenesis of ALL. Evidences show that numerous tumor suppressor genes are inactivated by promoter region methylation in different malignancies like p15INK4B gene was frequently inactivated by promoter methylation in myelodysplastic syndrome and AML [42]. Similarly FHIT gene methylation has been depicted in few solid tumors, like GIT tumors, lung and breast cancers, where it was linked to dysregulated expression [43, 44]. Further, loss or reduced expression of FHIT protein due to its altered methylation was shown to be connected to progression of disease in certain malignancies. [45, 46] In the current study, the results showed FHIT mRNA expression was significantly decreased in those ALL cases that exhibited with hypermethylated FHIT sequences (p = 0.0008). In concordance to our results, study by Bahari et al. [47] showed that the FHIT gene hypermethylation was significantly abundant in ALL cases with FHIT expression seen significantly lower in ALL patients.

The genetic and epigenetic modification in critical regulatory genes may affect gene expression or the structure and function of specific gene products which can lead to leukemia development. In this investigation, hypermethylation of the FHIT gene was found to be considerably greater in ALL patients than in healthy controls. Our findings show that epigenetic changes and abnormal gene expression of FHIT in ALL patients may play a role in the disease’s progression. Consistent with our findings, Malak et al. [32] found a substantial decrease in the expression of FHIT in ALL compared to healthy controls. Furthermore, another study demonstrated that when compared to controls, ALL samples had significantly lower expression of FHIT with an increase in methylation frequency [31]. The association becomes crucial according to several studies as DNA methylation is the most often identified change in individuals with ALL. Further, FHIT gene deregulation in particular the decreased expression due to promoter gene methylation has been related to solid tumors for disease progression [33, 48]. In accordance to our report, a study by Hallas et al. [22] established the role of FHIT gene alteration in ALL where loss of FHIT mRNA and its product in majority of cases in contrast to their presence in almost all healthy control samples specified its plausible contribution to the pathogenesis ALL. FHIT gene as reported is emerging as a putative therapeutic target not only for ALL but other cancer types as well. A meta-analysis conducted by Wu et al. [16] where it was shown that the inactivation of FHIT gene by hypermethylation is associated with an elevated risk of non-small cell lung cancer. Therefore, the aberrant methylation patterns along with decrease in the expression of FHIT observed as seen in our study and other ones cannot be interpreted as simple secondary event, but rather must play a critical role in determining the malignant phenotype.