Prognostic relevance of telomere length and telomerase reverse transcriptase variant (rs2242652) on the multiple myeloma patients

Abstract Background The search for enhancement of multiple myeloma prognostic tools is an area of current research. This study aimed to assess the clinicopathological impact of telomere length and telomerase reverse transcriptase (TERT) polymorphic variant, rs2242652, on multiple myeloma (MM) patients. Methods Fifty MM patients and 50 healthy controls were included. Relative telomere length (RTL) and rs2242652 genotype polymorphic variants of TERT were analyzed using real‐time polymerase chain reaction (PCR). The MM patients' group was categorized into stage I (n = 16); stage II (n = 12), and stage III (n = 22). Results The median telomere length was significantly longer in MM patients' group (0.78) as compared to controls (0.43) (P = .001). Multivariate regression analysis revealed that MM patients with RTL < 0.5 had significant poor response for induction remission therapy with odds ratio 26.45. On the other hand, TERT genotyping analysis of rs2242652 revealed insignificant difference between cases and controls (P = .234), regarding to induction remission response. Survival analysis using Kaplan‐Meier curve revealed that patients with shorter telomere length and those with TERT genotype GA had shorter overall survival. Conclusion Telomere length and TERT rs2242652 genotype polymorphism could be used for refining risk stratification of MM patients.

size). The presence of at least one of these markers is considered sufficient for a diagnosis of multiple myeloma, regardless of the presence or absence of symptoms or CRAB features. 2 Telomeres are non-coding repetitive nucleotide sequences at the ends of all chromosomes, protecting the end of the chromosome from deterioration or from fusion with neighboring chromosomes. For vertebrates, the sequence of nucleotides in telomeres is TTAGGG, with the complementary DNA strand being AATCCC. 3 Telomere length (TL) is maximum at birth and decreases progressively with advancing age and thus is considered as a biomarker of chronological aging. 4 Telomere elongation is a common molecular feature of advanced malignancies. 5,6 Telomerase is a ribonucleoprotein consisting of two main components, telomere RNA component (TERC) and telomere reverse transcriptase (TERT), which act as a reverse transcriptase in the elongation of telomere length. 7 Telomerase activity has been found in myeloma cells of 90% of the newly diagnosed and relapsed patients, while in only 13% of patients in remission. 8 It is reported that genetic variations in TERT and TERC are involved in the many types of cancers, 9 such as lung cancer, 10,11 breast cancer, 12 colon cancer, 13 and melanoma. 14 The variant of TERT gene (rs2242652) which located on 5p15, intron 4 of TERT (encoding telomerase reverse transcriptase), seems to be associated with cancer risk as this SNP has been associated with multiple cancers, including breast, ovarian cancer, 15 and prostate. 16 The aim of the present study was to assess the relative telomere length and TERT gene polymorphism and their impact on survival and response to therapy in a cohort of Egyptian myeloma patients.

| Sampling
Two mL of EDTA peripheral blood samples were collected from patients and controls for complete blood count (CBC) and DNA extraction. Five mL of blood samples were left to clot to obtain sera to be tested for protein electrophoresis, serum immunofixation, LDH, B2M, creatinine, calcium, and albumin. Two mL of citrated blood samples were collected for ESR. Bone marrow aspirate and bone marrow biopsy specimens were collected from patients for morphologic and immunophenotypic diagnosis.

| DNA extraction
DNA was extracted using Thermo scientific Gene JET Whole Blood Genomic DNA Purification Kit according to the protocol of manufacturer's instructions. The extracted DNA was stored frozen at −20°C.
The DNA samples were quantified by NanoDrop instrument, and the samples were measured 17-45 ng/μL.

| Genotyping analysis
The detection of rs2242652 variant of TERT gene on chromosome 5 was followed the protocol of Custom TaqMan ® SNP Genotyping Assays manufacturing kits. The order of DNAs from cases and controls was randomized on 96-well plate over two runs with duplication of 4 samples all over the runs for quality control purpose. PCR plates were read on DNA-Technology DT Prime4 real-time instrument, and software v7.6 was used to determine the genotyping.

| Relative telomere length measurement
Leukocyte telomere length was measured using a real-time quan-

| Relative telomere length calculation
Relative T/S values were calculated according to 2 −ΔΔ Ct

| Statistical analysis
Data were analyzed with SPSS version 21. The normality of data was first tested with one-sample Kolmogorov-Smirnov test. Association between categorical variables was tested using chi-square test.
Continuous variables were presented as mean ± SD (standard deviation) for parametric data and median for non-parametric data. The two groups were compared with Student t test (parametric data) and Mann-Whitney test (non-parametric data). ANOVA test was used for comparison of means of more than two groups in parametric data. Spearman correlation used to correlate continuous non-parametric data.
The results were considered significant when the probability of error P-value < .05 and highly significant when the P-value < .001.

| RE SULTS
The patient's characteristics in comparison with controls are shown in  Table 6).
The impact of TL on the MM patient's OS is presented in Table 7 and Figure 1. MM patients with shorter TL (P < .5) had shorter OS as compared to those with long TL (P = .049). Moreover, TERT genotype GA had shorter OS as compared to GG and AA genotypes (P = .042) ( Table 8 and Figure 2).

| D ISCUSS I ON
Cumulative evidence suggests that genetic factors are involved in MM pathogenesis. 19 Association of telomere length in malignancy is a matter of debate. The genetic variability of the telomerase reverse transcriptase (TERT) could play a role in MM etiology as this gene is responsible for telomere homeostasis and their polymorphic variants have been incriminated in several human cancers. 12,20,21 Few studies were done concerning the impact of telomere length and genetic polymorphic pattern association as risk factor in those patients.
The main interesting finding in our study was that the telomeres were significantly longer in MM patients as compared to controls in spite the fact of short telomere with age. This finding was in agreement with that reported by Campa et al 22  In the present study, there were highly significant differences in age with relative telomere length. The older patients' age was associated with RTL < 0.5 and RTL > 1 was associated with younger patients' age. This result is parallel with previous findings that stated that telomere is shortened with age. 4,40,41 The limitation of the present study is few patients number. We recommended extension of this study on large cohort group to validate this finding.

| CON CLUS ION
Telomere length and TERT genotype (rs2242652) could be used for refining risk stratification of MM patients.

E TH I C A L A PPROVA L
This study did not include animals. Informed consent was taken from all subjects who participated in this study.

O RCI D
Salah Aref https://orcid.org/0000-0002-4822-5204 F I G U R E 2 Impact of TERT genotypes on overall survival of MM patients