The Egyptian Journal of Haematology

ORIGINAL ARTICLE
Year
: 2019  |  Volume : 44  |  Issue : 2  |  Page : 91--97

Serum HOX transcript antisense RNA expression as a diagnostic marker for chronic myeloid leukemia


Muhammad T AbdelGhafar1, Alzahraa A Allam2, Sara Darwish3,  
1 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt
3 Department of Oncology, Faculty of Medicine, Tanta University, Tanta, Egypt

Correspondence Address:
Muhammad T AbdelGhafar
Department of Clinical Pathology, Faculty of Medicine, El-Gash St, Medical Campus, Tanta University, Tanta, 31511
Egypt

Abstract

Background The diagnosis of chronic myeloid leukemia (CML) is complex based mainly on the presence of Philadelphia chromosome with morphological characteristics and leukocyte alkaline phosphatase (LAP) score. However, it can be confused and should be differentiated from different disorders including specifically the leukemoid reaction (LR). HOX transcript antisense RNA (HOTAIR) expression may be a useful biomarker for detection of cancer. Objective This study was aimed to explore the accuracy of HOTAIR as a diagnostic marker for CML. Patients and methods During the period from 1 February 2017 till 31 December 2018, patients with CML and LR were enrolled in this study. The LAP score was assessed for all patients by cytochemistry on their peripheral blood smear. HOTAIR expression was relatively quantified using the RT-qPCR technique. The diagnostic accuracy of serum HOTAIR expression in differentiating both groups was evaluated. Results A total of 66 patients were included and assigned into two groups; CML patients (group I) and LR patients (group II). The serum HOTAIR expression level was significantly higher in the CML group than the LR group (P<0.001). The area under curve (AUC) of the serum HOTAIR was detected as 0.883 with a sensitivity of 67.7% and a specificity of 85.7% at a cutoff more than 2.8 for CML diagnosis which is slightly higher than that for the LAP score (AUC difference=0.0369; P=0.334). The increased serum HOTAIR expression is independently related with poor clinical, laboratory features of CML and the decreased LAP score. The diagnostic accuracy of HOTAIR was detected to be increased if combined with the LAP score (AUC=0.900). Conclusion Our study findings suggested that the serum HOTAIR can serve as a potential marker for the CML with high diagnostic accuracy specifically if combined with the LAP score.



How to cite this article:
AbdelGhafar MT, Allam AA, Darwish S. Serum HOX transcript antisense RNA expression as a diagnostic marker for chronic myeloid leukemia.Egypt J Haematol 2019;44:91-97


How to cite this URL:
AbdelGhafar MT, Allam AA, Darwish S. Serum HOX transcript antisense RNA expression as a diagnostic marker for chronic myeloid leukemia. Egypt J Haematol [serial online] 2019 [cited 2020 Mar 28 ];44:91-97
Available from: http://www.ehj.eg.net/text.asp?2019/44/2/91/271075


Full Text



 Introduction



Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by reciprocal translocation between chromosomes 9 and 22 defined as the Philadelphia chromosome with consecutive activation of the BCR-ABL tyrosine kinase that promotes CML pathogenesis and progression [1]. Newly raised epidemiological estimates reported that the average annual number of newly diagnosed CML ∼100 000 cases globally, which present this chronic disorder as a critical health issue [2]. According to the recent diagnostic criteria of the WHO [3], CML is confirmed through the detection of the BCR-ABL1 fusion gene by means of molecular techniques in addition to peripheral blood (PB). However, a bone marrow (BM) aspirate is considered essential for morphological evaluation of the CML phase and also for the genetic study [4].

CML should be differentiated from the leukemoid reaction (LR) which is considered a reactive condition to infection or systemic inflammation and is characterized by leukocytosis with a shift to the left and neutrophilia mainly mature [5]. The differentiation is based on certain criteria as the absence of immature cells, basophilia or monocytosis, and the BCR-ABL translocation with elevated leukocyte alkaline phosphatase (LAP). As, some of these criteria is not conclusive with the need of BM sample for the BCR/ABL gene, new markers with high diagnostic accuracy is needed to detect individuals with CML, and differentiate them for the LR.

Long noncoding RNAs (lncRNAs) are a class of noncoding RNA molecules consisting of more than 200 nucleotides [6]. They affect gene expression by multiple mechanisms involving histone modification, transcription factor activation, and competing with microRNAs [7]. Evidences suggested their role in cancer cell proliferation, invasion, and metastasis [8] and detected its expression in the body fluid of cancer patients [9]. Thus, lncRNAs are promising novel biomarkers for cancer diagnosis.

HOX transcript antisense RNA (HOTAIR) is a polyadenylated lncRNA of 2.2 kb in length with six exons, which is transcribed from the HOXC gene located on chromosome 12q13.13 [10]. HOTAIR seems to control the chromatin dynamics via interaction with histone methyltransferase enhancer of zeste homolog 2 and the lysine-specific histone demethylase 1A that modulates the methylation of lysine 4 of histone H3 (H3K4) [11]. This leads to histone methylation as the promotor of certain genes resulting in epigenetic changes and transcriptional regulation. Moreover, HOTAIR can competitively antagonize the activity of certain miRNAs as miR-130a [12], miR-331-3p [13], miR-124 [14], miR-218 [15], and miR-193a [16]. HOTAIR had been extensively studied and found to be expressed in relation to various solid tumors [17],[18],[19],[20],[21],[22],[23],[24],[25]. Therefore, HOTAIR expression may be a useful biomarker for the detection of cancer [26]. However, few studies investigate its relation to hematological malignancy. To our knowledge, no data is available till now for the association of HOTAIRE with CML.

As, the HOTAIRE expression was detected to be associated with malignancy, and was found to be expressed in body fluids and serum, it could be useful as a marker in hematological malignancies as CML. This study aimed to explore the value of HOTAIRE as a potential diagnostic marker in patients with CML.

 Patients and methods



Study design

This study was of cross-sectional, retrospective design. It was conducted at the Tanta University Hospitals during the period from 1 February 2017 till 31 December 2018. It was adopted to validate the diagnostic accuracy of HOTAIRE relative expression in patients with CML. The study protocol was approved by our national ethics committee and the study was performed in accordance with Helsinki Declaration. All patients were needed to be informed and consented before participation.

Study cohort

All patients with CML diagnosed according to the recent WHO diagnostic criteria for myeloproliferative neoplasms 2016 [3] and positive for Philadelphia chromosome (BCR-ABL major P210) were eligible for this study in addition to patients with LR diagnosed based on PB morphology as leukocytosis, toxic granulation, normal platelets count, high C-reactive protein, absence of splenomegaly with improvement of blood picture after a 3-month follow-up. According to our eligibility criteria, 31 cases of CML from the Hematology Unit of the Internal Medicine Department with 35 cases with LR from the ICU were recruited in a consecutive way out of 71 totally screened cases as three cases with other types of malignancies were excluded and two cases refused participation ([Figure 1]).{Figure 1}

Clinical evaluation

All cases were asked about their disease history and their medical records were reviewed. Furthermore, they were subjected to full clinical examination specifically for evaluation of the presence splenomegaly. Abdominal ultrasongraphy was performed for all patients to assess the presence of splenomegaly.

Sampling

From each participant, ∼10 ml of venous blood was taken by standard venipuncture using a sterile syringe and further placed into two plain vacutainer tubes for lactate dehydrogenase (LDH), uric acid estimation, and serum HOTAIR quantitation, then into sodium heparin tube for LAP score determination and then into K3 EDTA tube for complete blood count. The samples were sent immediately to the laboratory whereby serum separation from the blood sample in plain tube was done via centrifugation for 15 min at 3000 rpm. The serum samples were preserved in sterile RNase free tubes at −80°C till investigated. The PB smears were prepared from the both EDTA and heparin blood.

Laboratory investigations

Complete blood count was performed on ERMA PCE-210 automated cell counter, Japan with PB smears were stained by Giemsa stain and microscopically examined. LDH was measured by enzyme kinetic method and uric acid by colorimetric methods on Konelab 60 I Thermo Scientific auto-analyzer (Thermo Scientific, Vantaa, Finland), using commercial kits supplied by Thermo Fisher Scientific (Waltham, Massachusetts, USA). LAP activity score was semi-quantitatively assessed for all patients on their PB smears. The smears were prepared using the heparinized blood samples and cytochemically stained by LAP using kit provided by Sigma-Aldrich Inc. (St Louis, Missouri, USA) according to manufacturer instruction. The alkaline dye mixture containing sodium α-naphthyl phosphate as substrate, and fast blue RR as the diazonium salt was placed over the blood smears after fixation for 30 min and then rinsed by deionized water. After that, Mayer’s hematoxylin solution was added for 10 min. The slides were examined microscopically and 100 consecutive segmented neutrophils and band form were selected and rated from 0 to 4+ based on quantity and intensity of precipitated dye within the cytoplasm of these cells. The score was calculated via multiplying the cell rating degree by their number for each degree and score value ranging from 0 to 400 can be obtained.

Molecular detection of serum HOX transcript antisense RNA relative expression

The RNA was extracted from the serum using miRNeasy serum/plasma RNA extraction kit, Qiagen (Hilden, Germany) according to the manufacturer protocol. Reverse transcription was performed using miScript II reverse transcription kit (Qiagen). 20 μl of reaction mixture containing about 5 μl RNA yield (100 ng), 2 μl miScript reverse transcriptase mix, 4 μl 5× miScript HiFlex buffer, 2 μl 10× miScript nucleics mix was incubated for 60 min at 37°C then for 5 min at 95°C for enzyme inactivation.

The HOTAIR was further amplified by PCR using TaqMan RNA assay kit provided by Thermo Fisher Scientific. About 1.5 μl of RT product (45 ng RNA) was added to 10 μl 2× TaqMan universal PCR master mix and 1.0 μl 20× TaqMan RNA assay mix to reach a final reaction volume of 20 μl. The HOTAIR and U6 (reference gene) primers were supplied in the assay mix as: F:

5′-GGAAAGATCCAAATGGGACCA-3′; R: 5′-CTAGGAATCAGCACGAAGCAAA-3′ and F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′; R: 5′-CGCTTCACGAATTTGCGTGTCAT-3′, respectively. The cycling conditions were performed on Applied Biosystem real time PCR (Foster City, California, USA), step I version as initial holding at 95°C for 5 min; 40 cycles, 95°C for 30 s, 62.5°C for 30 s, and 72°C for 30 s. The cycle threshold (CT) was determined and the HOTAIR expression was relatively quantified using the 2ΔΔCt equation [27] whereby ΔΔCt=ΔCt (HOTAIR)−ΔCt (U6) for each sample.

Statistics

SPSS (IBM Corp, version 22.0, Armonk, NY, USA), MedCalc 18 (MedCalc Software, Ostend, Belgium) and GraphPad Prism version 8.0 (San Diego, California, USA) softwares were used for the statistical analysis and graph building of this study results. The CML and LR groups’ data were compared via t-test for the numerical normally distributed data, Mann–Whitney test for non-normally distributed data, and χ2-test for the nominal data. The HOTAIR expression level was correlated with clinical and laboratory characteristics of the CML group using Pearson correlation. All of the significant parameters in the univariate correlation were assessed by multivariate logistic regression analysis. The diagnostic performance of HOTAIR was assessed via the receiver operating characteristic (ROC) curve analysis. All P values less than 0.05 were considered as significant.

 Results



Basic characteristics of the study cohort

This study was conducted at Tanta University Hospitals on ∼66 patients. The CML patients (group I) was 31 cases, aged 48.19±3.79 years, presented with huge splenomegaly, and mean hemoglobin 8.57±0.66 g/dl, platelet count 522 387±136 162/μl and total leukocytic count 123 580±40 964 cells/μl with median blast cells 2.0%. Their median LAP score was 21.0. The LR patients (n=35) were assigned to group II and aged 48.49±3.70 years. Their mean hemoglobin 10.79±1.03 g/dl, platelets count 348 285±101 763/μl and leukocytic count 37 555±14 906 cells/μl. No blast cells were found in any of these patients. Their median LAP score was 113.0. The demographic, clinical and laboratory characteristics of the studied groups are further demonstrated in [Table 1].{Table 1}

Serum HOX transcript antisense RNA expression between the studied groups

In this study, the relative serum HOTAIR expression level was demonstrated in [Table 2] and [Figure 2] and found to be significantly higher in CML group than the LR group (P<0.001).{Table 2}{Figure 2}

Relative HOX transcript antisense RNA expression and group characteristics

Univariate Pearson correlation analysis was performed between the serum HOTAIR expression and the different demographic, clinical and laboratory characteristics of all study cohorts ([Table 3]). The serum HOTAIR expression was found to correlate positively with the presence of splenomegaly, LDH, uric acid, platelets, white blood cells, blast cells% and negatively with hemoglobin level and LAP score. All significant parameters were analyzed by multiple logistic regressions which revealed that the increased serum HOTAIR expression was still independently related to the presence of splenomegaly, increased white blood cells count, elevated blast cells% and decreased LAP score.{Table 3}

HOX transcript antisense RNA diagnostic accuracy

In order to assess the diagnostic accuracy of serum HOTAIR relative expression in CML patients, we use the ROC curve analysis ([Table 4] and [Figure 3]). The area under curve (AUC) of relative serum HOTAIR expression was estimated as 0.883 on differentiating the CML patients from the LR patients. The cutoff suggested was more than 2.8 which achieve 67.7% sensitivity, 85.7% specificity, 80.8% positive predictive value and 75.0% negative predictive value. The AUC of relative serum HOTAIR expression was found to be higher than AUC of the LAP score with no significant difference (AUC difference=0.0369; 95% confidence interval (CI): −0.0379 to 0.112; P=0.334). The diagnostic accuracy serum HOTAIR was found to be increased if combined with the LAP score (AUC=0.900).{Table 4}{Figure 3}

 Discussion



CML is a chronic hematological neoplasm. The diagnosis of CML is based mainly on the presence of PCR/ABL fusion gene besides the morphological characteristics of BM and PB. However, it can be confused with different chronic myeloproliferative disorders as well as with benign condition as the LR. Despite, LAP score is used to differentiate between both disorders, it offers a low diagnostic efficacy. HOTAIR as a member of the noncoding RNA may act as a marker for diagnosis of different tumors either solid or hematological neoplasms. However, it is extensively investigated in different tumors; unfortunately, its diagnostic role in CML had not been investigated yet.

In this study, we aimed to validate the accuracy of serum HOTAIR expression as a potential diagnostic marker for CML. We recruited two conflicting case series of CML and LR and the diagnostic value of the serum HOTAIR in differentiating both disorders was investigated. The serum HOTAIR expression was relatively quantified in both cases by the RT-qPCR.

We detected that the serum HOTAIR relative expression level was significantly higher in CML cases if compared with LR cases. It may suggest its diagnostic value in CML cases. Several studies had previously conducted and support these results in various solid and hematological tumors. The expression level of HOTAIR was found to be higher in the plasma of non-small cell lung cancer (NSCLC) [19], glioblastoma multiforme [20], esophageal squamous cell carcinoma patients (ESCC) [21], colorectal carcinoma [18] and breast cancer (BC) [23],[24] patients, and in the saliva of pancreatic carcinoma [22], or in mononuclear cells in acute monocytic leukemia [28] than in healthy controls. The HOTAIR was detected to be overexpressed in different cancerous tissues such as cervical [19], breast [20], gastric [24] and cancer tissues if compared with adjacent nontumorous tissues.

In this study, multivariate analysis showed that the serum HOTAIR relative expression level was independently related to the decreased LAP score and the poor clinical and laboratory features as the presence of splenomegaly, increased leukocytic count, and blast cell percentage in the PB which suggest the predictive role besides its diagnostic value as for the already used LAP score. Several studies correlated the HOTAIR expression level with the tumor clinical, histopathological characteristics and found that it was correlated with a larger tumor size [18], enhanced invasion [17], higher grade in [17],[18],[19],[20],[21], and lymph node metastasis [17],[18],[19].

In this study, we detect that the diagnostic accuracy of serum HOTAIR relative expression was satisfactory and nonsignificantly exceeding the LAP score accuracy. However with the same specificity, serum HOTAIR relative expression offers a much higher sensitivity for CML diagnosis than assigned by the LAP score. Growing evidences had suggested the high diagnostic accuracy of HOTAIR in several tumors. On differentiating malignant patients from healthy controls, the AUC was found as 0.793 in ESCC [21], 0.791 in NSCLC [19], 0.803 in cervical cancer with 60% sensitivity, 87.2% specificity [18], 0.913 with 86.1% sensitivity and 87.5% specificity in glioblastoma multiforme [20], 0.80 with 69.2% sensitivity, 93.3% specificity, 93.3% in BC [24] and 0.709 with 64.62% sensitivity and 75.38% specificity [23]. The diagostic accuracy of HOTAIR were found higher than other routinely used markers as in BC which was higher than the carcinoembryonic antigen and carbohydrate antigen 15-3 [24] and in ESCC which was higher than carcinoembryonic antigen [21].

Moreover, a combination of the serum HOTAIRE with the LAP score offered greater accuracy than assigned by each of them separately. This is in accordance with the studies combined HOTAIR with CCAT1 in colorectal carcinoma [25] or carcinoembryonic antigen in NSCLC [19] and proved to present a higher diagnostic accuracy than exhibited by everyone solely.

 Conclusion



This study suggested that serum HOTAIR expression can offer high diagnostic accuracy for differentiating CML from LR specifically if combined with the LAP score. It is recommended as it is noninvasive, and easily assayed on a serum specimen.

Acknowledgements

All authors contributed in the writing of this manuscript. Muhammad T. AbdelGhafar participated mainly in the study design and the biochemical analysis. Alzahraa A. Allam and Sara Darwish contributed specifically in patients’ selection and analysis of data.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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