Blood Res 2017; 52(2):
Published online June 22, 2017
https://doi.org/10.5045/br.2017.52.2.106
© The Korean Society of Hematology
1Inflammation and Inflammatory Diseases Research Center, Faculty of Medicine, Mashhad University of Medical sciences, Mashhad, Iran.
2Hematology Department, Ghaem Hospital, Faculty of Medicine, Mashhad University of Medical Sciencess, Mashhad, Iran.
3Molecular Diagnostic Unit, Research and Education Department, Razavi Hospitals, Mashhad, Iran.
4Department of Medical Informatics, Molecular Medicine Department, Faculty of Medicine, Mashhad University of Medical sciences, Mashhad, Iran.
5Department of Modern Sciences and Technologies, Molecular Medicine Department, Faculty of Medicine, Mashhad University of Medical sciences, Mashhad, Iran.
Correspondence to : Reza Torshizi, Ph.D. Department of Modern Sciences and Technologies, Mashhad University of Medical Sciences, Mashhad, Razavi Khorasan 91388-13944, Iran. torshiziR901@mums.ac.ir
Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy with very poor prognosis and short survival, caused by the human T-lymphotropic virus type-1 (HTLV-1). The HTLV-1 biomarkers trans-activator x (TAX) and HTLV-1 basic leucine zipper factor (HBZ) are main oncogenes and life-threatening elements. This study aimed to assess the role of the
Forty-three HTLV-1-infected individuals, including 18 asymptomatic carriers (AC) and 25 ATLL patients (ATLL), were evaluated between 2011 and 2015. The mRNA expression of
Significant differences in the mean expression levels of
The HTLV-1 PVL and expression of
Keywords HTLV-1, ATLL,
Human T-cell leukemia virus type 1 (HTLV-1) is a lentivirus that belongs to the Retroviridae family in the genus Deltaretrovirus [1]. About 20 million infected people live in Africa, the Caribbean, Asia, and Central and South America [2].
The
According to the Revised European American Lymphoma (REAL) classification and World Health Organization (WHO), ATLL is classified into four subtypes (acute, lymphoma, chronic, and smoldering) based on the following symptoms: the persistence of flower-like cells in peripheral blood smear, elevated lactate dehydrogenase (LDH), hypercalcemia, skin lesions, organomegaly, and lytic bone injury [7,8]. The most aggressive ATLL types are the acute and lymphoma types. The acute type has the highest prevalence rate (60%) and shortest survival time (4–6 mo) among all ATLL types, and it shows some of the most aggressive symptoms (e.g., hepatosplenomegaly, lymphadenopathy, a high frequency of atypical lymphocytes with nuclear hypersegmentation, as well as high levels of LDH and calcium in serum). The lymphoma type has a prevalence rate of 20% and a 9–10-month survival period, which demonstrates the aggressiveness of ATLL. The chronic and smoldering types have a survival period of 2–5 years and are less frequently associated with aggressive clinical conditions [2,9]. The primary infection is followed by a long-term latency period (20–40 yrs). Then, leukemogenesis of infected T-cells occurs in a multistep process via genetic and epigenetic alterations [10].
Although the exact pathogenic mechanism of ATLL is unknown, it is found that clonal expansion of infected T-cells increases the number of viral progeny, leading to plus-strand transcription of the HTLV-1 genome. In addition to the major gene described above, the genome also encodes other factors including accessory genes (open reading frames I and II), regulatory genes (
Transactivation of the viral LTR and functional cellular genes is synchronized by TAX; however, TAX does not bind to DNA, but may upregulate the expression of cell survival factors such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), which function as anti-apoptotic elements [11]. Previous studies have suggested that TAX protein promotes downregulation of the expression of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) and activating transcription factor (ATF) signaling proteins, as well as DNA repair proteins (e.g., P53 and retinoblastoma [Rb]). Therefore, TAX may play a critical role in malignant T-cell proliferation [12,13]. Regarding the TAX antigen structure, a strong immune response in HTLV-1-infected patients can eliminate or suppress TAX expression [14]. However, genetic alteration of HTLV-1 by methylation, deletion, or mutation in the 3' LTR region can lead to low TAX protein expression [15]. These mechanisms may allow ATLL cells to escape detection by the host immune system, enabling latent activation of the HTLV-1 genome [16]. The
A case series study was conducted on 43 HTLV-1-infected individuals in the Departments of Hematology and Oncology of Mashhad University of Medical Sciences, Mashhad, Iran, and in Ghaem and Imam Reza Hospitals from July 2011 to November 2015. Patients with suspected ATLL were investigated, and on the basis of symptoms and laboratory test results, 25 patients (12 males and 13 females) were diagnosed with ATLL and placed in the ATLL group. The remaining 18 patients (7 male and 11 female) were considered healthy, asymptomatic HTLV-1-infected carriers, and placed in the control group. HTLV-1 seropositive reactivity was checked for all subjects, and the presence of HTLV-1 was confirmed by conventional PCR. ATLL patients were further divided into four groups: acute, lymphoma, chronic, and smoldering, based on the criteria reported by Shimoyama [18].
cDNA was synthesized using random primers and reverse transcriptase (Bioneer, South Korea) according to the manufacturer's instructions. The reaction conditions were 12 cycles of 30 seconds at 24℃, 4 minutes at 44℃, and 30 seconds at 55℃. Glyceraldehyde-3-phosphate dehydrogenase (
After obtaining the desired sequences for the
Real-time PCR (TaqMan method) was performed in a Rotor-Gene 6000 cycler (Corbett, Hilden, Germany). Duplicate samples were included in the analysis, and expression is reported relative to that of a housekeeping gene (β2-microglobulin). The primers and probes used to amplify the corresponding genes are shown in Table 1. The PCR conditions were as follows: 95℃ for 4 minutes, followed by 45 cycles of denaturation at 94℃ for 15 seconds, annealing at the optimum temperature 20 seconds, and extension at 72℃ for 20 seconds.
To evaluate the HTLV-1 PVL, PBMCs were isolated from ethylenediaminetetraacetic acid (EDTA)-treated blood samples, as described above under “Isolation of PBMCs and extraction of RNA,” and DNA was extracted from the PBMCs, using a DNA extraction kit. Real-time PCR was performed using a commercial absolute quantification kit (Novin Gene, Iran) to measure the PVL of HTLV-1, using specific primers and a fluorogenic probe. HTLV-1 copy number was reported as the actual amount of cellular DNA quantified, using the albumin gene (
Different therapies were administered to the study participants according to the type of disease. The most common first-line treatment for T-cell lymphoma was combination chemotherapy, such as cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or cyclophosphamide, vincristine, doxorubicin, and dexamethasone (CVAD), which was administered to most patients [19,20].
Data were analyzed using SPSS version 18, and the results are reported as mean±standard deviation (SD). The Kolmogorov-Smirnov test was conducted to check variable for normal distribution. The Mann-Whitney U test was performed to analyze the two groups. Spearman's rank correlation coefficient was used to determine the relationship between study parameters. Kaplan-Meier analysis was performed to evaluate survival.
In this study, 25 ATLL patients (13 female and 12 male) and 18 HTLV-1 carriers (11 female and 7 male) were evaluated. The mean age of the subjects in the ATLL group was 53.92±2.17 years (95% confidence interval [CI], 35.42–88.29), while the mean of the carrier group was 38.94±2.04 years (95% CI, 25.13–53.43).
The frequencies of various clinical findings in ATLL patients were as follows: hepatomegaly (12%), splenomegaly (24%), lymphadenopathy (80%), edema (40%), and skin lesions (60%). The ATLL patients were further classified into four subtypes: acute (6), lymphoma (16), chronic (2), and smoldering (1). The mean calcium level was 10.44±0.56 mg/dL (95% CI, 7.23–16.83) in the ATLL group and 9.52±0.34 mg/dL (95% CI, 8.64–10.31) in carriers. Blood calcium levels in the acute, lymphoma, and smoldering groups were 13.13±0.98 (95% CI, 10.24–16.81), 9.14±0.31 (95% CI, 7.45–11.25), and 8.43, respectively. These data showed a significantly higher calcium blood level in patients with acute ATLL than in patients with other types of ATLL (
White blood cell (WBC) counts in ATLL patients and carriers were 191,340±6,789 cells/mL (95% CI, 1,200–132,000) and 4,933±392.3 cells/mL (95% CI, 4,400–5,700;
The HTLV-1 PVL was investigated in all 43 study subjects (18 carriers and 25 ATLL patients). The mean HTLV-1 PVL in ATLL patients was 18,100±4,918.9 copies/104 cells (95% CI, 53–91,183), and the percentage of HTLV-1-infected PBMCs in ATLL group was 118±49% (95% CI, 0.5–911). The mean HTLV-1 PVL in the carrier group was 531.23±119.2 copies/104 (95% CI, 2–1,329) in the carrier group, which indicated that 5.3±1.1% (CI: 95% 0.02–13.1) of PBMCs were infected with HTLV-1 (
The expression of each gene was divided by the expression of the β2-microglobulin gene to normalize the gene expression values. The mean
A significant correlation was observed between the HTLV-1 PVL and
Survival was investigated in all 25 ATLL patients. The variables evaluated in this analysis were age, gender, treatment (such as CHOP or CVAD), and viral gene expression (
The progression of ATLL is a polyphasic, long-term process, and the outcome of HTLV-1 infection is affected by host-virus interaction as well as cellular division and function [21]. The TAX and HBZ proteins are the two main viral components that interfere with the immune response and cause malignancy [16]. The HTLV-1
As previously mentioned, ATLL is a multi-step malignancy, and it seems that TAX is used by the virus to initiate disease progression. This process is followed by transactivation of infected T-cells, during which
Constant expression of the
Most infected T-cells are resistant to common chemotherapy combinations, and they continue to proliferate, with the exception of cells infected with the HIV retrovirus [24,25]. In the present study, the mean WBC count was higher in ATLL patients than in HTLV-1 carriers, which is indicative of aggressive HTLV-1-induced leukemogenesis.
A significant positive correlation was observed between PVL and
Two major life-threatening events occur in ATLL caused by the high levels of blood calcium, which can lead to heart attacks and opportunistic infections [26,27]. Hypercalcemia in acute ATLL patients might result from osteolytic bone marrow metastases through hyperactivation of osteoclastogenesis and resorption. It was previously shown that overexpression and secretion of parathyroid hormone-related protein (PTHrP) and macrophage inflammatory protein 1 alpha (MIP-1α) from infected T-cells leads to osteoclast development in bone marrow, resulting in the an osteolytic condition in ATLL patients with high numbers of infected lymphocytes [28].
In this study, a negative relationship was observed between the survival time of ATLL patients and HTLV-1 PVL. This result suggests the prognostic potential of PVL for monitoring ATLL patients. In addition,
We thank all the individuals who took part in this study and shared their experiences for the benefit of others. We also extend our gratitude to the authors who contributed to the manuscript and the staff of the departments of Hematology and Oncology at Ghaem and Imam Reza hospitals, for their cooperation in this study.
Association between survival time and proviral load (PVL) in adult T-cell leukemia/lymphoma (ATLL) patients (five PVL groups were defined to examine the impact of HTLV-1 PVL on survival among patients with ATLL).
Table 2 Clinical manifestations and survival time of adult T-cell leukemia/lymphoma (ATLL) patients.
Table 3 TAX and HBZ expression in adult T-cell leukemia/lymphoma (ATLL) patients and asymptomatic carriers.
a)
Abbreviations: SD, standard deviation; SEM, standard error of the mean.
Blood Res 2017; 52(2): 106-111
Published online June 22, 2017 https://doi.org/10.5045/br.2017.52.2.106
Copyright © The Korean Society of Hematology.
Mohammad Mehdi Akbarin1, Abbas Shirdel2, Alireza Bari2, Seyedeh Tahereh Mohaddes2, Houshang Rafatpanah1, Ehsan Ghayour Karimani3, Kobra Etminani4, Amin Golabpour4, and Reza Torshizi5
1Inflammation and Inflammatory Diseases Research Center, Faculty of Medicine, Mashhad University of Medical sciences, Mashhad, Iran.
2Hematology Department, Ghaem Hospital, Faculty of Medicine, Mashhad University of Medical Sciencess, Mashhad, Iran.
3Molecular Diagnostic Unit, Research and Education Department, Razavi Hospitals, Mashhad, Iran.
4Department of Medical Informatics, Molecular Medicine Department, Faculty of Medicine, Mashhad University of Medical sciences, Mashhad, Iran.
5Department of Modern Sciences and Technologies, Molecular Medicine Department, Faculty of Medicine, Mashhad University of Medical sciences, Mashhad, Iran.
Correspondence to:Reza Torshizi, Ph.D. Department of Modern Sciences and Technologies, Mashhad University of Medical Sciences, Mashhad, Razavi Khorasan 91388-13944, Iran. torshiziR901@mums.ac.ir
Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy with very poor prognosis and short survival, caused by the human T-lymphotropic virus type-1 (HTLV-1). The HTLV-1 biomarkers trans-activator x (TAX) and HTLV-1 basic leucine zipper factor (HBZ) are main oncogenes and life-threatening elements. This study aimed to assess the role of the
Forty-three HTLV-1-infected individuals, including 18 asymptomatic carriers (AC) and 25 ATLL patients (ATLL), were evaluated between 2011 and 2015. The mRNA expression of
Significant differences in the mean expression levels of
The HTLV-1 PVL and expression of
Keywords: HTLV-1, ATLL,
Human T-cell leukemia virus type 1 (HTLV-1) is a lentivirus that belongs to the Retroviridae family in the genus Deltaretrovirus [1]. About 20 million infected people live in Africa, the Caribbean, Asia, and Central and South America [2].
The
According to the Revised European American Lymphoma (REAL) classification and World Health Organization (WHO), ATLL is classified into four subtypes (acute, lymphoma, chronic, and smoldering) based on the following symptoms: the persistence of flower-like cells in peripheral blood smear, elevated lactate dehydrogenase (LDH), hypercalcemia, skin lesions, organomegaly, and lytic bone injury [7,8]. The most aggressive ATLL types are the acute and lymphoma types. The acute type has the highest prevalence rate (60%) and shortest survival time (4–6 mo) among all ATLL types, and it shows some of the most aggressive symptoms (e.g., hepatosplenomegaly, lymphadenopathy, a high frequency of atypical lymphocytes with nuclear hypersegmentation, as well as high levels of LDH and calcium in serum). The lymphoma type has a prevalence rate of 20% and a 9–10-month survival period, which demonstrates the aggressiveness of ATLL. The chronic and smoldering types have a survival period of 2–5 years and are less frequently associated with aggressive clinical conditions [2,9]. The primary infection is followed by a long-term latency period (20–40 yrs). Then, leukemogenesis of infected T-cells occurs in a multistep process via genetic and epigenetic alterations [10].
Although the exact pathogenic mechanism of ATLL is unknown, it is found that clonal expansion of infected T-cells increases the number of viral progeny, leading to plus-strand transcription of the HTLV-1 genome. In addition to the major gene described above, the genome also encodes other factors including accessory genes (open reading frames I and II), regulatory genes (
Transactivation of the viral LTR and functional cellular genes is synchronized by TAX; however, TAX does not bind to DNA, but may upregulate the expression of cell survival factors such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), which function as anti-apoptotic elements [11]. Previous studies have suggested that TAX protein promotes downregulation of the expression of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) and activating transcription factor (ATF) signaling proteins, as well as DNA repair proteins (e.g., P53 and retinoblastoma [Rb]). Therefore, TAX may play a critical role in malignant T-cell proliferation [12,13]. Regarding the TAX antigen structure, a strong immune response in HTLV-1-infected patients can eliminate or suppress TAX expression [14]. However, genetic alteration of HTLV-1 by methylation, deletion, or mutation in the 3' LTR region can lead to low TAX protein expression [15]. These mechanisms may allow ATLL cells to escape detection by the host immune system, enabling latent activation of the HTLV-1 genome [16]. The
A case series study was conducted on 43 HTLV-1-infected individuals in the Departments of Hematology and Oncology of Mashhad University of Medical Sciences, Mashhad, Iran, and in Ghaem and Imam Reza Hospitals from July 2011 to November 2015. Patients with suspected ATLL were investigated, and on the basis of symptoms and laboratory test results, 25 patients (12 males and 13 females) were diagnosed with ATLL and placed in the ATLL group. The remaining 18 patients (7 male and 11 female) were considered healthy, asymptomatic HTLV-1-infected carriers, and placed in the control group. HTLV-1 seropositive reactivity was checked for all subjects, and the presence of HTLV-1 was confirmed by conventional PCR. ATLL patients were further divided into four groups: acute, lymphoma, chronic, and smoldering, based on the criteria reported by Shimoyama [18].
cDNA was synthesized using random primers and reverse transcriptase (Bioneer, South Korea) according to the manufacturer's instructions. The reaction conditions were 12 cycles of 30 seconds at 24℃, 4 minutes at 44℃, and 30 seconds at 55℃. Glyceraldehyde-3-phosphate dehydrogenase (
After obtaining the desired sequences for the
Real-time PCR (TaqMan method) was performed in a Rotor-Gene 6000 cycler (Corbett, Hilden, Germany). Duplicate samples were included in the analysis, and expression is reported relative to that of a housekeeping gene (β2-microglobulin). The primers and probes used to amplify the corresponding genes are shown in Table 1. The PCR conditions were as follows: 95℃ for 4 minutes, followed by 45 cycles of denaturation at 94℃ for 15 seconds, annealing at the optimum temperature 20 seconds, and extension at 72℃ for 20 seconds.
To evaluate the HTLV-1 PVL, PBMCs were isolated from ethylenediaminetetraacetic acid (EDTA)-treated blood samples, as described above under “Isolation of PBMCs and extraction of RNA,” and DNA was extracted from the PBMCs, using a DNA extraction kit. Real-time PCR was performed using a commercial absolute quantification kit (Novin Gene, Iran) to measure the PVL of HTLV-1, using specific primers and a fluorogenic probe. HTLV-1 copy number was reported as the actual amount of cellular DNA quantified, using the albumin gene (
Different therapies were administered to the study participants according to the type of disease. The most common first-line treatment for T-cell lymphoma was combination chemotherapy, such as cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or cyclophosphamide, vincristine, doxorubicin, and dexamethasone (CVAD), which was administered to most patients [19,20].
Data were analyzed using SPSS version 18, and the results are reported as mean±standard deviation (SD). The Kolmogorov-Smirnov test was conducted to check variable for normal distribution. The Mann-Whitney U test was performed to analyze the two groups. Spearman's rank correlation coefficient was used to determine the relationship between study parameters. Kaplan-Meier analysis was performed to evaluate survival.
In this study, 25 ATLL patients (13 female and 12 male) and 18 HTLV-1 carriers (11 female and 7 male) were evaluated. The mean age of the subjects in the ATLL group was 53.92±2.17 years (95% confidence interval [CI], 35.42–88.29), while the mean of the carrier group was 38.94±2.04 years (95% CI, 25.13–53.43).
The frequencies of various clinical findings in ATLL patients were as follows: hepatomegaly (12%), splenomegaly (24%), lymphadenopathy (80%), edema (40%), and skin lesions (60%). The ATLL patients were further classified into four subtypes: acute (6), lymphoma (16), chronic (2), and smoldering (1). The mean calcium level was 10.44±0.56 mg/dL (95% CI, 7.23–16.83) in the ATLL group and 9.52±0.34 mg/dL (95% CI, 8.64–10.31) in carriers. Blood calcium levels in the acute, lymphoma, and smoldering groups were 13.13±0.98 (95% CI, 10.24–16.81), 9.14±0.31 (95% CI, 7.45–11.25), and 8.43, respectively. These data showed a significantly higher calcium blood level in patients with acute ATLL than in patients with other types of ATLL (
White blood cell (WBC) counts in ATLL patients and carriers were 191,340±6,789 cells/mL (95% CI, 1,200–132,000) and 4,933±392.3 cells/mL (95% CI, 4,400–5,700;
The HTLV-1 PVL was investigated in all 43 study subjects (18 carriers and 25 ATLL patients). The mean HTLV-1 PVL in ATLL patients was 18,100±4,918.9 copies/104 cells (95% CI, 53–91,183), and the percentage of HTLV-1-infected PBMCs in ATLL group was 118±49% (95% CI, 0.5–911). The mean HTLV-1 PVL in the carrier group was 531.23±119.2 copies/104 (95% CI, 2–1,329) in the carrier group, which indicated that 5.3±1.1% (CI: 95% 0.02–13.1) of PBMCs were infected with HTLV-1 (
The expression of each gene was divided by the expression of the β2-microglobulin gene to normalize the gene expression values. The mean
A significant correlation was observed between the HTLV-1 PVL and
Survival was investigated in all 25 ATLL patients. The variables evaluated in this analysis were age, gender, treatment (such as CHOP or CVAD), and viral gene expression (
The progression of ATLL is a polyphasic, long-term process, and the outcome of HTLV-1 infection is affected by host-virus interaction as well as cellular division and function [21]. The TAX and HBZ proteins are the two main viral components that interfere with the immune response and cause malignancy [16]. The HTLV-1
As previously mentioned, ATLL is a multi-step malignancy, and it seems that TAX is used by the virus to initiate disease progression. This process is followed by transactivation of infected T-cells, during which
Constant expression of the
Most infected T-cells are resistant to common chemotherapy combinations, and they continue to proliferate, with the exception of cells infected with the HIV retrovirus [24,25]. In the present study, the mean WBC count was higher in ATLL patients than in HTLV-1 carriers, which is indicative of aggressive HTLV-1-induced leukemogenesis.
A significant positive correlation was observed between PVL and
Two major life-threatening events occur in ATLL caused by the high levels of blood calcium, which can lead to heart attacks and opportunistic infections [26,27]. Hypercalcemia in acute ATLL patients might result from osteolytic bone marrow metastases through hyperactivation of osteoclastogenesis and resorption. It was previously shown that overexpression and secretion of parathyroid hormone-related protein (PTHrP) and macrophage inflammatory protein 1 alpha (MIP-1α) from infected T-cells leads to osteoclast development in bone marrow, resulting in the an osteolytic condition in ATLL patients with high numbers of infected lymphocytes [28].
In this study, a negative relationship was observed between the survival time of ATLL patients and HTLV-1 PVL. This result suggests the prognostic potential of PVL for monitoring ATLL patients. In addition,
We thank all the individuals who took part in this study and shared their experiences for the benefit of others. We also extend our gratitude to the authors who contributed to the manuscript and the staff of the departments of Hematology and Oncology at Ghaem and Imam Reza hospitals, for their cooperation in this study.
Survival time of adult T-cell leukemia/lymphoma (ATLL) patients according to subtype.
Association between survival time and proviral load (PVL) in adult T-cell leukemia/lymphoma (ATLL) patients (five PVL groups were defined to examine the impact of HTLV-1 PVL on survival among patients with ATLL).
Table 1 . Primers and probes for
Table 2 . Clinical manifestations and survival time of adult T-cell leukemia/lymphoma (ATLL) patients..
Table 3 . TAX and HBZ expression in adult T-cell leukemia/lymphoma (ATLL) patients and asymptomatic carriers..
a)
Abbreviations: SD, standard deviation; SEM, standard error of the mean..
Survival time of adult T-cell leukemia/lymphoma (ATLL) patients according to subtype.
|@|~(^,^)~|@|Association between survival time and proviral load (PVL) in adult T-cell leukemia/lymphoma (ATLL) patients (five PVL groups were defined to examine the impact of HTLV-1 PVL on survival among patients with ATLL).