|Year : 2021 | Volume
| Issue : 1 | Page : 384-390
Diagnostic and prognostic value of perforin-1 mRNA expression in acute myeloid leukemia
Eman S El Deen Arafat1, Suzy F Gohar2, Safa M Saber3, Iman Fawzy4, Eman M Abd El Gayed1
1 Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Oncology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Chemistry, Faculty of Science, Menoufia University, Menoufia, Egypt
4 Department of Clinical Pathology, Mansoura Central Laboratories, Ministry of Health, Mansoura, Egypt
|Date of Submission||29-Sep-2020|
|Date of Decision||25-Oct-2020|
|Date of Acceptance||28-Nov-2020|
|Date of Web Publication||27-Mar-2021|
Suzy F Gohar
Department of Clinical Oncology, Faculty of Medicine, Menoufia University, Shebin Elkom City, Menoufia Governate 32511
Source of Support: None, Conflict of Interest: None
To study diagnostic value of perforin-1 gene mRNA expression and its relation to patients' outcomes in acute myeloid leukemia (AML).
AML is a molecularly heterogeneous hematological malignancy with variable response to treatment and characterized by bone marrow and tissue infiltration with abnormally differentiated cells of hematopoietic origin. Perforin-1 is a glycoprotein responsible for pore formation in cell membranes of target cells. It has a role in immune regulation. Without perforin-1, cytotoxic T cells and natural killer cells show reduced or no cytolytic effect on target cells.
Patients and methods
Expression levels of perforin-1 mRNA were assessed by reverse transcriptase PCR and correlated with patients' features of response and survival.
Patients with AML had significantly lower perforin expression compared with the control group (P < 0.001). Only seven patients, representing 11.7% of the studied patients had low perforin expression which was significantly related to poor disease cytogenetics and poor response (P = 0.018 and 0.043, respectively). However, logistic regression analysis revealed that older age and poor cytogenetics were considered as independent risk predictors for poor response in studied AML cases. At the end of follow-up period, 55% of patients were alive. The relation between perforin expression and overall survival revealed that there was no significant relation between perforin expression and overall survival in the studied patients (P = 0.09).
Lower perforin-1 mRNA expression was significantly associated with some poor prognostic features in patients with AML, such as unfavorable cytogenetics and poor response. It is also associated with shorter survival; however, this relation was not statistically significant.
Keywords: cytogenetics, leukemia, myeloid, perforin, prognosis
|How to cite this article:|
El Deen Arafat ES, Gohar SF, Saber SM, Fawzy I, Abd El Gayed EM. Diagnostic and prognostic value of perforin-1 mRNA expression in acute myeloid leukemia. Menoufia Med J 2021;34:384-90
|How to cite this URL:|
El Deen Arafat ES, Gohar SF, Saber SM, Fawzy I, Abd El Gayed EM. Diagnostic and prognostic value of perforin-1 mRNA expression in acute myeloid leukemia. Menoufia Med J [serial online] 2021 [cited 2021 Aug 4];34:384-90. Available from: http://www.mmj.eg.net/text.asp?2021/34/1/384/312051
| Introduction|| |
Acute myeloid leukemia (AML) is one of the most common types of leukemia diagnosed in adults. Although many advancements have been made in the understanding of the genetic components of AML, the 5-year survival rate for all types of AML is still less than 25% .
Most of the clinical manifestations of AML reflect the accumulation of malignant, poorly differentiated myeloid cells within the bone marrow, peripheral blood, and infrequently in other organs .
Most patients present with a combination of leukocytosis and signs of bone marrow failure such as anemia and thrombocytopenia. Fatigue, anorexia, and weight loss are common complaints; lymphadenopathy and organomegaly are not typically seen. If left untreated, death usually ensues within months of diagnosis secondary to infection or bleeding .
Perforin-1 is a glycoprotein responsible for pore formation in cell membranes of target cells. Natural killer (NK) cells and CD8-positive T-cells are the main source of perforin-1. However, CD4-positive T cells are also able to express a low amount of perforin-1 when classic cytotoxicity is ineffective or disturbed .
Once released from cytolytic granule, perforin binds to target cell membrane and forms a pore, leading to activation of cell cytotoxicity. Perforin contributes in two ways to the elimination of target cells. After binding to the membrane of target cells, monomeric perforin molecules incorporate, in the presence of calcium, into the plasma membrane of target cells forming pores through the membrane, directly causing necrosis via lysis .
Perforin also is inserted into the plasma membrane and serves as a transport medium for granzymes like granzyme B, which activates caspase-8 and caspase-3, causing apoptosis .
Perforin mutations usually result in the decrease or absence of the protein or its activity and lead to abnormal cellular proliferation or lymphoproliferative disorders, may be owing to the defect of cell-mediated cytotoxic function and transformed cell elimination .
Resistance to perforin-mediated membrane damage has been found to occur also in leukemic cells from some patients with AML and not in acute lymphocytic leukemia (ALL) cells .
The aim of the current study was to assess diagnostic and prognostic value of perforin-1 mRNA expression in AML.
| Patients and methods|| |
This study was carried out in the Department of Medical Biochemistry and Molecular Biology in collaboration with the Hematology Unit in Clinical Oncology Department, Faculty of Medicine, Menoufia University. The study was approved by ethical committee of Faculty of Medicine, Menoufia University.
Patients were recruited for the study from January 2018 to December 2019. All patients with suspected acute leukemia who presented to the emergency room in Clinical Oncology Department were subjected to thorough history, physical examination, and investigations in the form of complete blood count, differential count platelet count, comprehensive metabolic panel, lactate dehydrogenase, and uric acid. A fresh bone marrow aspirate and biopsy for morphologic assessment (blood and bone marrow), conventional cytogenetic analysis (i.e. karyotype), appropriate molecular-genetic and/or fluorescence in-situ hybridization testing, and flow cytometry immunophenotyping were done. The flow cytometry panel should be enough to distinguish AML (including acute promyelocytic leukemia), T-ALL (including early T-cell precursor leukemias), B-cell precursor ALL (B-ALL), and AL of ambiguous lineage for all patients diagnosed with AL.
Classification of AML was done according French American-British (FAB) classification. The FAB system is based on morphology and cytochemistry .
Based on cytogenetic analysis, patients were classified into patients with favorable risk cytogenetics [inversion (16) and translocation t (16;16), t (8;21), and t (15: 17)], intermediate risk normal cytogenetics [+8 alone, t (9;11)] and poor risk cytogenetics [complex ≥ 3 clonal chromosomal abnormalities, monsoonal karyotype -5, 5q-, -7, 7q-, 11q23, other than t (9;11), inv (3), t (3;3), t (6;9), and t (9;22)].
Patients with proved diagnosis of AML of any FAB type and with good performance status (<2) were included in the study.
Patients with performance status (≥2), organ failure, antecedent hematologic disorder or therapy-related AML, and patients aged more than 60 years with unfavorable cytogenetic were excluded.
After written consent, 60 patients with AML were included in addition to 60 age-matched and sex-matched healthy controls.
Further investigations were done in the form of coagulation profile (prothrombin time, activated partial prothrombin time, and fibrinogen level) and imaging studies, including dental survey and computed tomography scan of the chest and abdomen, or chest radiograph and abdominal ultrasound.
Computed tomography of brain without contrast was done if central nervous system (CNS) hemorrhage was suspected. Brain MRI with contrast was done if leukemic meningitis was suspected and lumbar puncture if indicated (to assess CNS involvement or exclude meningitis).
Many commonly used induction regimens contain an anthracycline. Therefore, assessment of cardiac risk factors and assessment of myocardial function (by echocardiogram) were done.
Measurement of perforin-1 mRNA expression was performed using reverse transcriptase PCR using real-time PCR for all participant at the time of recruitment.
Overall, 6 ml of blood samples was taken from each participant and divided into three tubes: 4 ml was put into two EDETA-containing tubes, where one of them was used for complete blood count and the other for detection of perforin-1 mRNA expression.
Complete blood picture was measured with Pentra-80 automated blood counter (ABX–France, Rue du Caducee, Paris Euromedecine-BP-7290.34a184 Montpellier-Cede × 4). Estimation of perforin-1 mRNA expression was performed using real-time reverse transcriptase PCR.
RNA was isolated from peripheral blood leukocytes using QIAamp RNA Blood MiniKit (2013; Qiagen, USA), and then the first step of PCR was performed: complementary DNA was synthesized using QuantiTect Reverse Transcription Kit (2012; Qiagen, Applied Biosystems, USA), followed by second step of PCR (real-time PCR step): it was performed using QuantiTect SYBR Green PCR Kit with ready-made quantiTect Primer Assay, Qiagen. For measurement of perforin-1 mRNA levels, the following primers were used: forward primer 5′-CGCCTACCTCAGGCTTATCTC-3′and reverse primer 5′- CCTCGACAGTCAGGCAGTC-3′.
Forward and reverse primers for human glyceraldehyde-3-phosphate dehydrogenase were 5′-CGGAGTCAACGGATTGGTCGTAT-3′ and 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′, respectively. PCR was conducted under the following conditions: each reaction was performed in a final volume of 20 μl, containing 10 μl SYBR Green 2× QuantiTect PCR Master Mix, 3 μl cDNA, 1 μl forward primer, 1 μl reverse primer, and 5 μl RNase-free H2O. The mix was incubated at 94°C for 3 min, followed by 55 cycles: denaturation at 94°C for 30 s, annealing at 55°C for 40 s, and extension at 72°C for 31 s. Data analysis was done in Applied Biosystems 7500 software, version 2.0.1. The relative quantification of gene expression was performed using comparative ΔΔCt method . Perforin-1 mRNA was normalized to the mRNA levels of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. Melting curve was done to confirm specificity of the amplification and absence of primer dimers.
All patients with de novo non-APL-AML, aged less than 60 years, with favorable or unfavorable cytogenetics, or aged more than or equal to 60 years, with favorable cytogenetics or molecular markers), received chemotherapy as follows: days 1–3, an anthracycline (daunorubicin 60–90 mg/m2 intravenous OR idarubicin 12 mg/m2), and days 1–7, cytarabine 100–200 mg/m2 continuous intravenous.
Patients with APL AML received induction therapy in the form of all-trans retinoic acid 45 mg/m2 in divided doses until clinical remission + daunorubicin 50 mg/m2 × 4 days + cytarabine 200 mg/m2 × 7 days
All patients were evaluated by bone marrow biopsy 14 days after completion of induction therapy.
Patients with marrow blasts less than 5%, no Auer rods, no extramedullary disease (e.g., CNS and soft tissue disease), normal maturation of all cellular components in the bone marrow, neutrophils more than or equal to 1000/μl, platelets more than or equal to 100 000/μl, and if patient became transfusion independent for more than 4 weeks were considered in morphological complete remission and classified as good responders. However, patients with persistence of cytopenia and blasts more than 5% were considered as incomplete remission and classified as poor responders.
All patients were followed up for 6 months after diagnosis. Progression-free survival was calculated from time of diagnosis till progression or last follow-up visit. Fate of the patients at the end of follow-up period was reported as either dead or alive.
Data were analyzed using IBM SPSS statistics, version 20 (SPSS Inc., Chicago, Illinois, USA). Quantitative data were expressed as mean and SD. χ2 test was used to examine the relation between qualitative variables. For quantitative data, comparison between two groups was done using either Student t test or Mann–Whitney test (nonparametric t test) as appropriate. The receiver operating characteristic (ROC) curve provides a useful way to evaluate the sensitivity and specificity for quantitative diagnostic measures that categorize cases into one of two groups. Spearman's correlation method was used to test correlation between numerical variables. Regarding regression analysis, logistic regression analysis was used for prediction of risk factors, using generalized linear models. A P value less than 0.05 was considered significant.
| Results|| |
This prospective case–control study included 60 patients as the case group and 60 apparently normal age-matched and sex-matched patients as the control group. Compared with the control group, the patients had significantly lower perforin expression (mean ± SD, 27.7 ± 2.9 in patients vs. 34.7 ± 1.3 in control group; P value less than 0.001). Moreover, there was a significant difference between the studied groups regarding total leukocytic count, hemoglobin levels, and platelet counts [Table 1] and [Figure 1].
|Table 1: Comparison between the two studied groups according to different laboratory parameters|
Click here to view
|Figure 1: Amplification plot of perforin-1 mRNA gene expression [normalized fluorescence signal (ΔRn) plotted versus cycle number].|
Click here to view
ROC curve of perforin expression was conducted for discrimination between AML cases and control groups. Using area under curve of 0.978, cutoff values of less than or equal to 30.9, P value less than 0.001, and confidence interval 95%=0.951–1.000, perforin had sensitivity of 88.3, specificity of 98.3, positive predictive value of 98.1, and negative predictive value of 89.4 to predict AML [Figure 2].
|Figure 2: ROC curve for perforin expression to diagnose AML group. AML, acute myeloid leukemia; ROC, receiver operating characteristic.|
Click here to view
Based on the estimated cutoff point, perforin was downregulated (≤30.9) in 53 patients, representing 88.3% of the studied patients. Perforin expression had significant relation with disease cytogenetics (P = 0.018) and achievement of complete remission at the end of induction phase (response) (P = 0.043).
At the end of induction phase, only 46.7% (28 patients) of the patients experienced complete remission and were categorized as good responders and the rest were considered as poor responders. The relation between response and other patient features revealed significant relation between good response and higher perforin expression, older age, presence of splenomegaly, hepatomegaly, FAB categories other than M3, lower hemoglobin level, and patient's fate at the end of follow-up period. [Table 2].
|Table 2: Relation between response and different parameters for acute myeloid leukemia group (n=60)|
Click here to view
Logistic regression analysis was conducted for prediction of incomplete response using age, sex, splenomegaly, hepatomegaly, poor cytogenetics, CNS involvement, lymph node involvement, M3, total leukocytic count, and perforin expression as covariates. Older age and poor cytogenetics were considered as independent risk predictors for poor response in studied AML cases [Table 3].
|Table 3: Univariate and multivariate analyses for the parameters affecting response|
Click here to view
The mean survival of the studied patients was 5.23 months. At the end of follow-up period, 55% of patients were alive. The relation between patient factors and perforin expression revealed that there was no significant relation between perforin expression and overall survival in the studied patients [Table 4].
|Table 4: Relation between overall survival and different parameters for acute myeloid leukemia group (n=60)|
Click here to view
| Discussion|| |
After viral or other infections, activated effector T and NK cells form an immunologic synapse with target cells and then release specialized granules that mediate cellular cytotoxicity. Mutations in genes that encode key components of the CD8 T cell and NK cell cytolytic pathway can lead to defects of cytotoxic function and failure of elimination of infected and malignant cells .
NK cells can kill AML cells through release of cytoplasmic granules containing perforin and granzymes, antibody-dependent cellular cytotoxicity, and expression of proteins from the tumor necrosis factor TNF family. These processes are activated without sensitization or need for HLA-compatibility. Nonantigen-specific cellular therapies using NK and cytokine-induced killer cells represent an attractive alternative in this disease .
One of the basic functions of NK cells is immuno-surveillance of the body. Several in vitro studies on mammalian cells, including human cells, and in vivo studies in mice and rats prove that NK cells recognize tumor cells as targets and can control tumor growth and metastasis diffusion in vivo. Tumor immuno-surveillance role of NK cells has also been implicated in controlling the growth of B-cell lymphomas that spontaneously arise in mice lacking both perforin and b2-microglobulin .
Increase perforin and granulysin expression in NK cells at the levels of mRNA and protein is involved in regulation of apoptosis and the activation-induced cell death pathway .
NK cells in patients with AML may experience phenotypic changes and lose their function and be unable to recognize and kill AML cells . Based on compelling evidence that NK activity is drastically altered in leukemic conditions, multiple immunotherapeutic strategies directed at enhancing effective NK cell function are being prospectively evaluated as AML therapy . NK cells do not require sensitization or human leukocyte antigen-compatibility to kill tumor cells .
To our knowledge, there is no current study that explored the relation between perforin-1 expression and AML. The aim of the present study was to assess the diagnostic value of perforin-1 gene mRNA expression and its relation to patients' features and outcomes in AML.
Cases showed significantly lower expression of perforin-1 gene compared with control, and it was downregulated in most of the studied patients with AML, suggesting possible role of perforin-1 downregulation in pathogenesis of the disease by impairing the cytotoxic effect of NK cell against tumor cells. These findings augment the theory by Dulphy et al. regarding the role of NK cell physiology in development of AML.
Moreover, patients experienced significantly lower hemoglobin and platelet counts in comparison with the controls. This is related to the nature of the disease, as accumulation of malignant cells in the bone marrow is manifested by bone marrow failure signs.
In the current study, ROC curve analysis revealed cutoff with good sensitivity, specificity, and accuracy, suggesting the possible diagnostic role of perforin gene expression level.
Relation between expression and patients' features was estimated and revealed that there was a significant relation between perforin expression and cytogenetics and ability to achieve complete response at end of induction phase, as low expression of perforin gene was associated with unfavorable cytogenetics and poor response, suggesting its poor prognostic value. Most patients with high perforin expression were alive at the end of the follow-up period.
Response analysis revealed that older age, presence of splenomegaly and hepatomegaly, low hemoglobin level, poor cytogenetics, non-M3 AML, and lower perforin expression were associated with poor response. However, multivariate analysis revealed that only older age and poor cytogenetics were the independent risk factors for poor response.
Regarding survival analysis, although lower perforin-1 expression was associated with shorter survival, this relation was not significant; however, this suggests poor prognostic value. On the contrary, older age, presence of splenomegaly and hepatomegaly, CNS involvement, poor cytogenetics, M1, and M6 were significantly related to poor survival.
| Conclusion|| |
Perforin-1 mRNA expression was significantly associated with poor prognostic features in patients with AML such as unfavorable cytogenetics and poor response. It is also associated with shorter survival; however, this relation was not statistically significant. The authors recommend further studies of NK cell dysfunction and perforin mRNA level on a larger scale of patients, as cellular therapy with NK cells is a promising field.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Nieto M, Demolis P, Béhanzin E, Moreau A, Hudson I, Flores B, et al
. The European Medicines Agency Review of Decitabine (Dacogen) for the treatment of adult patients with acute myeloid leukemia: summary of the scientific assessment of the committee for medicinal products for human use. Oncologist 2016; 21
Döhner H, Estey EH, Amadori S, Appelbaum FR, Büchner T, Burnett AK, et al
. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood 2010; 115
Osińska I, Popko K, Demkow U. Perforin: an important player in immune response. Cent Eur J Immunol 2014; 39
Mandal A, Viswanathan C. Natural killer cells: in health and disease. Hematol Oncol Stem Cell Ther 2015; 8
Carlsten M, Järås M. Natural killer cells in myeloid malignancies: immune surveillance, NK cell dysfunction, and pharmacological opportunities to bolster the endogenous NK cells. Front Immunol 2019; 10
Yang L, Liu H, Zhao J, Da W, Zheng J, Wang L, et al
. Mutations of perforin gene in Chinese patients with acute lymphoblastic leukemia. Leuk Res 2011; 35
Otten HG, Van Ginkel WG, Hagenbeek A, Petersen EJ. Prevalence and clinical significance of resistance to perforin-and FAS-mediated cell death in leukemia. Leukemia 2004; 18
Kumar CC. Genetic abnormalities and challenges in the treatment of acute myeloid leukemia. Genes Cancer 2011; 2
Soliman SE, Habib MS, El-Dien MM, Gohar SF, Alhassanin SA. Evaluation of receptor for advanced glycation end product/high-mobility group bo×1 (RAGE/HMGB1) expression status and its prognostic value in breast cancer. Egypt J Biochem Mol Biol 2019; 37
Ghosh S, Carmo M, Calero-Garcia M, Ricciardelli I, Ogando JC, Blundell MP, et al
. T-cell gene therapy for perforin deficiency corrects cytotoxicity defects and prevents hemophagocytic lymphohistiocytosis manifestations. J Allergy Clin Immunol 2018; 142
Hansrivijit P, Gale RP, Barrett J, Ciurea SO. Cellular therapy for acute myeloid leukemia–current status and future prospects. Blood Rev 2019; 37
Petranovic D, Pilcic G, Valkovic T, Tokmadzic VS, Laskarin G. Perforin-and granulysin-mediated cytotoxicity and interleukin 15 play roles in neurocognitive impairment in patients with acute lymphoblastic leukaemia. Med Hypotheses 2014; 83:122–126.
Stringaris K, Sekine T, Khoder A, Alsuliman A, Razzaghi B, Sargeant R, et al
. Leukemia-induced phenotypic and functional defects in natural killer cells predict failure to achieve remission in acute myeloid leukemia. Haematologica 2014; 99
Przespolewski A, Szeles A, Wang ES. Advances in immunotherapy for acute myeloid leukemia. Future Oncol 2018; 14
Tan Y, Wu Q, Zhou F. Targeting acute myeloid leukemia stem cells: current therapies in development and potential strategies with new dimensions. Crit Rev Oncol Hematol 2020; 152
Dulphy N, Chrétien AS, Khaznadar Z, Fauriat C, Nanbakhsh A, Caignard A, et al
. Underground adaptation to a hostile environment: acute myeloid leukemia vs. natural killer cells. Front Immunol 2016; 7
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]