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ORIGINAL ARTICLE
Year : 2023 | Volume
: 12
| Issue : 1 | Page : 44-49
Evaluation of plasma progranulin level and the estimation of its prognostic role in adult patients with de novo acute myeloid leukemia
Farah Ghani Hussein1, Abeer Anwer Ahmed2
1 Department of Hematology, The National Center of Teaching Laboratory, Baghdad, Iraq
2 Department of Pathology, College of Medicine, Mustansiriyah University, Baghdad, Iraq
Correspondence Address:
Dr. Farah Ghani Hussein
Medical City, Baghdad
Iraq
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ijh.ijh_58_22
BACKGROUND: Acute Myeloid leukemia (AML) is a clonal, malignant disease of hematopoietic tissues that is characterized by the accumulation of blast cells, principally in the marrow, and impaired production of normal blood cells. Progranulin (PGRN) is a multifunctional secreted glycoprotein implicated in tumorigenesis, development, inflammation, and repair. High PGRN expression was reported as a prognostic marker in many types of nonhematological and limited hematological malignancies.
AIM OF STUDY: To evaluate the level and prognostic significance of PGRN in adult patients with de novo acute myeloid leukemia.
PATIENTS, MATERIALS AND METHODS: This analytic cross-sectional study was conducted on 60 adult de novo AML patients who were newly diagnosed from December 2021 to October 2022 in the Haematology Department of Baghdad Teaching Hospital in Medical City. A total of 28 healthy individuals were included in this study as a control group. The diagnosis was based on morphology, immunophenotyping, and genetic studies of the peripheral blood and/or bone marrow aspirate samples in the National Center of Teaching Laboratories of the Medical City in Baghdad. Measurement of plasma PGRN level was done by double-sandwich enzyme-linked immunosorbent assay (ELISA) technique using PGRN ELISA kit.
RESULTS: Plasma PGRN level was significantly higher in AML patients than in controls, and also was higher in patients who did not achieve remission. Plasma PGRN level shows a strong positive correlation with the peripheral and bone marrow blast percentages and insignificant correlation with age, gender, total leukocyte count, hemoglobin level, and platelets. There was a statistically significant difference in the median of plasma PGRN level between M3 and non-M3 groups.
CONCLUSIONS: PGRN is higher in AML patients at diagnosis than in the control group, with plasma level more in those with poor response to treatment and may be used as an independent risk factor in those patients.
Keywords: Acute myeloid leukemia, enzyme-linked immunosorbent assay, plasma progranulin level
Acute myeloid leukemia (AML) is a malignancy originating in the hematopoietic stem cell or a closely related multipotential hematopoietic cell. It is characterized by the clonal proliferation of abnormal blast cells in the marrow and impaired production of normal blood cells, resulting in anemia; thrombocytopenia; and low, normal, or high white cell counts depending on the concentration of leukemic cells in the blood. Acute myeloid leukemia is the most common acute leukemia in adults. AML accounts for 80% of acute leukemia in adults. Approximately 21,500 new cases of AML occur annually, representing approximately 32% of the new cases of leukemia in the United States each year. Approximately 11,000 patients with AML in the United States die each year as a result of the disease.[1]
Progranulin (PGRN) is a multifunctional secreted glycoprotein implicated in tumorigenesis, development, inflammation, and repair.[2] PGRN, also known as granulin epithelin precursor (GEP), PC-cell-derived growth factor, proepithelin, and acrogranin, is an autocrine growth factor[3] Structurally, PGRN consists of seven-and-a-half tandem repeats of the granulin/epithelin module (GEM), several of which have been isolated as discrete 6-kDa GEM peptides.[4] PGRN is expressed in rapidly cycling epithelial cells, leukocytes, neurons, and chondrocytes and plays a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing, inflammation, host defense, and cartilage development and degradation. Some human cancers also express PGRN and PGRN contributes to tumorigenesis in breast cancer, ovarian carcinoma, and glioma and also PGRN acts as a neurotrophic factor and mutations in the Granulin gene cause frontotemporal dementia.[3] In hematopoietic malignancy, the overexpression of PGRN has been investigated in chronic lymphocytic leukemia and malignant lymphoma, and elevated serum PGRN level is associated with decreased overall survival (OS), disease-free survival, relapse-free survival, and progression-free survival.[5]
Aims of the study
Assess the plasma PGRN level in de novo adult acute myeloid leukemia patientsComparison of plasma PGRN level at diagnosis in relation to remission statusCorrelate between plasma levels of PGRN with some hematological parameters at presentation, AML subtypes, and extramedullary involvement. Patients, Materials and MethodsThis cross-sectional study was conducted on 60 adult patients with de novo AML from December 2021 to October 2022 in the Haematology Department of Baghdad Teaching Hospital in Medical City.
The diagnosis was based on morphology, immunophenotyping, and genetic studies of the peripheral blood (PB) and/or bone marrow aspirate (BMA) samples in the National Centre of Teaching Laboratories of the Medical City in Baghdad.
Thirty-one AML patients included in this study received the remission induction therapy protocol of “3 + 7” (Daunorubicin plus Cytosine arabinoside), 15 patients on (Decitabine and Venetoclax) and the remaining APL patients (14 patients) were on (ATRA, Daunorubicin, and Arsenic trioxide).
For patients who were on “3 + 7,” examination of PB and BMA samples were done on day (21–28) from the start of remission induction chemotherapy to assess the remission status. Those who were on “Decitabine and venetoclax” assessment were done after four cycles of treatment. APL patients, examination of PB and BMA samples were done at day 36 from the start of remission induction. Complete remission (CR) after AML therapy is defined as (neutrophil count >1 × 109/L, platelet count >100 × 109/L, and <5% blasts in the marrow by microscopy, the absence of extramedullary AML). Follow-up for AML patients was done for 5 months from the diagnosis to assess the disease outcome (whether the patient is still alive or dead).
This study was approved by the Ethics Committee of the Iraqi council for medical specialization, and Informed written consent was obtained from all of the patients who participated in the study.
Data by a questionnaire including the main symptoms and physical signs, especially the presence of extramedullary features, which include lymphadenopathy, splenomegaly, hepatomegaly, mediastinal widening and CNS involvement, besides hematological parameters, were obtained from each patient. The included patients were newly diagnosed adult patients with de novo AML who were aged <80 and >15 years and randomly collected concerning gender. The control group of 28 healthy adults was included in this study. The age ranged between 19 and 73 years, and they were 15 males and 13 females.
Blood sample collection and preparation
From each patient and control included in this study, a venous blood sample for complete blood count and blood smear, the remaining anticoagulated blood was centrifuged within 30 min of collection to obtain plasma, the plasma was stored at − 80°C at the National Centre of Teaching Laboratories of the Medical City then used for measuring plasma PGRN level by double-sandwich enzyme-linked immunosorbent assay (ELISA) technique using PGRN ELISA kit from MYBIOSOURCE.[6]
ResultsDemographic characteristics of the study population
The mean age of the newly diagnosed patients was 42.38 ± 17.27 years (range 15–73 years) which did not differ significantly from that of the control group (mean = 45 ± 14.79 years, range 19–73 years). The frequency of males in the newly diagnosed and control group was 35 (58%) and 15 (53%), respectively, with no significant differences [Figure 1].
Hematological and clinical characteristics of the newly diagnosed patients
The mean hemoglobin (Hb) concentration was 8.12 ± 1.94 g/dL (range 3.60–13.00 g/dL). The majority of the patients (96%) were anemic, with those having Hb <7 mg/dL accounting for 33.3% of the patients. The total WBC count was 47.16 ± 53.56 × 109/L as a mean (range 0.90–284.40 × 109/L) with 73.3% of patients had leukocytosis with WBC ≥11 × 109/L, while 13.3% of patients had normal total leukocyte count. The mean platelet count was 45.09 ± 31.46 × 109/L (range 1.00–132.00 × 109/L). 66.6% of patients had severe thrombocytopenia with platelets count <50 × 109/L. The mean blast percentage in PB and bone marrow was 62.90 ± 27.23% (range 9%–98%) and 75.05 ± 17.40% (range 21%–98%), respectively. 98.3% of cases had a PB blast percentage >20%, with only one case having a PB blast percentage below that (9%). Half of the patients (50%) demonstrated a bone marrow blast percentage ≥80%. The majority of patients (65%) achieved remission, while (35%) did not achieve remission. The number of patients who were alive after 5-month follow-up was 50/60 (83.3%), while 10 patients were dead (16.6%) [Table 1].
Table 1: Hematological characteristics of the newly diagnosed acute myelogenous leukemia patientsPallor was the most common manifestation of the 60 newly diagnosed patients and was present in 60% of them, followed by fever (55%), extramedullary disease (50%), and bleeding (40%),
Plasma concentration of progranulin in patients and control and its correlation with other variables
Plasma PGRN concentration was higher in patients than in controls (median 1092.8 pg/mL, [range 467.79–2093.8 pg/mL] versus median = 661.79 pg/mL, [range 164.99–913.93 pg/mL]) with highly significant differences P < 0.001 [Figure 2].
Figure 2: Comparison of serum progranulin levels in patients and controlsThe PGRN level did not correlates with age, gender, total leukocyte count, Hb, and plt, but showed a significant positive correlation with serum lactate dehydrogenase (S. LDH), PB, and BM blasts percent. Moreover, the mean plasma PGRN level didn't show statistically significant difference between patients with comorbidities and those without (P=0.135), patients with extramedullary disease and those without (P=0.674) and cytogenetic risk groups. In contrast, the plasma PGRN level was higher in the French–American–British (FAB) M3 subtype than non-M3 subtypes as shown in [Table 2].
Table 2: Correlations of progranulin at presentation with other hematological parameters in acute myelogenous leukemia patient's groupAssociation of plasma progranulin at diagnosis in relation to remission status
By using an unpaired test (Mann–Whitney U-test), AML patients' group who achieved a response (39/60 patients had CR), the median PGRN level was 923.80 pg/ml, while that for AML patients who did not achieve remission (21/60 did not achieve CR) was 1228.80 pg/ml (P = 0.034). After 5 months duration of follow-up from the diagnosis of AML patients, it was found that the median plasma PGRN level before treatment in the patient group who died (1264.56 pg/ml) were higher than that of the patients' group who remained alive (1035.05 pg/ml), but this observation did not show any statistical significance (P = 0.204).
DiscussionAML is the most common acute leukemia in adults. The classic treatment is cytarabine-based chemotherapy, targeted drug therapy, and hematopoietic stem cell transplantation; the 5-year OS rate for patients is still around 30%. In addition, although chemotherapy and targeted drug therapy can prolong the survival of AML patients, there are still some patients who cannot obtain a good prognosis, still have drug resistance and relapse, or even death.
In this study, the mean age and range of AML patients included were 42.38 ± 17.27 (mean ± SD) years and 15–73 years; those results were comparable to Iraqi studies,[7],[8],[9] while in the united states and European acute myeloblastic leukemia patients, the highest incidence was reported in elderly patients.[10],[11] This disparity may be explained by the effect of ethnic, geographical, and environmental factors and the higher mean age in Western countries compared to Iraq. The patients were randomly selected concerning gender, and the male-to-female ratio was 1.4:1. Similarly, many other studies revealed that males predominate in AML.[7],[9],[12],[13]
Out of the 60 patients included in this study, M3 was the most common subtype of AML (23%), that is comparable to local studies done by Tawfiq et al.[7] Mohammad et al.[9] and Hussein[14] the most common symptom for AML patients was pallor. This is comparable to a local study done by Tawfiq et al.[7] and Pouls et al.,[15] but differ from other studies done by Kulsoom et al.[16] where fever being the most common cause of presentation. Pallor is an important feature in acute leukemia and in the majority of cases, is due to BM infiltration by malignant cells along with a reduction in normal red blood cells and bleeding.
The extramedullary manifestations were found in 50% of the 60 AML patients. This result is comparable to Muhsin and Al-Mudallal.[8] Bleeding was the least presenting features in this study and accounts for 40% of cases. This was comparable to a local study done by Tawfiq et al.[7]
At presentation, the median WBCs count was 32.65 × 109/l, and this is higher than what was described in Egypt and other parts of Iraq.[7],[17] However, comparable to Hussein[14] and Pouls et al.[15] The median Hb level at diagnosis was 8.1 g/dl, which is close to what was found in Egypt, a median of 8.4 g/dl[17] and in the local study in Iraq.[7],[15] The median platelet count was 39 × 109/l. This is comparable to local studies in Iraq.[7],[15] Most previous studies have indicated that S. LDH is an independent prognostic factor for patients with AML.[18] In this study, the median S. LDH level was 465 U/L, which is comparable to a study by Xiao et al.,[18] Sorror et al.[19]
This study evaluated the plasma PGRN expression in AML patients by ELISA. The median plasma PGRN level was 1092.8 pg/ml, range (467.79–2093.8 pg/ml) for AML patients, while for controls was 661.79 pg/ml, with a range (164.99–913.93 pg/ml). These results indicated that high plasma PGRN was a common occurrence in AML patients, and the plasma level of PGRN in AML was significantly higher than that in healthy controls (P < 0.001), which is in agreement with two studies.[20],[21] Previous studies demonstrated the same results in other hematological and solid tumors.[22],[23],[24],[25],[26],[27] PGRN was demonstrated to be a regulator of carcinogenesis as it promotes cell proliferation, migration, invasion, angiogenesis, malignant transformation, resistance to anticancer drugs, and immune evasion.[28]
When followed up the patients in this study at the time of assessment of remission, AML patients achieved CR 65%, while 35% did not achieve CR. The median plasma PGRN level at diagnosis was significantly higher in AML patients not in remission than in those with CR (P = 0.034). Similar results are found in patients with malignant lymphoma[25] and some solid tumors.[29] The possible reason for the PGRN increase was that high expression of PGRN was associated with cancer development and progression and the dynamic changes of PGRN levels may show the chemotherapy response rate to the AML patients that high expression of PGRN in AML patients had low chemotherapy response rate.
In this study, the correlations between the PGRN level and patient age, gender, total leukocyte count, Hb, Plt were insignificant, which agrees with many other studies,[20],[21],[26] also the mean plasma PGRN level did not differ between male and females which is comparable to study done by Qin et al.[20] and El-Ghammaz et al.,[26] but disagrees with Azazzi et al.[21]
There was a significant correlation between plasma PGRN level and the proportion of immature cells in the PB and bone marrow (r = 0.384, P = 0.002) (r = 0.445, P < 0.001), respectively. This result is similar to a study done by Qin et al.[20] and This is in line with Göbel et al., who found a clear positive association in patients with CLL between increasing leukemic cells and PGRN plasma levels[22] and solid tumors, in which the expression of PGRN is related to the histological grading.[30],[31] However, this disagrees with a study by Azazzi et al.[21] and El-Ghammaz et al.[26] In breast cancer, the overexpression of PGRN is more common in invasive ductal adenocarcinoma and is closely related to tumor grading, growth index, and P53 expression.[32],[33]
The plasma PGRN concentrations significantly correlated with S. LDH activity (P < 0.001), which reflects the quantity of tumor burden and is considered independent prognostic factor. This is in agreement with the study done by Azazzi et al.[21] and Yamamoto et al.[25] There were no statistically significant differences between plasma PGRN at diagnosis and comorbidities (P = 0.135) which agrees with Azazzi et al.[21] and El-Ghammaz et al.[26]
In this study, there were statistically significant differences in plasma levels of PGRN between M3 and non-M3 groups at the time of diagnosis (P = 0.021). This means there is an association between plasma PGRN level and FAB classification; this finding disagrees with Azazzi et al.[21] and Qin et al.[20] The possible explanation is that the granulin modules can be excised from PGRN and occur in tissues and biological fluids as individual polypeptides. These polypeptides were called granulins because they were isolated from the granular fraction of human granulocytes.[34]
Newly diagnosed AML patients were subdivided into three categories according to the European Leukemia Net risk classification: higher risk, intermediate, and low risk. Accordingly, 5 (8.3%) patients had high-risk AML, 23 (38.3%) had intermediate-risk AML, while 32 patients (53.3%) had low-risk AML and showed no statistically significant differences with a mean rank of plasma PGRN level at diagnosis this is in agreement with Azazzi et al.[21] and Qin et al.[20]
ConclusionPlasma PGRN is high in AML patients at the time of diagnosisPatients with high-plasma PGRN levels at the time of diagnosis have a poor response to chemotherapyThere is a significant association between plasma PGRN level at diagnosis and the remission status after induction therapyThe plasma level of PGRN is higher in M3 than in the non-M3 groupHigh plasma PGRN level is significantly correlated with a high percentage of peripheral and marrow blast cells while not correlated with total leukocyte count, Hb, and platelet at the time of diagnosis.Acknowledgment
The authors would like to thank Mustansiriyah University/faculty of medicine, particularly the department of pathology and forensic medicine, for their support during the work.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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