Implications of Multimerin-1 (MMRN-1) expression in adult de novo acute myeloid leukemia: a preliminary study in Delta Egypt

ABSTRACT Adult acute myeloid leukemia (AML) is challengeable disease with poor heterogeneous outcome. Refining risk stratification is important for decision making and tailoring of therapy, multimerin-1(MMRN1) has been identified as a differentially expressed gene (DEG) in various cancers and it has been proposed as a possible cancer biomarker so, we aim to address prognostic value of Multimerin-1 in adult AML. This study was conducted on 240 AML, 40 healthy control, Taq man gene expression by RT PCR. Higher expression of Multimerin-1 was significant associated with failure of complete remission, relapse, short survival, highly significant association with minimal residual disease (MRD) positivity, molecular FLT3 (p 0.004, p.008) unfavorable cytogenetic (0.013). Cut off > 2.38 shows significantly short Overall Survival (OS), Disease-Free Survival (DFS). Finally, it could be independent poor risk for short survival; relapse thus may help in refine AML risk-stratification and tailoring therapy toward personalized medicine.


Introduction
Acute myeloid leukemia is a challenging disease with heterogeneous outcomes that vary widely between patients.Cytogenetic abnormalities and somatic mutations are the most important and provide the framework for diagnostic classification and risk stratification schemes [1].
According to the nearest survey, AML is common cancer in adults and the second most common leukemia in children, with relatively higher rates observed in countries with high development rates in North America, Oceania, and Europe [2].In 2021, More than 20,240 new cases of AML were estimated in the United States (1.1% of all new cancer cases).The estimated death rate from AML was 11.400 (1.9% of all cancer deaths) [3].The cure rates have increased by up to 15% in patients over 60 and by about 40% in patients under 60 according to recent improvements in management guidelines.Despite improvements in treatment modalities, the outlook for the older population is still very bad [4].
The extracellular matrix (ECM), megakaryocytes, endothelial cells (Weibel-Palade cells), and platelets all contain large amounts of the protein multimerin-1 (MMRN1), a member of the EMILIN/multimerin family.MMRN1 has been suggested as a potential cancer biomarker in recent years due to its identification as a differentially expressed gene (DEG) in a number of tumors [5].Out of 22 different cancer types, the expression of MMRN1 is markedly downregulated in 17 (bladder, breast, colon, esophagus, liver, lung, ovary, prostate, rectum, renal (cell carcinoma and papillary cell carcinoma), skin, stomach, testis, thyroid, and uterine carcinosarcoma and uterine corpus endometrial carcinoma) and markedly increased in pancreatic cancer and acute myeloid leukemia [6].
The activity of the oncoprotein MLL-AF6, which is produced when the genes for the histone methyl transferase mixed lineage leukemia (MLL) and the cytoplasmic protein AF6 combine, increases the expression of MMRN1, one of the genes linked to acute myeloid leukemia.The expression of the gene MMRN1 [7] distinguishes leukemia stem cells from leukemia progenitor cells, which are important in therapy resistance and disease relapse.A few studies were done; no further research was done in Egypt.Therefore, the current study aimed to assess the predictive value of MMRN1 expression in patients with adult acute myeloid leukemia from Delta Egypt and its relation to clinicopathological features of the disease.

Patients and methods
This cohort analytical study was conducted on 240 newly diagnosed AML patients in a tertiary hospital specialized oncology center at Mansoura University from April 2020 to March 2022.The patients were subjected to morphological examination of peripheral and bone marrow smears (blast cells ≥ 20%) that follow the updated WHO classification [8], which was confirmed by immunophenotyping through a standardized and optimal multiple fluorochrome panel for a more specific lineage of leukemic blasts.Immunophenotyping evaluation was done on next-generation multicolor 3-laser 10-color flow cytometry using different monoclonal antibodies (MoAbs) panels 7-8 into each tube with different matched staining index fluorochromes to increase sensitivity and specificity of diagnosis.Cytogenetic t(8; 21); t (15; 17); inv16; t (16; 16); t (6; 9); in addition to conventional karyotyping and genetic assessments (FLT3; NPM), were done for the aim of risk stratification.All patients received cytosine arabinoside plus anthracycline-based regimens as described previously by The International Working Group for Therapeutic Trials in AML's updated recommendations were used to evaluate the treatment's effectiveness [8].The AML patients included in this study had a 24-month follow-up period.
In addition to 40 healthy volunteers matched in age as a reference control, newly diagnosed AML patients with no previous treatment were included, while patients with a history of exposure to chemotherapy or radiotherapy and secondary AML patients and pediatric AML were excluded from study.

Ethical issues
All patients included in this study gave informed consent in accordance with relevant guidelines and regulations of the Declaration of Helsinki.

Sampling &methods
Three ml of peripheral venous blood was drawn from every patient for routine laboratory biochemistry investigation and complete blood counting (CBC), 1 ml on an EDTA purplecapped vacutainer tube for routine immunophenotyping from bone marrow, 1 ml on Na heparinized green capped vacutainer for cytogenetic study, another one ml on EDTA tube from bone marrow for molecular DNA extraction for Nucleophismin and FLT3.Lastly, one ml on EDTA from peripheral blood for RNA extraction to study gene expression by RT-PCR.
Total RNA was purified and extracted using the QIAamp RNA Mini Kit (QIAGEN Ⓡ Austin, Texas, USA, catalog no.52304).Total RNA concentration and purity were assessed using a spectrophotometer (Nanodrop, Quawell, Q-500, Scribner, USA).The concentration of RNA was obtained by measuring the absorbance at 260 nm.Purity was determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm (A260/A280).The pure RNA is kept at −80°C until further analysis and has an A260/A280 ratio of 1.9-2.1.Reversetranscription is conversion of the isolated RNA into complementary DNA (cDNA).In accordance with the manufacturer's instructions, this was carried out using the Applied Biosystems TM High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific's, USA), amplification for cDNA and the detection of the MMRN1 gene were carried out using the quantitative real-time polymerase chain reaction (qPCR) method and the TaqMan gene expression assay (Cat :HS00201182,Lot P181227) through the Step One Plus TM Real Time PCR system from Applied Biosystems (USA).The computerized thermocyclers were programmed to run at 95°C for 10 s, 95°C for denaturation for 0.15 s, and 60°C for annealing for 1.0 s (40 cycles).

Relative gene expression determination
The Ct was obtained for the target gene (Cttarget) for MMRN1 and for the reference gene B-actin (Ctref) for each well of the PCR run.ΔCtpatient was calculated for patient samples by subtract the Ctref from Cttarget for the patient samples and also ΔCtcontrol was calculated for control samples by subtract the Ctref from Cttarget for the control samples.
After calculating the Δ Ct control for all control samples in the run, the average of them was calculated as CT avgcont, and then the CT was calculated for each patient sample by subtracting the CT avgcont from the CT patient for this sample as follows: ΔΔCt = Δct patient -Δct avgcont The fold changes in the levels were determined by 2 −∆∆CT (cycle threshold) method and expressed as fold change (FC) using Step One software (Applied Biosystems, USA) [9].

Molecular assay of FLT3-ITD by conventional PCR
Thermo Scientific Gene JET Whole Blood Genomic DNA Purification Kit was used to extract DNA in accordance with the manufacturer's instructions.The extracted DNA was stored frozen at −20°C.The DNA samples were quantified by a Nano-Drop instrument, and the samples were measured at 17-45 ng µl.Apply 1 µl of extracted DNA with 12.5 µl ready master mix, 0.1 l of 100 pmol of each primer (Forward: 5/GCA ATT TAG GTA TGA AAG CCA GC 3/, Reverse: 5'CTT TCA GCA TTT TGA CGG CAA CC 3'), and a protocol of 95°C for 3 min followed by 35 cycles of 95°C for 30 s, 66°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 7 min.

Molecular assay of Nucleophismin by real-time PCR
The Ipsogen NPM1 Muta Screen Kit (Qiagen, REF 677,013, Germany) uses specific primers and an internal double dye probe with a reporter and a quencher (FAM TM, TAMRA TM) for amplification reactions in order to detect wild-type and mutant NPM (Mutants A, B, and D) by oligonucleotide hydrolysis principle.In addition, a 3-end modified phosphate oligonucleotide is used that perfectly matches the wild-type NPM1 gene and does not allow polymerization with amplification thermal cycle protocols of 50°C for 2 min, 95°C for 10 min, 95°C for 15 s, 60°C for 90 min for 40 cycles) Using DT Prime 4/DNA technology (SN: ASD312, USA)

Minimal residual disease (MRD) by next generation flow cytometry
Detection of abnormal leukemic blast in bone marrow sample after patient finish induction chemotherapy protocols and undergoing hematological remission through multiple, optimized fluorochromes 7-8 colors with matched staining index into tube to differentiate normal bone marrow cells from leukemic blast depending on guidelines of European leukemia net (ELN), this was carried out on leukemia associated phenotypes (Laps) or aberrant expression on initial phenotypes at diagnosis [15].

Statistical analysis
The collected data was revised, coded and tabulated using statistical package for Social Science (IBM Corp. Released 2017.IBM SPSS Statistics for Windows, Version 25.0.Armonk, NY: IBM Corp.).Data were presented and suitable analysis was done according to the type of data obtained for each parameter.Numerical data were expressed as mean and standard deviation.Qualitative data were expressed as frequency and percentage.Chi-square test was used to compare the nominal data in different groups.p value is considered significant if < 0.05 at confidence interval 95%.The ROC Curve (receiver operating characteristic) provides a useful way to evaluate the sensitivity and specificity for quantitative diagnostic measures that categorize cases into one of two groups.The optimum cut off point was defined as that which maximized the AUC value.Endpoints definitions: Overall survival is defined as the time from diagnosis until death or end of the study.DFS is defined as the time from remission until disease relapse, death or end of the study [16]

Results
Demographic data as well as laboratory data of studied cohort of AML patients are shown in Table 1 Their ages range from 23 to 81 years who had a median age of 41 years, a male represents 51.7%, while female 48.3%.When compared to the control group, the median MMRN1 gene was significantly higher in the AML group (median = 2.38 versus 0.9, p = 0.024), range of (AML 0.0003-588,13 while range in control 0.203-4.595).According to morphological analysis and FAB classification, the bulk of the samples in our study were classified as M1-2, 28.3% as M4-5, 8.3% same as M3, and at least 1.7 as M0.The bone marrow blast is 70%, compared to the typical peripheral blast of 30%.A molecular genetics investigation reveals that 95/240 (39.5%) of cases had FLT3 positivity, while 145/240 (60.4%) of the cases have Nucleophismin positivity.
Next- generation multicolor flow cytometry was used to assess the minimal residual disease following induction protocol medication.32/240 (13.3%) were positive, while negative 150 (62.5%).some cases were not applicable due to the lack of availability of some markers or difficulty in interpreting due to similar phenotypes of normal myeloblasts or sometimes dysplasia changing phenotypic patterns.(Table 2).
Regarding the clinical outcome of AML cases and its association with MMRN1 gene expression level, complete remission (CR) was achieved in 56.7% of cases, while 43.3% failed to achieve CR.Out of those who achieved CR, 29.4% relapsed.32 cases died (53.3%) during the study period.A higher MMRN1 gene level was significantly associated with the following: 1) failure of CR when compared to CR among AML cases (p = 0.001); 2) relapse when compared to non-relapse among AML cases (p 0.001); and 3) non-survivors when compared to survivors during the study period among AML cases (p 0.001) (Table 3).
ROC curve analysis of MMRN1 gene showed moderate accuracy (AUC = 0.724) for discrimination between AML cases and control groups.At the MMRN1 gene's best cutoff value of 1.189, the diagnosis of AML had a sensitivity of 71.7%, a specificity of 70%, a PPV of 28.5%, an NPV of 93.7%, and an accuracy of 70.2%.For the prediction of non-remission, the MMRN1 gene showed a moderately accurate AUC (AUC = 0.745).At the MMRN1 gene's best cutoff value of 2.675%, the prediction of non-remission had a sensitivity of 73.1%, a specificity of 73.5%, a PPV of 67.8%, an NPV of 78.1%, and an accuracy of 73.3%.In addition, for the prediction of relapse, the MMRN1 gene showed a high accuracy AUC (AUC = 0.900).At the MMRN1 gene's best cutoff value of 1.365, the prediction of relapse had a sensitivity of 81.3%, a specificity of 90%, a PPV of 86.7%, an NPV of 85.7%, and an accuracy of 86.1% (Table 4).As regards correlation with hematological parameters, the MMRN1 gene expression level showed significant positive correlations with LDH and BM blasts.But not with age, TLC, Hb, or platelets in the AML group (Table 5).Highly significant association between minimal residual disease (MRD) positivity and molecular genetic FLT3 positivity (P 0.004, P 0.008), in more highly significant association with unfavorable cytogenetic risk (P 0.013) (Table 6) When comparing overall survival (OS) and disease-free survival (DFS) according to median MMRN1 gene level in the AML group.The AML cases with a high MMRN1 gene subgroup (MMRN1 gene > 2.380) showed significantly shorter OS and shorter DFS when compared to the low MMRN1 gene subgroup (MMRN1 gene < 2.380) (Table 7, Figures 1 and 2).According to Cox regression analysis, shorter OS and shorter DFS in AML cases were independently associated with greater MMRN1 gene expression levels (Tables 8 and 9).
The current study revealed significant increase in MMRN1 expression in adult AML than control.This is the first study on adult AML at Delta Egypt.This finding in agreement with Laszlo et al. [11] Who found increased expression in pediatric AML while, dissimilar finding with Zhang et al. [13] Who found down expression       of MMRN1 in multiple myeloma patients by proteomic analysis and western blot, however he concluded in his study that it could be potential biomarker for diagnosis multiple myeloma and therapeutic follow up by changes protein concentration.On other hand, Saini et al. [12] reported that decreased expression in breast tissue than normal.The variability in gene expression by up and down regulation in different hematological and solid tumor increasing clinical value of this marker, wide spread in tissues.
ROC curve analysis of MMRN1 gene showed moderate accuracy (AUC = 0.724) for discrimination between AML cases and control groups.
At the MMRN1 gene's best cutoff value of 1.189, the diagnosis of AML had a sensitivity of 71.7%, a specificity of 70%, a PPV of 28.5%, an NPV of 93.7%, and an accuracy of 70.2%.For the prediction of non-remission, the MMRN1 gene showed a moderately accurate AUC (AUC = 0.745).At the MMRN1 gene's best cutoff value of 2.675%, the prediction of non-remission had a sensitivity of 73.1%, a specificity of 73.5%, a PPV of 67.8%, an NPV of 78.1%, and an accuracy of 73.3%.In addition, for the prediction of relapse, the MMRN1 gene showed a high accuracy AUC (AUC = 0.900).At the MMRN1 gene's best cutoff value of 1.365, the prediction of relapse had a sensitivity of 81.3%, a specificity of 90%, a PPV of 86.7%, an NPV of 85.7%, and an accuracy of 86.1% (Table 4).As regards correlation with hematological parameters, the MMRN1 gene expression level showed significant positive correlations with LDH and BM blasts.But not with age, TLC, Hb, or platelets in the AML group (Table 5).Highly significant association between minimal residual disease (MRD) positivity and molecular genetic FLT3 positivity (P 0.004, P 0.008), in more highly significant association with unfavorable cytogenetic risk (P 0.013), When comparing overall survival (OS) and disease-free survival (DFS) according to median MMRN1 gene level in the AML group.
The AML cases with a high MMRN1 gene subgroup (MMRN1 gene > 2.380) showed significantly shorter OS and shorter DFS when compared to the low MMRN1 gene subgroup (MMRN1 gene < 2.380).According to Cox regression analysis, shorter OS and shorter DFS in AML cases were independently associated with greater MMRN1 gene expression levels.
Interesting findings in the current study found that a higher MMRN1 gene level was significantly associated with the failure of CR when compared to CR among AML cases.Higher MMRN1 gene levels were significantly associated with relapse when compared to non-relapse among non-survivors.This finding is in agreement with Laszlo et al. [11]'s report that the highest relative MMRN1 expression fared worse than the lowest expression.According to Numata et al. [7], this association between high MMRN1 expression and relapse may be explained by the fact that MMRN1 is one of the genes that is upregulated by the activity of the oncoprotein MML-AF6, which is  created when the genes for the histone methyltransferase mixed lineage leukemia (MLL) and the cytoplasmic protein AF6 fuse.MMRN1 expression distinguishes leukemia stem cells, which contribute to therapy resistance and disease relapse, from leukemia progenitor cells [19].In view of this finding and others, MMRN1 is assumed to be a player in cell adhesion through integrin and may play a significant role in the bone marrow matrix with stem cell progenitors that contribute to relapse and failure of chemotherapy.The focus of the researcher on the bone marrow matrix as targeted therapy increases the clinical utility of this marker as a therapeutic hope for many solid malignancies and AML [20].
More interesting findings in our study were stratified according to the median level of the MMRN1 gene.AML cases with a high MMRN1 gene subgroup > 2.380 showed significantly shorter OS when compared to cases with a low MMRN1 gene subgroup (p 0.001).Moreover, AML cases with a high MMRN1 gene subgroup showed significantly shorter DFS when compared to cases with a low MMRN1 gene subgroup (p = 0.001).
The current study used age, gender, TLC, marrow blasts, LDH, cytogenetic risk, and MMRN1 gene expression level as covariates to conduct Cox regression analysis to assess the potential function of MMRN1 expression as an independent predictor of OS in AML cases.Higher levels of MMRN1 gene expression were thought to be a poor independent predictor of shorter OS in AML cases.In line with our findings, Laszlo et al. [11] discovered that high MMRN1 expression was linked to worse OS and EFS over the long term; patients with high MMRN1 expression were less likely to have a low-risk disease and more likely to have standard-risk and high-risk disease.Thus, it may not be surprising that univariate analyses showed high MMRN1 expression to be statistically significantly associated with lower CR rates, shorter survival estimates, and a higher risk of relapse.However, even after adjustment for cytogenetic and molecular disease risk, high MMRN1 remained statistically significantly associated with inferior OS, EFS, and RR, indicating that MMRN1 expression is an independent biomarker of poor outcome in adult AML.
Our study assumes the hypothesis that MMRN1 is present in a granule of platelets at rest.The host defense mechanism is weakened by the rising function of platelets in the development of micrometastatic niches in leukemia and solid malignancies as well as their promotion of neo vasculature, according to current research and predictions for the future.Thus, given that inhibition of E selectin causes disruption of the study's vascular niches, this could explain the involvement of MMRN 1 in leukemia relapse following conventional chemotherapy and Open Avenue as a therapeutic target for integrin [17].The discrepancies in the findings of the above-mentioned studies can be explained by a number of factors, including differences in genetic background and geneenvironment interactions, the stage of AML, dissimilar populations, the selection of patients, and limited sample size.

Conclusion
According to our findings, higher MMRN1 expression is an independent adverse predictive marker in adult AML, who are much more likely to experience first-treatment failure, relapse, and poor leukemia-free survival.
MMRN1 May be helpful for prescribing more potent chemotherapy, employing allogeneic hematopoietic cell transplantation in future studies, or offering therapeutic hope for targeting therapy in order to further individualize treatment options for AML.

Figure 1 .
Figure 1.OS according to MMRN1 gene level in AML group.

Figure 2 .
Figure 2. DFS according to MMRN1 gene level in AML group.
The study has been approved by the Mansoura Faculty of Medicine Local Ethics Committee.(IRB code No. MS. 19.05.651).

Table 1 .
Demographic data and MMRN1 gene expression level among studied groups.

Table 2 .
Hematological data at diagnosis of AML cases.

Table 4 .
Validity of MMRN1 gene for discrimination between AML cases and control groups, prediction of nonremission and for prediction of relapse among AML cases achieved CR.

Table 5 .
Correlations between MMRN1 gene expression level and hematological data in AML cases.

Table 6 .
Association of MMRN1geneexpressionlevel with cytogenetic, molecular stratification and MRD pattern in AML.

Table 7 .
Comparison of OS and DFS according to median MMRN1 gene level in AML group.

Table 8 .
Cox regression analysis for prediction of OS in AML group.

Table 9 .
Cox regression analysis for prediction of DFS in AML group.