Value of the Assessment of Interleukin 6 level in Allogenic T-cell Replete Haploidentical peripheral blood haematopioetic stem cell transplantation using post-transplant cyclophosphamide as a predictor for the occurrence of acute Graft versus Host Disease

INTRODUCTION

Allogeneic hematopoietic cell transplantation (HSCT) is one of the curative therapeutic options for hematological malignancies (Henden et al., 2015). The host hematopoietic system is reconstituted with donor hematopoietic system and donor lymphocytes, especially T cells that have the potential to exert Graft Versus Tumor effects concurrently with graft-versus-host disease (Mussetti et al., 2017). Clinical data have shown that the severity of GVHD is negatively correlated with the chance of relapse (Greco et al., 2017). Acute GVHD is a major side effect of allogeneic HSCT.

Large number of pro inflammatory cytokines are involved in the pathophysiology of GVHD such as interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-g) (Drobyski et al., 2018). These cytokines are elevated after allogeneic BMT and affect GVHD through either direct cytotoxic effects on host tissues or by activation of immune effector cells (Toubai et al., 2016). Blockade of these cytokines modulated the severity of GVHD.

IL-6 is a cytokine that have broad effects, such as maturation and activation of B cells, T cells, and macrophages (Tvedt et al., 2017). IL-6 has been directly implicated in many human inflammatory diseases. Blockade of IL-6 signaling with anti–IL-6R monoclonal antibody Tocilizumab, has been shown to have therapeutic effects in many human diseases, such as rheumatoid arthritis, Castleman’s disease, and juvenile idiopathic arthritis (Roddy et al., 2016).

 IL-6 plays an important role in the severity of GVHD and that its blockade can ameliorate GVHD without leading to a complete loss of Graft Versus Tumor responses (Varelias et al., 2015). Given the safety and efficacy of IL-6 blockade in the treatment of several immunologic diseases in humans, there is data suggesting that targeting IL-6 with tocilizumab might be an immediately testable strategy in humans to decrease acute GVHD without losing the significant antitumor benefits of allogeneic BMT.

The Aim of the Study

Aim of this study is the early identification of patients at increased risk of HSCT-related complications, with a focus on acute Graft versus Host Disease, according to IL6 level as a potential biomarker. Serum IL6, measured before conditioning and 7 days after allo-HSCT, to predict main transplant outcomes with PT-Cy.

PATIENTS AND METHODS

This study was conducted on 40 patients with Hematological diseases from Maadi Military hospital and Ain Shams University hospital, BMT unit, who subjected to allogeneic stem cell transplant from Haplo-identical donars, after obtaining a written informed consent from the patients with median follow up one year.

Inclusion Criteria: Age range between 18 till 60 years old. Patients with haematological diseases indicated for allogeneic peripheral blood stem cell transplantation.  Patients with malignant haematological disorder should be in complete remission before proceeding to stem cell transplantation. Patients with available haploidentical stem cell donor with 5/10 to 8/10 matching in the HLA-A, HLA-B, HLA-C, HLA-DR and HLA-DQ loci. Patients receiving reduced intensity conditioning 

Method:  Peripheral blood samples will be collected from the study participants twice. Samples will be obtained on day -7 (by the time of initiation of conditioning regimens) and day +7 post-stem cell infusion. Measurement of interleukin-6 (IL-6) level will be performed by Enzyme Linked Immunosorbent assay (ELISA).

Study Interventions 

All patients included in this study will be subjected to the following: Full medical history. Thorough clinical examination. Routine pre-transplant assessment including: Human leucocyte antigen typing of HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1 using intermediate resolution typing. Donor specific allo-antibodies (DSA). Panel reactive allo-antibodies. Complete blood picture, blood group and Rh. Liver profile which includes: liver enzymes, serum albumin, total proteins, total and direct bilirubin and LDH level. Renal profile which includes: blood urea nitrogen, serum creatinine, serum uric acid, serum Na, serum K and corrected creatinine clearance. Random blood sugar. Lipid profile which includes: Total cholesterol level, triglycerides, LDL and HDL cholesterol. Coagulation profile which includes: PT, PTT, INR. Viral markers which include: HCV PCR, HBV PCR, HBsAg, HBsAb, HBeAg, HBeAb, HBcIgM, HBcIgG, HCVAb, CMVIgM, CMVIgG, CMV PCR, EBVIgM, EBVIgG, Toxoplasma IgM and IgG, HSVIgM, HSVIgG. TB gold Quantiferon test. Bone marrow aspirate+/- trephine biopsy, and minimal residual disease assessment. Dental examination. Cardiopulmonary assessment which includes: ECG, echocardiography, chest X ray, pulmonary function test and diffusion lung capacity of carbon monoxide (DLCO). Other radiological studies which include: CT chest, CT neck, CT pelvi abdomen, CT brain and skeletal survey if indicated.

Transplant procedure: All patients will receive reduced intensity conditioning regimen. Graft versus host disease prophylaxis will be composed of cyclophosphamide 50 mg/kg /day on days +3 and +4, mycophenolate mofetil (MMF) 15 mg/kg/day capped at 3 gm per day divided in three doses starting on day +5 and cyclosporine 3-5 mg/kg/day aiming at a trough serum level between 200-400 ng/mL. Both MMF and cyclosporine will be given 24 hours after the last dose of cyclophosphamide.

Post-Transplant workup: Patients will be followed up for the occurrence of acute graft versus host disease (aGvHD). Timing and severity will be recorded using the Glucksberg staging system and International Bone Marrow Transplantation Registry (IBMTR) grading. Peripheral blood IL-6 measurement using ELISA will be performed at the onset of aGvHD. Acute GvHD cases will be managed according to the severity of the disease. The outcome of the treatment will be reported as well. Follow up of the disease using bone marrow aspirate, minimal residual disease assessment using multi-parameter colour flowcytometry or polymerase chain reaction (if molecular abnormality have been reported earlier in the disease course), chimerism analysis will be performed according to the regular follow up of cases in the host institution.

Methods:- Human interleukin 6 ELISA kit. This kit is used to assay the Interleukin 6 (IL-6) in the sample of human’s serum, blood plasma, and other related tissue Liquid.

Principle of the Assay:- The kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to assay the level of Human Interleukin 6 (IL-6) in samples. Add Interleukin 6 (IL-6) to monoclonal antibody Enzyme well which ispre-coated with Human Interleukin 6 (IL-6) monoclonal antibody, incubation; then, add interleukin 6 (IL-6) antibodies labeled with biotin, and combined with streptavidin-HRP to form immune complex; then carry out incubation and washing again to remove the uncombined enzyme. Then add Chromogen Solution A, B, the color of the liquid changes into the blue, And at the effect of acid, the color finally becomes yellow. The chroma of color and the concentration of the Human Substance Interleukin 6 (IL-6) of sample were positively correlated.

Materials supplied in the Test Kit:

Materials required but not supplied: 

1. 37 ℃ incubator
2. Standard Enzyme reader
3. Precision pipettes and Disposable pipette tips
4. Distilled water
5. Disposable tubes for sample dilution
6. Absorbent paper 

Precautions: Beening taken out from the 2-8℃ environment,thekit should be balanced. 30 minutes in the ambient temperature then use. If the Coated plates of Enzyme haven’t been used up after opened, the remaining plates should be stored in Sealed bag. For each step, add Sample with sample injector which should be calibrated frequently, in order to avoid unnecessary experimental tolerance. The operation shall be carried out accordance to the instructions strictly. And test results must be based on the readings of the Enzyme reader. In order to avoid cross-contamination, it is forbidden to re-use the suction head and seal plate membrane in your hands. All samples, washing buffer and each kind of reject should according to infective material process. The idle agents shall be put up or covered. Do not use reagent with different batches. And use them before expired date. The substrate B is light-sensitive. Prolonged exposure to light is forbidden.

Washing method: Manually washing method: shake away the remaining liquid in the enzyme plates; place some bibulous papers on the test-bed, and flap the plates on the upside downstrongly. Inject at least 0.35 ml after-dilution washing solution into the well, and marinate 1~2 minutes. Repeat this process according to your requirements. Automatic washing method: if there is automatic washing machine, it should only be used in the test when you are quite familiar with its function and performance.

Specimen requirements: Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active. Extract as soon as possible after Specimen collection, and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can’t, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles. Serum – coagulation at room temperature 10-20mins，centrifugation 20- min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again. Plasma - use suited EDTA or citrate plasma as an anticoagulant, mix 10-20 mins, centrifugation 20-min at the speed of 2000-3000 r. p.m. remove supernatant, If precipitation appeared, Centrifugal again. Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, if precipitation appeared, Centrifugal again. The Operation of Hydrothorax and cerebrospinal fluid Reference to it. Cell culture supernatant-detect secretory components, collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, detect the composition of cells, Dilut cell suspension withPBS（PH7.2-7.4), cell concentration reached 1million/ml, repeated freeze-thaw cycles, damage cells and release of intracellular components, centrifugation 20-minat the speed of2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again. Tissue samples- After cutting samples, check the weight, add PBS PH7.2-7.4), rapidly frozen with liquid nitrogen, maintain samples at 2-8℃ after melting, add PBS（PH7.4）, Homogenized by hand or Grinders, centrifugation 20-minat the speed of 2000-3000r.p.m. removesupernatant.

Note: Grossly hemolyzed samples are not suitable for use in this assay

Assay procedure: 

Standard dilution: This test kit will supply one original Standard reagent, please dilute i t by yourself according to the instruction.

The quantity of the plates depends on the quantities of to-be-tested samples and the standards. It is suggested to duplicate each standard and blank well. Every sample shall be made according to your required quantity, and try to use the duplicated well as possible Inject samples: Blank well: don’t add samples and IL-6 -antibody labeled with biotin, Streptavidin-HRP, only Chromogen solution A and B, and stop solution are allowed; other operations are the same. Standard wells: add standard50μl,Streptavidin-HRP 50μl(since the standard already has combined biotin antibody, it is not necessary to add the antibody); To be test wells: add sample 40μl, and then add both IL-6 - antibody 10μl and Streptavidin-HRP 50μl. Then seal the sealing memberance, and gently shaking, incubated 60 minutes at 37 ℃. Confection: dilute 30 times the 30×washing concentrate with distilled water as standby. Washing: remove the memberance carefully, and drain the liquid, shake away the remaining water. Add chromogen solution A 50μl, then chromogen solution B 50μl to each well. Gently mixed, incubate for 10 min at 37℃ away from light. Stop: Add Stop Solution 50μl into each well to stop the reaction (the blue changes into yellow immediately). Final measurement: Take blank well as zero, measure the optical densit (OD) under 450 nm wave length which should be carried out within 15 min after adding the stop solution. According to standards’ concentration and the corresponding OD values, calculate out the standard curve linear regression equation, and then apply the OD values of the sample on the regression equation to calculate the corresponding sample’s concentration. It is acceptable to use kinds of software to make calculations. 

Summary procedures:

Calculate:

Take the standard density as the horizontal, the OD value for the vertical, draw the standard curve on graph paper, find out the corresponding density according to the sample OD value by the sample curve (the result is the sample density) or calculate the straight line regression equation of the standard curve with the standard density and the OD value, with the sample OD value in the equation, calculate the sample density.

Sensitivity and Assay range:

Sensitivity: 2.112 ng/L

(The sensitivity of this assay, was defined as the lowest protein concentration that could be differentiated from zero. It was determined by sub tracting two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.)

Assay range: 3 ng/L→600ng/L

Validity & Storage: six months (2-8℃)

Statistical analysis 

The collected data was revised, coded, tabulated and introduced to a PC using Statistical package for Social Science (SPSS 26). Data was presented and suitable analysis was done according to the type of data obtained for each parameter.

Descriptive statistics: Mean, Standard deviation (± SD) and range for parametric numerical data, while Median and Interquartile range (IQR) for non-parametric numerical data. Frequency and percentage of non-numerical data.

Analytical statistics:  Mann Whitney Test (U test) was used to assess the statistical significance of the difference of a non-parametric variable between two study groups. Chi-Square test was used to examine the relationship between two qualitative variables. Fisher’s exact test was used to examine the relationship between two qualitative variables when the expected count is less than 5 in more than 20% of cells. Wilcoxon signed rank test was used assess the statistical significance of the difference of an ordinal variable (score) measured twice for the same study group. Correlation analysis (using Spearman's rho method) to assess the strength of association between two quantitative variables. The correlation coefficient denoted symbolically "r" defines the strength (magnitude) and direction (positive or negative) of the linear relationship between two variables. r= 0-0.19 is regarded as very weak correlation. r=0.2-0.39 as weak correlation. r=0.40-0.59 as moderate correlation. r=0.6-0.79 as strong correlation. r= 0.8-1 as very strong correlation. 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. P- value: level of significance: P>0.05: Non significant (NS). - P< 0.05: Significant (S).

RESULTS

Table (1): Demographic data of the study group (n=40)

Table (2): Acute GVHD grading in the study group.

Table (3): Acute GVHD stages in the study group.

Table (4): IL-6 level distribution in the study group.

Table (5): Pre transplant disease status in the study group.

 Table (6): Engraftment status in the study group.

Table (7): Pre transplant CMV distribution in the study group.

Table (8): Infection and VOD in the study group.

Table (9): NRM in the study group.

Table (10): Major post-transplant complications in the study group.

Table (11): Relapse in the study group.

Table (12): Acute GVHD

*One Way ANOVA test of significance (f):

*Post-hoc Bonferroni test was significant between: Group 3 Vs. (Groups 1 & 2).

Table (13): Relation between IL-6 level and incidence of infection in the study group by ANOVA test (n=40)

Table(14):Relation between IL-6 level and incidence of skin acute GVHD in the study group by ANOVA test (n=40).

Table (15): Relation between IL-6 level and incidence of liver acute GVHD in the study group by ANOVA test (n=40).

Table (16): Relation between IL-6 level and incidence of GIT acute GVHD in the study group by ANOVA test (n=40).

Table (17): Roc curve of IL-6 +7 to predict acute GVHD.

Table (18): Progression Free Survival.

Table (19): Overall survival for the study group (N= 40).

­

Overall survival (OS) gradually decrease with time, as highest survival proportion at day 11 (95.0%) and lowest survival proportion at day 365 (52.5%) with mean survival 252.1 days.

DISCUSSION 

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment option for many malignant and non-malignant hematological disorders, still limited by severe complications and transplant-related mortality (TRM) (Gooley et al., 2010).

Acute Graft-vs.-Host Disease (aGvHD) is a leading cause of morbidity and TRM after allo-HSCT. Despite prophylactic treatment with immunosuppressive agents, historically 20– 80% of recipients develop aGvHD after allo-HSCT. Post-transplant cyclophosphamide (PT-Cy) has emerged as a promising pharmacological strategy in the setting of allo-HSCT, thanks to its safety profile and effectiveness in reducing GvHD and finally TRM (Greco et al., 2017).

New diagnostic and therapeutic tools are still needed to customize the administration of immunosuppressive drugs for patient care optimization. To that end, there has recently been considerable research effort devoted to the discovery and validation of GvHD-relevant biomarkers (Levine et al., 2012).

Biomarkers that are GvHD and target-organ specific may improve the diagnosis, management, and prognosis of post-transplant complications. Potential applications include predicting response to treatment, defining new risk stratification that incorporates biomarker values, and initiating preemptive therapy before onset of clinical symptoms (Greco et al., 2017).

Patients who are predicted to have low-risk aGVHD may benefit from lower doses and shorter courses of immune suppression. In addition, because not all cases of aGVHD progress in the same way or have the same outcome, the therapy should be tailored not only to the severity of the disease, but also to the predicted rate of progression (Mcdonald et al., 2015).

As a result, numerous researchers have examined whether adding novel plasma biomarkers at different time points before and after transplantation can add to the accuracy of prediction compared with other prognostic tools. Timely recognition of patients who are at high risk for aGVHD or who would likely demonstrate resistance to steroids early in the course of transplantation may lead to more stringent monitoring, better preventive care, and introduction of alternative and more effective immunosuppressive treatments earlier in the course of treatment (Tatekawa et al., 2016).

It is reasonable to assume that plasma proteins involved in the complex pathophysiology of aGVHD might be altered in these patients. For the past 20 years, various groups have been investigating potential biomarkers to enhance the early and more accurate diagnosis and risk stratification of patients with aGVHD. Recent research has applied proteomics technologies to identify aGVHD biomarkers. This has led, in a short period, to the identification of novel biological pathways and biomarkers predictive of and associated with aGVHD. Nevertheless, no single biomarker or panel of biomarkers has been validated for clinical use via large multicenter trials (Hartwell et al., 2018).

The balance between pro- and anti-inflammatory cytokines influences the risk of aGvHD. Interleukin-6 (IL6) is a cytokine associated with several inflammatory diseases and a modulator of the immune responses involved in aGvHD pathogenesis (Narazaki et al., 2018).

IL6 can be targeted with a selected inhibitory strategy based on anti-IL6 receptor antibody tocilizumab (TCZ) (Narazaki et al., 2018). Moreover, IL6 could be easily and rapidly tested by many centers as routine clinical practice, thanks to the availability of commercial assays. Certainly this represent an important additional value as compared to other proposed biomarkers for GvHD, validated in large clinical trials but still hardly accessible on large scale (Hartwell et al., 2018). However, available data on its potential role as systemic biomarker predictive of GvHD are still limited and conflicting (Mcdonald et al., 2015).

In This study we conducted on 40 patients with Hematological diseases from Ain Shams University hospital and Maadi Military hospital, BMT unit, (22 male, 18 female) with age range (18 - 44) years old (Table 1) who underwent Haplo allogeneic stem cell transplant, after obtaining a written informed consent from the patients.

Aim of this study is the early identification of patients at increased risk of HSCT-related complications, with a focus on acute Graft versus Host Disease, according to IL6 level as a potential biomarker. Serum IL6, measured 7 days before conditioning and 7 days after allo-HSCT, to predict main transplant outcomes with PT-Cy

Our patients were 40 (22 male 55% and 18 female 45 %) 10 patients had B-ALL 25%, 8 patients had AML 20%, 8 patients had Aplastic anemia 20%, 6 patients T-ALL 15%, 4 patients Biphenotypic acute leukemia 10%, 2 patients Sickle cell disease 5% and 2 patients MDS EB type 2 represent 5%.

40 patients received Haplo related HSCT (3\6 or more antigen mismatch) with RIC regimens. 

All the study population received PTCY (50mg\kg D+4& D+5), Cyclosporine (5mg\kg\day starting initial oral dose them follow up according to serum cyclosporine level (3 times) weekly) starting from Day zero and MMF (30mg\kg\day starting from D+ 6 for 1 month) as acute GVHD prophylaxis

The diagnosis of aGVHD can be made on clinical grounds in patients presenting with a rash, diarrhea and elevation of bilirubin within the first several weeks of transplant. However, the diagnosis is frequently not straightforward. The role of skin and liver biopsies may be helpful in making the diagnosis but this is still controversial (Döring et al., 2015).

In our study 28 patients had Acute GVHD, 18 patients had grade 1 & 2 Acute GVHD 64.3% while, 10 patients had grade 3 & 4 Acute GVHD 35.7% as in.

The median range of IL-6 level at day -7 before infusion of stem cells (85 ng\L) while, IL-6 level at day 7 after infusion of stem cells (107.5 ng\L).

In relation to IL 6 level pre-Transplant (Day -7) and post-Transplant (D+7), Acute GVHD grade III-V is higher than acute GVHD grade I-II, P. value (<0.001, 0.011) respectively.

There is Statistically significance detected between IL 6 level (in Day -7 before infusion of stem cells and day +7 after infusion of stem cells) and GVHD severity with P.Value (0.006, <0.001) respectively.

Grade 3-4 Acute GVHD patients had high Post-transplant and pre- transplant IL 6 level than patients developed Grade 1-2 GVHD with Mean (331\131) than (113.89\86) with statistically significant difference P value (<0.001, 0.006) respectively. 

Roc curve was done for IL-6 +7 to detect occurrence of acute GVHD, greater Area under the curve (AUC) was 0.765 means that the diagnostic test is perfect in the differentiation between the patients had the disease, p-value was less than 0.001 it is highly significant, So we can use IL-6 +7 to detect occurrence of acute GVHD. Cut-off value was more than 110, so if IL-6 +7 value more than it this patient more liable to have the disease with sensitivity (53.6%) and specificity (100%). The percentage of cases had IL-6 +7 value more than 110 and had the disease was (+PV) was 100, while the percentage of cases had IL-6 +7 less than or equal 110 and hadn’t the disease was (-PV) was 48. Negative likelihood ratio (-LR) was 0.46.

Our study results were matched with Greco et al. who collected samples from 166 consecutive allo-HSCT patients. By ROC analysis, they identified a threshold of 2.5 pg/ml for pre-transplant IL6 and 16.5 pg/ml for post-transplant IL6. Both univariate and multivariate analyses confirmed the ability of high baseline IL6 levels to predict grade II–IV acute GvHD (p = 0.04), and of high post-transplant IL6 to predict higher risk of grade II–IV (p < 0.01) and grade III–IV acute GvHD (p < 0.01). 

In our study Skin acute GVHD stage 3-4 patients had high Post-transplant and pre- transplant IL 6 level than patients developed stage 1-2 skin acute GVHD with Mean (345\155) than (105\84) with statistically significant difference P value (0.006, 0.006) respectively. 

Our results are matched with Döring et al. (2015) on skin GvHD. They observed a total of 15 (24.59 %) of the 61 pediatric patients experienced an acute GvHD of the skin. 6 (40 %) out of 15 patients had a skin GvHD stage I. 8 (53.3 %) patients suffered from skin GvHD stage II, while 1 (6.67 %) patient experienced acute skin GvHD stage III. The 9 patients with a skin GvHD stage II and III, developed significant increases of IL-6 (P= 0.0010) 8 of the 9 patients with skin GvHD stage II and III had an increase in IL-6 a few days before (median 2 days) the appearance of exanthema of the skin. The patients with skin GvHD stage I had only an increase of IL-6.

In our study Liver acute GVHD stage I-II patients in our study had high Post-transplant IL 6 level with Mean (243.75) with statistically significant difference P value (0.006) while, Liver acute GVHD stage I-II patients had Pre-transplant IL 6 level with Mean (113.13) with statistically insignificant difference P value (0.052).

Post-transplant IL 6 results were augmented with Döring et al. (2015) results on liver GvHD on two the pediatric patients experienced liver GvHD stage III and stage IV, respectively. In both cases, a clear increase in the level of IL-6 could be observed. In one patient, these increases occurred two days before laboratory chemical changes were seen for direct and indirect bilirubin and the transaminases ALT and AST. In the other patient, the cytokine levels and the laboratory chemical markers changed simultaneously. However, due to the small number of cases, it was not possible to detect any statistical significance.

In our study GIT acute GVHD stage 3-4 patients had high Post-transplant IL 6 level than patients developed stage 1-2 GIT acute GVHD with Mean (226.67) than (198.89) with statistically significant difference P value (0.003) while, GIT acute GVHD stage 3-4 in our study patients had low pre-transplant IL 6 level than patients developed stage 1-2 GIT acute GVHD with Mean (86.67) than (108.78) with statistically insignificant difference P value (0.624). 

Döring et al. (2015) Intestinal GvHD: In 9 (14.8 %) of the 61 patients an acute intestinal GvHD was observed. Acute intestinal GvHD stage I occurred in 2 (22.2 %) patients, while intestinal GvHD stage II was seen in 6 (66.7 %). One patient (11.1 %) suffered from intestinal GvHD stage III. At the onset of the first clinical symptoms of acute intestinal GvHD with an increase of feces quantity, significant increases of IL-6 (p= 0.0010) observed in all pediatric patients with acute intestinal GvHD stage II and III. These results were matched with our study results on post-transplant IL 6 level.

Hematopoietic stem cell transplantation (HSCTs) involves the intravenous infusion of hematopoietic stem and progenitor cells (HSPCs) to restore the bone marrow (BM) function after intensive cancer-eradicating treatments or to replace defective bone marrow with normal bone marrow in non-malignant hematologic disorders (Passweg et al., 2016).

In our study     there is statistically significant difference of the incidence of infection in the study group with highest distribution of bacterial infection (55%), and lowest distribution with viral and fungal infection (5%). There’s statistically difference between the mean IL 6 level at day -7 and day +7 and the development of infection with p value (0.001, 0.074) respectively. 

Song et al. (2019) Serum IL-6 levels could discriminate sepsis (area under the curve [AUC], 0.83–0.94, P< 0.001; cut-off value, 52.60pg/mL, 80.4% sensitivity, 88.9% specificity) from controls and could distinguish septic shock (AUC, 0.71–0.89; cutoff value, 348.92pg/mL, 76.1% sensitivity, 78.4% specificity) from sepsis. Twenty-eight-day mortality was significantly higher in the group with high IL-6 (≥ 348.92pg/mL) than in the group with low IL-6 (<348.92pg/mL) (P=0.008). IL-6 was an independent risk factor for 28-day mortality among overall patients (hazard ratio, 1.0004; 95% confidence interval, 1.0003–1.0005; p=0.024). In septic shock patients, both the initial and follow-up PTX3 levels were consistently significantly higher in patients who died than in those who recovered (initial p=0.004; follow-up P< 0.001). These results matched with our results.

Döring et al. (2015) they did a study on Cytokine serum levels during post-transplant adverse events in 61 pediatric patients after hematopoietic stem cell transplantation. 11 (18.0 %) of the 61 patients developed sepsis. 5 (8.2 %) patients had a bacteremia with positive blood cultures. In 21 (34.4 %) out of 61 patients 25 localized bacterial infections were detected over the course of the observation period. Bacterial infections appeared in the urine (n=11), feces (n=8), throat (n=5) and bronchoalveolar lavage (n= 1). In the case of sepsis a significant increase of IL-6 (P= 0.0020), IL-8 (P=0.0020), sIL-2R (P=0.0156), and TNF-α (P=0.0078) was observed in all pediatric patients (n=8) for which an analysis of the cytokine levels was performed on the day of the occurrence of sepsis. These patients also had a body temperature ≥38.3 °C at that point in time. In the remaining 3 patients that experienced a sepsis, the final analysis of cytokine levels was done more than 24 h prior. Fever was not present at the time blood was taken. There was no change in the analyzed cytokine levels at this time. In all 5 (10.42 %) of the 61 patients diagnosed with bacteremia, a significant increase of IL-6 (P<0.0001) and IL-8 (P=0.0006) was observed. These results matched with our results.

Overall survival is the length of time from either the date of diagnosis or the start of treatment for a disease, such as cancer, that patients diagnosed with the disease are still alive. 

In our study overall survival (OS) gradually decrease with time, as highest survival proportion at day 11 (95.0%) and lowest survival proportion at day 365 (52.5%) with mean survival 252.1 days.

In our study cases with Interleukin 6 level ≤ 110 ng/L are significantly associated with higher mean PFS (280.79) in comparison to cases with Interleukin 6 > 110 ng/L, that have lower mean PFS (142.4), 

Incidence of high PFS is significantly higher 1.97 times in cases with interleukin 6 ≤ 110 ng/L than cases with interleukin 6 > 110 ng/L (P value 0.003). 

CONCLUSION 

Grade 3-4 Acute GVHD patients had high Post-transplant and pre- transplant IL 6 level than patients developed Grade 1-2 GVHD with Mean (331\131) than (113.89\86) with statistically significant difference P value (<0.001, 0.006) respectively. 

Cases with Interleukin 6 level ≤ 110 ng/L are significantly associated with higher mean PFS (280.79) in comparison to cases with Interleukin 6 > 110 ng/L, that have lower mean PFS (142.4). Incidence of high PFS is significantly higher 1.97 times in cases with interleukin 6 ≤ 110 ng/L than cases with interleukin 6 > 110 ng/L (P value 0.003).