The prognostic impact of different subsets of natural killer cells among Iraqi patients with AML

Abstract

Background: Acute myeloid leukemia (AML) is a diverse disease characterized by a wide range of genetic alterations and molecular abnormalities that influence clinical outcomes and provide prospective therapeutic targets. The immune evasion and active immune suppression by AML are due in part to the leukemia's inherent make-up as well as mechanisms acquired via subsequent mutation under selection pressure. Natural killer (NK) cells perform critical roles in the body's first-line innate defenses against viral infections, tumor progression, and metastatic dissemination. The constitutive production of effective, ready-to-use lytic machinery, as well as the quick release of interferon-gamma (IFN-α) and tumor necrosis factor-alfa (TNF-α) upon cell activation, allow for early intervention by NK cells, resulting in target cell destruction and the induction of an inflammatory response. NK cells depend on the balance of both inhibitory and activating receptor signals to select which target cells will be attacked or tolerated

Patients and methods: Thirty AML patient at diagnosis enrolled in this study from Baghdad teaching hospital, follow up these patients after 14 days of treatment and get result of remission after 30 days of treatment, twenty healthy looking persons considered as control subject. The technique used to detection of NK cells and their subsets was Flowcytometry.

Result: Decrease number of total NK in AML patients. The NK cells subset appear in patient was CD56^bright CD16^dim.

Conclusion: NK cells and their subset can be used as diagnostic and prognostic biomarker for AML patients.

Full Text

Introduction:

Acute myeloid leukemia (AML) is a clonal disorder characterized by the proliferation and the accumulation of myeloid precursors in the bone marrow (BM) as result of recurrent genetic, epigenetic, and metabolic abnormalities that show aberrant differentiation patterns leading to hematopoietic impairment ^24.

AML is a very variable illness that can manifest as "a de novo or secondary disease" (therapy-related or post-antecedent hematologic condition). The incidence of onset rises with age, and age is also linked to a larger frequency of high-risk cytogenetic and molecular abnormalities^35.

Although AML accounts for only about 1% of all cancers with an incidence of 4 per 100,000 a year, it is the most common acute leukemia in adults ^19,8,15.  AML is often associated with elderly persons and is uncommon before the age of 45, while it can occur at any age. The typical age upon diagnosis is 68 years, with most individuals diagnosed between the ages of 65 and 74 years ⁽³⁰ and predominant in male more than female ^18,7.

The immune system plays a dual role in cancer: It can not only suppress tumor growth by destroying tumor cells or inhibiting their overgrowth but also induce tumor progression either by selecting for tumor cells that able to survive in an immunocompetent host or by establishing conditions within the tumor microenvironment that facilitate tumor overgrowth ^11,26.

Natural Killer cells ( NK ) are large granular lymphocytes arising from the lymphoid origin which are deemed as the third largest population of lymphocytes following T and B cells include (10-15%) of all peripheral blood lymphocytes involved in defense against certain virus-infected and malignant cells ³³. In contrast to T cells, NK cells elicit rapid anti-tumor responses based on signals from activating and inhibitory cell surface receptors (22. They also lyse target cells via antibody-dependent cellular cytotoxicity (ADCC), a critical mode of action of several therapeutic antibodies used to treat cancer ⁵. Without prior sensitization, NK cells are innate lymphoid cells that contribute to the immune system's first-line defense against infections and malignant cells ^32,10. NK cells are divided into two categories based on surface expression levels of CD56 and CD16 as determined by immunofluorescence intensity. The standard CD56^dimCD16^bright, inhibitory and/or activating receptors ⁶.  In general, the major subpopulations of NK cells are CD56^bright CD16−/+ (5-10% of NK cells) and CD56^dim CD16+ NK cells (90-95% of NK cells), is a potent producer of immunoregulatory cytokines including interferon (IFN)-γ, tumor necrosis factor (TNF)-α/β and interleukin IL-10 ^16,17(6,34). NK cells rely on the balance of both inhibitory and activating receptor signals to determine which target cells will be attacked or tolerated ^25,14.

Patients and methods:

Patients: Thirty patients diagnosed as AML divided to 19 male and 11 female and follow-up at day 14 at treatment and at day 30 after treatment were collected from Baghdad Medical City, from February 2021 to November 2021. Twenty healthy looking persons match age and sex with patient considered as control subject.

In this study, the inclusion criteria were that patients newly diagnosed with any type of acute myeloid leukemia except AML-M3. Bone marrow aspirate sample available for immunophenotyping study with multicolor FC, of any sex, age 18 - 65 years. The treatment 7+3 regimen.

Exclusion criteria were age under 18 years, AML -M3 patients, another treatment protocol.

The patients’ peripheral blood (PB) and bone marrow aspirate (BMA) samples were received as fresh as possible and for each patient the following analysis were performed in diagnostic settings: full blood count, peripheral blood film and BMA smear morphology with SBB staining if morphology of blast cells was not showing clear myeloid differentiation, BMA multicolor FC testing for Using multicolor FC to evaluate the expression of CD7, CD11c, CD13, CD14, CD19, CD33, CD34, CD45, CD64, CD117 and MPO in adult patients with morphologically, cytogenetic analysis and genetic mutation ( NPM-1 and FLT-3 ), newly diagnosed AML

Study design: This is a cohort study that was performed in diagnostic laboratory settings

Collection of blood samples from each patient and healthy-looking patients, 5 ml of vein puncture blood sample was taken by using a syringe, dispensed in 2 tubes containing EDTA each with 1.5 ml, one for complete blood count and flow cytometry analysis.

Detection of NK cells, their subsets by flowcytometry.

In present study 8-collor Flow cytometry BD FACSCANTO II instrument was used to detect Immunophenotyping NK cells and their subsets in patients with AML and control subject.

The Surface receptors used for detection NK cells is (CD45 conjugated Violate 500 (V500), CD3 conjugated Violate 450 (V450), CD56 conjugated allophycocyanin (APC), CD16 conjugated ( PerCP - Cyanines 5.5), The NK cells immunophenotype  is ( CD45+,CD3-,CD56+,CD16+).All antibodies were products either (Elabscience/ China) and Santa Cruz biotechnology/UK)

Sample processing for BD FACSCANTO II flow cytometer: Using the following techniques with the principle of Stain–Lyse–Wash.

Preparation of reagents

Reagents were prepared according to the manufacturer’s instructions as follow:  Red Blood Cell (RBC) Lysis Buffer: Prepared by Dilute 1ml of 10X RBC Lysis Buffer with 9ml distilled water to form 1x working concentration. Then warmed to room temperature prior to use.

 Flow cytometry staining of whole blood procedure:

 five μl of fluorochrome conjugated antibodies (CD45-V500, CD3-V450, CD56-APC, CD16- PerCP - Cyanines 5.5) were added to 100μl of anti-coagulated blood. Another tube had 100μl of whole blood used as unstained control. Tubes were incubated in the dark for 15 minutes at room temperature. Next, 2ml of 1X RBC lysis solution was added to both tubes (stained and unstained) then incubated in the dark for 10 minutes at room temperature. The tubes were centrifuged for 5 minutes at 2300 rpm, discarding the supernatant. Tubes were washed with 2ml of cell wash solution via centrifugation for 5 minutes at 2300rpm, discarding the supernatant. Pellets were resuspended in 0.5mL of 1X cell fix buffer. Then both tubes were acquired on flow cytometric analysis FACS Canto II (BD BIOSCENCES) and analyzed with DIVA software version 2016. At least 100,000 events were analyzed for each sample.

Gating:

Gating strategy used to identify NK cells and their subsets and receptors; The serial gating used in this study involved in the first plot gating on viable cells using forward scatter (FSC) versus side scatter (SSC) plot lymphocytes were selected by CD45 versus side scatter (SSC) Then Gating on CD56 verses CD3. NK cells were identified by the expression of CD56 and CD16 and the absence of CD3. CD56 ⁺ CD16 ⁺ cells were select NK cells subset according to the florescent emission either CD56 ^Bright CD16 ^dim (Cytokines producers) and CD56 ^dim CD16 ^bright (Cytotoxic NK cells) figures (1), (2), (3), (4), (5) and (6).

Figure 1: Flow cytometry analysis of AML patient at zero day. a)  dot plot to of unstained cells b) dot plot to choose singlets cells c) dot plot to identify CD45+  d) dot plot to identify CD3- CD56+ population e) dot plot to identify CD56+CD 16+  ( NK cells ) as well as detection of CD56^bright CD16^dim (p4) and  CD56^dim CD16^bright  (p3)

Figure 2: Hierarchy analysis data from cells stained with Anti-CD45, Anti-CD3, Anti-CD56, Anti-CD16.

Figure 3: Flow cytometry analysis of AML patient after 14 days treatment. a)  dot plot to of unstained cells b) dot plot to choose singlets cells c) dot plot to identify CD45+  d) dot plot to identify CD3- CD56+ population e) dot plot to identify CD56+CD 16+  ( NK cells ) as well as detection of CD56^bright CD16^dim (p4) and  CD56^dim CD16^bright  (p1)

Figure 4: Hierarchy analysis data from cells stained with Anti-CD45, Anti-CD3, Anti-CD56, Anti-CD16.

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Figure 5: Flow cytometry analysis of control subject. a)  dot plot to of unstained cells b) dot plot to choose singlets cells c) dot plot to identify CD45+  d) dot plot to identify CD3- CD56+ population e) dot plot to identify CD56+CD 16+  ( NK cells ) as well as detection of CD56^bright CD16^dim (p4) and  CD56^dim CD16^bright  (p1) .

Figure 6: Hierarchy analysis data from cells stained with Anti-CD45, Anti-CD3, Anti-CD56, Anti-CD16.

Statistical analysis:

Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) version 23.0.0. Analytic statistics was carried out using t-test with p-value of ˂0.05 was considered significant. Descriptive statistics was presented into tables, bar charts, stem and leaf plots, scatter plots and trend-line mainly in the form of range, mean and standard deviation.

Result:

General characteristics of the studied groups.

The patients enrolled in this study 30 patients diagnosed with AML at day zero of therapy and 24 out of 30 patients were followed up at day 14 after therapy,19 males and 11 females with male: female ratio 1.72:1, table (1), 6 patients out of 30 were deceased. 20 healthy looking persons considered as a control subjects.

Mean age of patients with AML was 40.2±15.2 years and range from 18 - 65 years, while the mean for the control subjects was 41.1±13.3 years and range from 24 - 64 years, table (2) and figure (7).

Table 1: Sex distribution of patients with AML:

Sex group	Patients (n=30)		Control subjects (n=20)
	No.	%	No.	%
Males	19	63	13	65
Females	11	27	7	25
Male: Female	1.72:1		1.86:1

Table 2: Age distribution of patients with AML:

Age group (years)	Patients (n=30)		Control subjects (n=20)
	No.	%	No.	%
16-25	8	26.6	2	10
26-35	4	13.3	6	30
36-45	7	23.4	6	30
46-55	5	16.7	2	10
56-65	6	20	4	20
Range	18-65		24-64
Mean ± SD	40.2 ± 15.2		41.1 ± 13.3

Figure 7: Age distribution of patients with AML at diagnosis and at 14 days after treatment.

Hematological abnormalities in AML patients' group

In this study, there were two or more abnormalities including anemia, thrombocytopenia, leucopenia or leukocytosis, neutropenia and presence of blast cells with newly diagnosed patients with AML. The results for the abnormalities of complete blood count (CBC) and presence of blast cells in peripheral blood film in patients with AML are presented in table (3).

Table 3: Abnormalities of CBC and presence of blast cells in peripheral blood film in patients with AML:

Parameter/ Patients (n=30)		At diagnosis (n=30)		At day 14 after treatment (n=24)
		No.	%	No.	%
Anemia	Present	27	90	22	91.7
	Absent	3	10	2	8.3
Platelet count (X109/l)	≥ 100	6	20	2	8.3
	< 100	24	80	22	91.7
WBC count (X109/l)	> 10.0	18	60	1	4.2
	4.0-10.0	4	13.3	2	8.3
	<4.0	8	26.7	21	87.5
Neutrophils (%)	>70	0	0	15	62.5
	40-70	5	16.7	4	16.6
	<40	25	83.3	5	20.9
Monocytes (%)	3 - 12	10	33.3	6	25
	<3.0	15	50	11	45.8
	>12.0	5	16.7	7	29.2
Lymphocytes (%)	<20	23	76.7	20	83.3
	20-40	0	0	0	0
	>40	7	23.3	4	16.7
Eosinophils (%)	1-5	8	26.7	6	25
	<1.0	22	73.3	18	75
Basophils (%)	0.1-1	2	6.7	16	66.7
	<0.1	28	93.3	8	33.3
Immature/ Blast cells	Present	30	100	7	29.2
	Absent	0	0	17	70.8
Alive/ Deceased	Alive	30	100	24	80
	Deceased	0	0	6	20

Hematological remission with patient of AML:

Hematological remission is defined as absence of clinical evidence of leukemia with bone marrow blast cells <5%, absence of Auer rods, neutrophil count ≥1*10⁹/l, and platelet count ≥ 100*10⁹/l ⁴. 15 (62.5%) out of 24 of patient with AML at day 30 after treatment were in remission, and 9 (37.5%) out of 24 patients were not in remission, table (4).

Table 4: Hematological remission of patients with AML at day 30 after treatment:

Patients (n=24)	No.	%
Remission	15	62.5
No remission	9	37.5

Presence of NK cells and their subsets.

According to data presented in table (5), there was a statistically significant difference in the proportion of cells in the CD45+ CD3- CD56+ CD16+ (Natural killer cells) in patients with AML at diagnosis and at day 14 after treatment (mean±SD) (2.82±2.96, 1.308±1.928) respectively, with p-value (0.04). While there was no statistically significant difference in patients with AML at diagnosis and at day14 after treatment, and control subject p-value (0.134, 0.56) .

For cytokine-producer NK cells (CD45+ CD3- CD56^bright CD16^dim) there were no statistically significant difference in patients with AML at diagnosis and day 14 after treatment (5.08 ± 5.06, 6.56 ± 1.928) respectively, with p-value (0.39). On the contrary, there were a statistically significant difference between patients with AML at diagnosis (5.08±5.06), and AML at day 14 after treatment (6.56±7.63) compared with control subjects (0.03±0.09), p-value (0.001, 0.001).

For cytotoxic NK cells (CD45+ CD3- CD56^dim CD16^bright) there were statistically significant difference in patients with AML at diagnosis (7.55±7.29) and AML at day 14 after treatment (3.64±2.97) compared with control subject (18.8 ±17.6) p-value (0.017,0.03,0.001) respectively.

Table 5: Comparison of presence of NK cells and their subsets in AML patients at diagnosis and after 14 days therapy in comparison to control subjects:

CD expression (%)	Range (Mean ± SD)
	Patients		Control subjects (n=20)	p-value compare to
	At diagnosis (n=30)	At day 14 (n=24)		at diagnosis and at 14 days after treatment	at diagnosis and control subjects.		at 14 days after treatment and control subjects.
CD45+ CD3- CD56+ CD16+	0.1 – 12.5 (2.82±2.96)	0.3 – 9.6 (1.308±1.928)	0.3 – 5.6 (1.66±2.03)	0.04	0.134		0.56
CD45+ CD3- CD56bright CD16dim	0 – 20.9 (5.08 ± 5.06)	0 – 25.1 (6.56 ± 7.63)	0 – 0.3 (0.03 ± 0.09)	0.39	<0.001		<0.001
CD45+ CD3- CD56dim CD16bright	0.7 – 34.9 (7.55 ± 7.29)	0.4 – 9.8 (3.64 ± 2.97)	1.3 – 48.7 (18.8 ±17.6)	0.017		0.03		<0.001
significant p value <0.05 highly significant p- value <0.01

Presence of NK cells and their subsets in patients with AML in relation to mortality.

According to the data presented in table (6), in relation to mortality, there were no statistically significant differences in the proportion of cells in the total NK cells, cytokine producer NK cells, cytotoxic NK cells.

Table 6: Comparison of presence of NK cells and their subsets in patients with AML, in relation to mortality:

CD expression (%) at diagnosis	Range (Mean ± SD)
	Alive (n=24)	Deceased (n=6)	p-value
CD45+ CD3- CD56+ CD16+	0 - 9.9 (2.39±2.53)	0.6 - 12.5 (4.4±4.16)	0.128
CD56bright CD16dim	0 - 20.9 (5.3±4.44)	0.5 - 7.7 (4.2±3.81)	0.648
CD56dim CD16bright	0.7- 17.4 (6.72±5.19)	1.7 - 34.9 (10.86±12.96)	0.219
significant p value <0.05 highly significant p- value <0.01

Presence of NK cells and their subsets in patients with AML in relation to remission at 30 days after treatment.

According to the data presented in table (7), In regarding to remission at 30 days after treatment, there was no statistically significant difference in the proportion of cells in the Total NK cells, cytokine producer NK cells, cytotoxic NK cells.

Table 7: Comparison of presence of NK cells and their subsets at diagnosis in patients with AML, in relation to remission at 30 days after treatment:

CD expression (%) at diagnosis	Range (Mean ± SD)
	Remission (n=15)	No remission (n=9)	p-value
CD45+ CD3- CD56+ CD16+	0.1-9.9 (2.07±2.62)	0.2 - 7.0 (2.7 ±2.39)	0.598
CD56bright CD16dim	0 - 8.8 (4.16±2.72)	1.0 - 20.9 (7.21 ±7.9 )	0.173
CD56dim CD16bright	0 - 14.3 (7.20 ± 5.34)	0.8 - 17.4 (5.92 ± 5.13)	0.569
significant p value <0.05 highly significant p- value <0.01

Discussion:

Acute myeloid leukemia is a bone marrow malignancy defined by the clonal proliferation and differentiation arrest of myeloid progenitor cells. In the United States (US), the incidence of AML is 4.3 per 100,000 per year ²⁷. In Iraq Abdulridha and colleges studied 3102 of leukemia patients in the Iraqi Center for Hematology in the City of Medicine in Baghdad between January 2018 and December 2019, AML was 37% of total leukemia in 2018 while in 2019 AML was 40.8 % of total leukemia¹

AML may affect people of all ages, from infants to the elderly ^13,2. In the United States, the median age of AML diagnosis ranged from 62 to 68 years ³¹. Similar variations were seen in Europe and Canada ^28,29. In Saudi Arabia, the median age at AML diagnosis seems to be lower than that reported for the United States and Europe, despite the fact that there is minimal data to support this notion. The current research, which included 30 patients with AML at the time of diagnosis and followed up 24 of those patients after 14 days of treatment, found that the mean age of patients with AML was (40.2±15.2 years, ranging from 18 to 65 years according to the exclusion criteria.

The AML patients in this study was 19 males and 11 females with male: female ratio 1.72:1. This result agrees with Juliusson and colleges as with most leukemia, AML is a more common hematological malignancy in males than in females ^13.

The present research examines the overall number of NK cells and their subsets, in AML patients.

According to this study showed significant differences in proportion of NK cells between patients with AML at diagnosis and at day 14 after treatment (p-value = 0.04), while there were no significant differences between patients with AML at diagnosis, at day 14 after treatment, and control subjects (p-value 0.134, 0.56)

For cytokine-producer NK cells (CD56^bright CD16^dim), there was no statistically significant difference in patients with AML at diagnosis and day 14 after treatment p-value (0.39). On the contrary, there was a statistically significant difference between patients with AML at diagnosis and AML at day 14 after treatment with control subjects, p-value (0.001, 0.001), respectively. For cytotoxic NK cells (CD56^dim CD16^bright), there was statistically significant difference in patients with AML at diagnosis, AML at day 14 after treatment with control subject p-value (0.017, 0.03, 0.001), respectively.

Several research reports reported today relate impaired NK cell function with alterations in NK cell subset makeup, phenotype, and the capacity to create a completely functioning immunological junction ²⁸. Natural killer cell deficiency (NKD) is a primary immunodeficiency in which CD56+CD3 natural killer (NK) cells that mediate cytotoxicity against malignancies are defective. The majority of cases are viral infections and cancers that keep coming back in children and teens ²⁰.

Leukemia cells in AML may evade NK cell-mediated detection due to NK cell abnormalities, immunosuppressive features of AML cells, or interactions between NK cells and other immune cells that facilitate immune evasion ¹⁷. On the basis of surface expression levels of CD56 and CD16, as determined by the intensity of immunofluorescence, they may be divided into two categories ³⁶. The classical CD56^dimCD16^bright NK cell subset constitutes about 90% of the overall population in peripheral blood and displays potent cytolytic activity by producing cytotoxic granules containing perforin and granzymes. CD56^brightCD16^dim NK cells are a prolific generator of immunoregulatory cytokines, including as interferon (IFN)-γ, tumor necrosis factor (TNF) α/β , and interleukin (IL-10^{) 6}. Rey et al. (2017) found that in both AML patients, the immature NK cell population was lost selectively. This suggests that NK cell maturation is messed up in myeloid malignancies. This loss of primitive NK cells is accompanied by an increased percentage of phenotypically more CD56^brightCD16^negative/dim NK cells in the peripheral blood of AML patients ²³ . But Aggarwal et al. (2016) found that the number of mature NK cells (CD56^dimCD16^bright) was lower in AML. NK cells from AML patients have defective cytolytic activities ³. With activating and inhibitory receptors, the function of NK cells is either inhibitory or activating depending on the balance between activating and inhibitory receptors on NK cells ¹⁶. For activating and inhibitory receptors, the function of NK cells can be either inhibitory or activating depending on the balance between activating and inhibitory receptors on NK cells.

In relation to mortality, there are no statistically significant differences in the proportion of cells in the total NK cells, their subsets, and inhibitory and activation receptors among AML at diagnosis and AML at day 14 after treatment. AML is a disease of older people, with a median starting age of 68 – 72 years. NK cell activity may not be a factor in AML morbidity ¹². Song et al. (2018) found that older age is consistently related to worse survival and increased treatment-associated morbidity ³¹. Both tumor biology and age-related patient factors that reduce treatment tolerance are responsible for the poor prognosis of AML in older individuals ²¹.

According to the data reported in table (9) There is no statistically significant difference in the number of total NK cells and their subsets among AML patients at diagnosis and those in remission at 14 days and 30 days following therapy. In remission, we cannot consider the subsets and receptors of NK cells to be an indication of prognosis due to alterations in NK cell-activating receptors and their ligands in malignancies, which is one of the most prevalent problems that may lead to a diminished treatment response and tumor relapse ⁹.

Conclusion;

The observations of the present study were that total NK cells decreased in AML patients, (CD56^bright CD16^dim) elevated vs. (CD56 ^dim CD16 ^bright) NK cell subset, so NK cells and their subsets can be used as diagnostic and prognostic biomarkers for AML patients.