Immunophenotyping in diagnosing pediatric acute leukemia after setting up the first flow cytometry unit in Mosul City in Iraq: an observational study of the project performed through a contribution from Japan

Introduction

Flow cytometry (FCM) is a worldwide routinely adopted laser-based technology used in clinical practice to provide simultaneous multi-parameter analysis of a single cell. FCM produces characteristic scattered and fluorescent light signals, which allow evaluation of cell populations and immunophenotyping. Over the last 30 years, FCM technology has improved dramatically to become a powerful tool in the immunophenotyping of acute leukemia (AL) and cancer biology (1-3).

AL includes acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). In pediatrics, ALL accounts for about one-quarter of all childhood cancers, and it constitutes approximately 80% of pediatric AL cases, as it is five times more common than AML. ALL has two main subtypes: B-lineage ALL (B-ALL), which represents about 85% of ALL cases, and T-lineage ALL (T-ALL) with 10–15% (4,5). Accurate and rapid diagnosis of ALL or AML and further subtyping are crucial steps for the timely selection of appropriate therapeutic protocol and for determining prognostic significance. Furthermore, correct management leads to better outcomes and ultimately improves survival (6). Therefore, immunophenotyping by FCM is vital in classifying AL types and subtypes along with other biological characteristics, as lineage identification can provide a confirmatory diagnosis or differential diagnosis and prognosis. Accordingly, treatment options would also be possible to decide (2).

Iraq, one of the Middle Eastern countries, has suffered for several decades from repeated wars, economic blockade, terrorism, and their aftermath, which resulted in the collapse of the health system (7-9). In the northwest of Iraq, in the Nineveh province, lies Mosul, the second-largest city in Iraq, with a population exceeding two million. In 2014, Mosul was occupied by the Islamic State of Iraq and the Levant (ISIL), and the city suffered extreme damage for more than 3 years; the medical system had colossal damage as well. Patients with cancer need advanced health services, which become impossible in such circumstances. Shortages in laboratory services, including molecular and biological analyses, severely impacted the diagnosis, management, and, ultimately, the outcome of diseases such as leukemia (10,11).

Universal health coverage, as promoted by the World Health Organization (WHO), means that everyone should have access to essential medical services without excessive financial burden. However, this is not feasible in many countries in the developing world, such as Iraq. Fortunately, non-profit organizations (NPOs) concerned with health-related issues from more developed countries could play an essential role in less developed countries in terms of strengthening the health systems by working in partnership with administrative health institutes to manage joint projects through collaborations to present opportunities for knowledge translation to be utilized on the ground, especially those related to a particular specialization. The WHO prioritized five medical topics: cancer, cardiovascular diseases, chronic respiratory diseases, diabetes, and mental health (12-14). The Japan Chernobyl Foundation (JCF) is a Japanese NPO dedicated to providing humanitarian aid, primarily through medical care to children with leukemia. JCF was established in 1991 to support the pediatric cancer victims of the Chernobyl disaster (15). Likewise, JCF has been at the forefront of sponsoring emergency and developmental medical projects in Iraq since 2004, with a particular focus on pediatric oncology (11,16). Within this collaborative framework, medical collaboration has been planned and achieved by accomplishing the FCM unit project for the first time in Mosul City, Iraq.

In this article, we aimed to highlight the role of a Japanese NPO, namely JCF, and its medical collaboration project in establishing the first FCM unit in Mosul City in Iraq through a fund from the Japanese government and to disclose the data of more than 3-year follow-up of the work along with the outcome of cases and in particular the childhood ALL cases from a scholarly point of view. We present this article in accordance with the STROBE reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-24/rc).

Methods

Setting up of the FCM unit

To enhance diagnostic capabilities, under the title (Strengthening the Diagnostic System in the Pediatric Oncology Center in Mosul), JCF submitted a plan to the Japanese Ministry of Foreign Affairs in 2019 for setting up an FCM Laboratory Unit at Ibn Al-Atheer Hospital for Children in Mosul, Iraq. The project target aligns with achieving the Sustainable Development Goals for ensuring healthy lives and promoting well-being for all ages. Once approved, funding of US dollar (USD) 234,010 was provided to JCF for a 1-year project to be performed from January 2020 to January 2021 (17).

Unfortunately, due to the circumstances of the coronavirus disease-2019 (COVID-19) pandemic, a significant delay occurred because of the closure of airports for several months in many countries, including Iraq. However, an FCM machine and other hematology-related devices, including an advanced microscope, centrifuge, cytospin, vortex, and pipettes, were eventually provided. Likewise, a refrigerator for storing the cluster differentiation (CD) markers, a computer unit with a printer, different kinds of CD markers, and other necessary materials were finally delivered. The unit was completely set up and in use by January 2021. JCF has also supported the purchase of the CD markers on two occasions to prevent work from stopping in case local financial resources are unavailable.

The FCM machine (BD FACSCanto™ II), with its innovative eight-color capabilities, is the third of its kind to be delivered to Iraq, but the first in Mosul City. Notably, Iraq had another 17 FCM machines distributed in other provinces within Iraq, mostly of older generations. Although the FCM unit was provided to the pediatric oncology unit, it is also used to analyze samples from adult cases and, thus, serves people of all ages in Mosul and the surrounding areas without charge.

Training of the staff

A 4-week training plan for two hematopathologists and a technician, to be conducted outside Iraq, had to be canceled due to the COVID-19 pandemic. However, since the hematopathologists from Mosul already had knowledge about FCM and immunophenotyping, and since the training included theoretical and practical refreshing sessions, an alternative plan was set for the training inside Iraq. It is noteworthy that the training was conducted free of charge through fruitful cooperation with experts from the Sulaymaniyah and Erbil provinces in northern Iraq, with continued support and follow-up after the end of the training and for several months.

Samples preparation

The Mosul FCM staff prepared a request form for doing the FCM analysis and emailed it to hospitals to be filled out upon referral. Bone marrow aspirate (BMA) and peripheral blood samples of cases with abnormal hematological findings or suggestive of AL were freshly received in ethylenediaminetetraacetic acid (EDTA) blood collection tubes, along with the filled request form, including a brief history, clinical notes about the patient, recent complete blood count (CBC), and the BMA results. One portion of each sample was initially checked for white blood cell (WBC) count with a differential and morphological evaluation before proceeding to the FCM. The EDTA blood samples stored at room temperature (20–25 ℃) were usually analyzed within less than 24 hours of staining, provided no more than 48 hours had passed from blood collection. The samples were stained, lysed, and then washed by utilizing the direct immunofluorescence procedure, and according to the manufacturer’s instructions, they were processed and analyzed using the FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA) (18,19).

Anonymous patient data, including age, sex, address, and initial CBC with the BMA results, along with the coded FCM reports, were prospectively collected from February 2021 to January 2024. In addition to the clinical notes on pediatric ALL cases, their treatment, and outcomes with a median follow-up of 26.2 (range, 0–44.3) months. Newly diagnosed ALL and AML cases who were 0–16 years old were included in the study.

FACSCanto II flow cytometer and steps of analysis

The machine is configured with three lasers to detect up to eight colors, featuring a fully integrated fluidics cart, a BD FACS loader option for automated sample acquisition, and BD FACSCanto Clinical Software (FACSDiva v.6) for analysis. The eight fluorochromes to which the monoclonal antibodies (MAbs) are conjugated included fluorescein isothiocyanate (FITC), phycoerythrin (PE), PE and cyanine dye 7 (PE-Cy7), allophycocyanin (APC), APC-H7, peridinin chlorophyll (PerCP)-Cy5-5, V450, and V500. All the MAb materials were purchased from Becton Dickinson Biosciences, San Jose, CA, USA.

In case of suspicion of AL, an AL orientation tube (ALOT), which included [cytoplasmic (c) myeloperoxidase (MPO), cCD79a, CD19, CD7, surface (s)SD3, cCD3, CD34, and CD45] was used according to the European Group for Immunological Characterization of Leukemia (EGIL) (2). Based on the results obtained from ALOT, the sample was further stained with a subsequent set of antibodies to interpret the results more accurately (Table 1).

A side scatter (SSC) vs. CD45 plot was used first to identify the blast population after doublet discrimination and removing debris based on light scatter characteristics. Then, the CD34 vs. SSC plot was used to gate on CD34-positive blasts. However, in the case of CD34-negative blasts, the blast region recognized by the SSC vs. CD45 plot was evaluated using other CD markers (18). Subsequent plots are needed to evaluate the cells of interest for further markers’ expression. Events with a dim-moderate expression of CD45 and low SSC were regarded as blasts, usually (lymphoblasts) of ALL; therefore, a panel of MAb was used to define the lineage of blasts. When CD19 and cCD79a were bright (positive), we proceeded with the B-ALL evaluation, and when cCD3 along with CD7 or sCD3 were positive, we considered the T-ALL assessment. Markers of immaturity, including CD34, human leukocyte antigen (HLA)-DR, and terminal deoxynucleotidyl transferase (TdT), were also used.

When cMPO was reported positive, AML was expected, and further types of MAb were utilized.

For B-ALL diagnosis and subtyping, along with CD19 and cCD79a, the panel included CD10, cCD22, and CD20, plus cytoplasmic immunoglobulin M (cIgM) and sIgM. Notably, the light chains of Ig Kappa and Lambda were used to determine clonality for diagnosing mature B-cell leukemia (Table 1). Lymphoblasts from de novo B-ALL were positive for CD19, cCD22, and cCD79a and negative for sIgM. Bright CD19 expression corresponds to B-ALL, provided that more than one of the following markers (cCD79a, CD10, or cCD22) were also strongly positive.

To diagnose cases with expected T-ALL, cCD3, and CD7 should be positive, along with negative or weak cMPO. cCD3 must be strong, or if reported as weak, CD2 and/or CD5 must also be positive. Early T-cell precursor (ETP)-ALL diagnosis required the utilization of CD1a, CD2, cCD3, sCD3, CD5, CD7, CD8, and the use of myeloid markers such as CD13, CD33, CD117, CD11b, and stem cell markers including CD34 and HLA-DR (20).

When cMPO was positive, CD13, CD33, CD64, CD117, CD16, CD11b, and CD14 were utilized for myeloid lineage analysis and other MAb, as needed. To diagnose AML, the positivity of two or more of (cMPO, CD13, CD33, CD64, CD117) was a must, provided that ALL criteria were not met.

Cases that showed intermediate-high SSC with moderate CD45 expression and negativity of both HLA-DR and CD34 with positive cMPO were reported as AML-M3 or acute promyelocytic leukemia (APL). Cases with intermediate SSC and bright expression of CD45, with positivity to two CD markers among the monocytic markers (including CD11b, CD14, CD36, CD64, or CD4 heterogeneous expression), were reported as blast corresponding to monocytic lineage (promonocytes and monoblasts). When CD45 was negative with low SSC, these cases were further evaluated using more markers, such as (CD36 and CD71 for proerythroblasts) or (CD41a for megakaryoblasts).

The anchor marker, CD45, was used with all tubes to identify important cell populations on a single plot. CD34, CD38, CD58, CD56, CD61, CD66c, CD105, HLA-DR, TdT, IREM2, kappa, and lambda markers were evaluated for further details accordingly to interpret the blast populations. Isotype antibodies served as a negative control in separate tubes.

The positivity of a sCD marker was approved when more than 20% of the gated population was positive. A cCD marker was reported as positive when more than 10% of the gated population was positive (21).

Two hematopathologists double-checked the results before issuing a report.

Ethical statement

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The scientific and ethics committees at the Directorate of Health in Mosul and the Ministry of Health in Iraq approved the project (IRB Nos. #5909 and #49562), respectively. Informed consent was obtained from the patients’ parents or legal guardians.

Statistical analysis

Statistical analyses were performed whenever needed using SPSS program v.28 (SPSS, IBM Corporation, Armonk, NY, USA). The Chi-squared test was used to assess the significance of differences between two independent groups, and the Mann-Whitney U-test was used in case the data were not normally distributed. Statistical significance was defined as a P value of <0.05. Kaplan-Meier was used for survival analysis; missing cases were considered events when evaluating the high-risk (HR) group.

Results

The EGIL was adopted for immunophenotyping to evaluate and diagnose the enrolled cases (Table 2).

Using the FCM machine, 192 cases were analyzed, including 113 pediatric and 79 adult cases. Among the 113 children, there were 106 cases of AL, including 84 ALL and 22 AML patients.

As we aimed to enroll only those with new diagnoses of ALL and AML, the follow-up samples (eight ALL and two AML cases) were excluded. Thus, the remaining diagnostic samples of 96 cases of AL (76 ALL and 20 AML cases) were evaluated here.

The 76 pediatric cases of ALL

Immunophenotyping using FCM disclosed (60/76, 78.9%) pediatric B-ALL and (16/76, 21.1%) T-ALL cases (Table 3). Most B-ALL were positive for CD10, HLA-DR, TdT, and CD38. Regarding CD34, it was commonly expressed; however, its expression was variable and usually dim or partial. At the same time, CD45 was usually dim or negative. CD20 was either negative or partially positive (Figure 1).

Figure 1 Immunophenotyping of ALL cases. (A-D) B-ALL (blast population in red) showing low SSC, negative to dim CD45, positive expression of CD19, CD34, CD10, CD38, CD58, and CD66c. (E-H) T-ALL (blast cells in red) with low SSC, intermediate to bright CD45, positive expression of CD5, cCD3, TdT (intermediate), CD99, CD1a, and weak expression of sCD3. ALL, acute lymphoblastic leukemia; APC, allophycocyanin; B-ALL, B-lineage ALL; c, cytoplasmic; CD, cluster differentiation; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PE-Cy7, PE and cyanine dye 7; PerCP, peridinin chlorophyll; s, surface; SSC, side scatter; T-ALL, T-lineage ALL; TdT, terminal deoxynucleotidyl transferase.

B-ALL subtyping was based on the stage of development, according to markers’ expression. Hence, B-ALL cases with negative CD10 were referred to as pro-B ALL (early stage) or EGIL B-I, and those with CD10 expression were classified as common-ALL or EGIL B-II, corresponding to the intermediate stage of development. Meanwhile, the B-ALL cases expressing cIgM were regarded as pre-B-ALL (late stage) consistent with EGIL B-III, and the expression of sIgM disclosed mature B-ALL or EGIL B-IV subtype. Our data revealed the presence of common-B, pro-B, pre-B, and mature-B, with frequencies of 46 (76.7%), 7 (11.7%), 6 (10.0%), and 1 (1.6%), respectively. Myeloid antigens (usually CD13 and CD33) can be aberrantly detected on the leukemic blasts from B-ALL. We had 20 cases of B-ALL with such findings.

T-ALL lymphoblasts were positive in most cases for CD5 and CD2 and variably expressed other pan T-cell markers (sCD3, CD4, and CD8) as well as immature markers such as CD1a and CD34. T-ALL subtyping included one case of ETP in our cohort, as the patient’s sample had not expressed CD1a, CD5, and CD8, whereas CD7, cCD3, CD2, CD117, CD34, and HLA-DR were all expressed. Other T-ALL subtypes included pro-T (T-I) with cCD3 and CD7 positivity and pre-T (T-II) with positivity for CD2, CD5, and CD8. The cortical type (T-III) has positive CD1a, while the medullary or mature type (T-IV) shows negative CD1a and strongly positive sCD3. CD79a, or myeloid markers such as CD13 or CD117, were sometimes expressed in our patients with T-ALL. The identified T-ALL subtypes among our patients included five cortical, four medullary, three pro-T, three pre-T-ALL, and one ETP-ALL cases.

For the 76 ALL series of cases, the median age was 6 (range, 0.2–15) years old, and the median WBC count was 19.5 (range, 1.3–925) ×10⁹/L. The male-to-female ratio (M/F) was 1.5, and 86.8% of the samples were of BM origin. One patient had Down syndrome. The HR criteria included (24/76, 31.6%) cases with WBC counts of ≥50×10⁹/L, in addition to (29/76, 38.2%) cases with ages of ≥10 years or ≤12 months (Table 4). The median WBC counts according to lineages in B-ALL and T-ALL were 16.5 (range, 1.3–333) ×10⁹/L and 184.5 (range, 7.3–925) ×10⁹/L, respectively. B-ALL had a median age of 5.5 (range, 0.2–15) years, and the M/F ratio was 1. On the other hand, T-ALL had a median age of 8 (range, 1–14) years, and the majority were males with an M/F of 15:1. About 30% of B-ALL and 36% of T-ALL cases were 10 years or older, and there was a substantial association between the high WBC and the male sex, with T-ALL subtype.

The risk stratification and treatment strategy for our ALL cohort were based on available clinical criteria, including initial WBC count, age, central nervous system involvement, BM morphological evaluation during and after the induction phase, and immunophenotypic diagnosis, using the UKALL-11 protocol (22). Patients with ALL were assigned to a standard-risk (SR) (38.2%) eligible for regimen-A treatment and the remaining (61.8%) to a HR group eligible for regimen-B treatment. Two cases that showed 5–25% BM blasts on the 29^th day of induction were escalated to receive regimen-C. Seven cases (9.2%) abandoned therapy at different stages of treatment (Figure 2). With a median follow-up of 26.2 (range, 0–44.3) months, the overall survival (OS) and the event-free survival (EFS) were 73.7% (95% CI: 66–86.2%) and 67.1% (95% CI: 57.4–82.8%), respectively (Figure 3). Unfortunately, without cytogenetic data, further refinement of the favorable risk or poor prognostic criteria could not be addressed.

Figure 2 Flow diagram showing the ALL cases with respect to risk stratification and a summary of the outcome. ALL, acute lymphoblastic leukemia; COVID-19, coronavirus disease-2019; HSCT, hematopoietic stem cell transplantation.

Figure 3 Kaplan-Meier survival curves for the SR and HR groups of the enrolled children with ALL from Mosul City. With a median follow-up of 26.2 (range, 0–44.3) months, the OS was 73.7% (95% CI: 66–86.2%), and the EFS was 67.1% (95% CI: 57.4–82.8%). ALL, acute lymphoblastic leukemia; CI, confidence interval; EFS, event-free survival; HR, high-risk; OS, overall survival; SR, standard-risk.

The 20 pediatric cases of AML

Regarding the pediatric AML cases, there were 20 de novo cases, including AML-M0, M1, M2, M3, M4, M5, M6, and M7, with a frequency of (1, 1, 3, 3, 5, 4, 2, and 1), respectively (Figure 4). Two APL cases were of the variant subtype, as it was reported that the co-expression of CD34 and CD2 was indicative of the APL variant (23).

Figure 4 Immunophenotyping of AML cases. (A-D) A case of AML-M0 (blast cells in purple), showing low SSC and dim CD45, with MPO negativity. The blast cells were positive for CD34, CD117, HLA-DR, CD13, and CD33. (E-H) A case of AML-M5 (blast cells in purple); in the CD45 vs. SSC plot, the monocytic population, depending on maturation, shows brighter CD45 and falls closer to the monocyte region. CD34 is prominently negative, and MPO is heterogeneous, indicating the variety of maturation for the cells. HLA-DR and CD64 are positively expressed. CD13 and CD11b are positive. AML, acute myeloid leukemia; APC, allophycocyanin; CD, cluster differentiation; FITC, fluorescein isothiocyanate; HLA, human leukocyte antigen; MPO, myeloperoxidase; PE, phycoerythrin; PE-Cy7, PE and cyanine dye 7; PerCP, peridinin chlorophyll; SSC, side scatter.

The median age for AML cases was 5.5 (range, 2–16) years, and the median WBC count was 38 (range, 3–220) ×10⁹/L. The M/F was 1.9.

Discussion

To our knowledge, this article is the first to disclose and stratify a childhood AL cohort from Mosul regarding B- or T-lineage immunophenotyping and their outcome, owing to the routine use of FCM analyses for all AL cases in the city for the first time. Despite the COVID-19 pandemic, JCF was able to provide funding through the Japanese government, and the project was closely followed up with collaboration between specialists from both Japan and Mosul.

The immunophenotyping results revealed that approximately 80% of childhood AL cases were ALL, while the remaining cases were AML. B-ALL and T-ALL represented 78.9% and 21.1% of ALL cases, respectively. Although still comparable, T-ALL in our cohort from Mosul is higher than our previous report from Iraq and studies from Pakistan, Japan, the UK, and the USA, with frequencies of 10–15% (4,18,24-27). Taken together, the HR group of about 62% in our study was significantly higher than a cohort from Iraq by Al-Hadad et al. of 34% (P<0.001) as well as the total enrolled ALL cohorts in the same study of 49% (P=0.032), and a report from the UK of 42% (P<0.001) (22,24). Meanwhile, a similar increase in HR cases of 67.6% was reported in an article from Vietnam (28). It is evident that the HR group constitutes a significant fraction of our study, even without the presence of biomarkers, which would further refine the risk criteria, as is the case with reports from the UK or Vietnam. This means that the HR cases are expected to increase when such details are disclosed.

Generally, 90% of the 76 newly diagnosed ALL cases were from Nineveh province, with 31 from Mosul City, 37 from the surrounding districts, and eight from other provinces. In addition, (11/16, 68.75%) of T-ALL cases were from the surrounding districts of Mosul City, and (2/16, 12.5%) were from other provinces. All the AML cases were from the Nineveh province. The median duration of illness prior to diagnosis was as short as 2 weeks for T-ALL, while it reached up to 1 month in the case of B-ALL.

Among 71 cases with available induction course results, 66 (93%) achieved complete remission (CR) in terms of morphological BM and extramedullary site evaluation but not using the minimal residual disease (MRD) technique. Meanwhile, 2 failed to achieve CR (2.8%), and 3 (4.2%) died either from infection in two cases or bleeding in one case, in agreement with the study from Baghdad, Iraq (139/1,415, 9.8%) (P=0.35) and the report from Vietnam (18/238, 7.6%) (P=0.27) (22,28). After excluding the seven patients who abandoned therapy or transferred to another center, 8 out of 69 (11.6%) cases had relapsed; 7 of them were from the HR group, and 4 of the latter relapsed within 18 months after therapy in BM. One of the relapsed patients underwent a hematopoietic stem cell transplantation (HSCT) and is currently in continuous CR. Whilst the relapse rate in our series was comparable to the result from Vietnam of (20/238, 8.4%) (P=0.57), it was significantly lower than the total of three cohorts from Baghdad of (278/1,218, 23%) (P=0.03); however, the number of cases and the duration of follow-up are less in the current study (22,28). Excluding induction mortality, death was reported in (17/69, 24.6%); 13 of them were from the HR category, including two who died from refractory disease, and five of the eight patients with relapse died as well. Furthermore, (9/62, 14.5%) who achieved CR died, and 4 of them proved to have severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) responsible for COVID-19. Such a death rate in CR was comparable to the study from Baghdad, which showed 12% (P=0.22) (22). Generally, the death rate was (20/76, 26.3%) in ALL, and septic death was the leading cause of mortality in half of the deceased patients, primarily in those younger than 2 years old, and 4 of them died during the maintenance course. Similarly, Kiem Hao et al. reported a mortality of (74/238, 31%) (P=0.43) in pediatric ALL cases in Vietnam, among them (32/74, 43%) (P=0.59) caused by infection (28). Although infection is a major cause of treatment-related mortality in childhood ALL, the frequency in our cases was significantly higher than the report from the UK of (8%), (P<0.001) (27). The abandonment rate (9.5%) in our series of cases was also comparable to Al-Hadad et al. 13% (P=0.29) but significantly higher than the rate reported by Kiem Hao et al. (7/238, 2.9%) (P=0.02).

Unfortunately, routine local cytogenetic studies are not yet available in most hemato-oncology centers in Iraq to define biomarkers and classify AL cases in accordance with the WHO Classification and its 2022 revision, which primarily relies on genetic findings and refinements in the definitions of entities (29). In fact, WHO classification that incorporated genetic findings into diagnostic algorithms of AL was adopted in the advanced world since 2001 and upgraded with each revision, while, in Iraq, until only a few years ago, Iraqi hematologists had to depend solely on morphology and the old French-American-British (FAB)-classification of AL due to the shortage of advanced medical equipment and dilapidated hospitals infrastructures due to wars and their aftermath.

Based on the remarkable advancements in molecular and genetic analysis in developed countries, the 5-year survival rate for pediatric ALL exceeds 90%. Meanwhile, there is scarce information about therapy results in low-middle-income countries. In Iraq, pediatric oncologists struggle to reach 70%, according to studies from the Children’s Welfare Teaching Hospital, a tertiary hospital with the main referral pediatric oncology center in Baghdad (4,22,24,30,31). Likewise, in the current cohort, the OS was 73.7%, and the EFS was 67.1% for cases of ALL. However, when the SR group of cases was evaluated separately, the OS was 86.2%, and the EFS was 82.8%. Of note, abandonment was reported among the HR group, and when considering abandonment as an event, the OS and EFS for the HR group were 38.5% and 29%, respectively. Although such an outcome was dismal, it was expected given the inadequacy of supportive care, such as the lack of an intensive care unit, insufficient infection control measures, inadequate antifungal treatment, and lack of salvage options such as allogenic HSCT and so on. Besides, there is ignorance among many of the patients’ families, leading to critical repercussions, for instance, delaying bringing the patient in case of fever or discontinuing treatment when the patient seems improved, expecting that their child has been cured. Additionally, this study faced another challenge related to the COVID-19 pandemic, which exacerbated the situation and contributed to increased mortality.

It is worth noting that pediatric cancer accounts for a higher percentage of cancer cases in developing countries, including Iraq, compared with higher-income countries due to the more considerable proportion of children in poorer nations, besides the higher frequency of cancer among the general population in such limited-resource countries. Since developing countries represent 78% of the global population and 86% of the world’s children, action is required. Hence, a sustainable approach to building local capabilities and capacity for cancer care is warranted (7,26,32-34). Therefore, Iraq also needs to obtain support from other developed countries, especially in terms of humanitarian and medical aspects, whether from governmental or non-governmental sources.

Before establishing any medical project, it is essential to know what is currently available on the ground, and collaborative efforts are needed to address the requirements and financial needs (7,32). Based on this concept, staff from JCF made several visits to Iraq between 2018 and 2019, meeting with the people in charge before establishing the FCM unit in Mosul. In addition, a workshop concerned with FCM results was arranged by the Japan-Iraq Medical Network (JIM-NET) and JCF in Erbil, Iraq, in March 2019, and several Iraqi hematopathologists and pediatric oncologists were invited, including those from Mosul and their colleagues from Erbil, Sulaymaniyah, Baghdad, and other provinces in Iraq (16). That meeting provided a valuable opportunity for specialists to discuss the benefits and challenges they face regarding FCM analysis in their centers. Thus, we began implementing our project plan based on the recommendations from that workshop and establishing a relationship among Iraqi doctors themselves in the field of hematopathology, particularly among the experts in FCM analysis, for further cooperation in the future and to improve their skills.

Remarkably, the Directorate of Health in Mosul has become responsible for meeting the FCM unit’s needs from its local budgets, and the unit has been integrated into a government hospital laboratory located near the newly established Al-Hadbaa Hospital for Hematology and Oncology. Thus, samples are transferred directly to the unit, and initial evaluation based on history and morphology before panel selection is much easier, and access to hematologists is possible (35). Accordingly, hemato-oncologists in Mosul can now receive the FCM results of their referred cases rapidly, and the test is free of charge, compared to before when they used to request FCM immunophenotyping to be done elsewhere outside Mosul. Some patients or their guardians agree to perform the test in other provinces in Iraq; however, many cannot afford the high expenses (around 800 USD) for a single test, plus the transportation costs.

Interestingly, the main point of strength in this collaboration was the presence of an Iraqi pediatric oncologist who had fled Mosul due to the ISIL invasion and settled in Japan. Al-Kzayer, who had worked as the first dedicated pediatric oncologist in Mosul since 2001, had established the first pediatric oncology center there. From Japan, she was able to build bridges with her colleagues in various parts of Iraq and contributed to several collaborative studies (11,16,24,36). Similarly, she coordinated humanitarian projects in collaboration with JCF, including the FCM in Mosul City. Such fruitful projects were made possible through overseas collaboration between colleagues who originated from the same hometown.

JCF also supported genetic studies conducted at Shinshu University School of Medicine in Nagano, Japan, to evaluate cases of AL in children from Iraq (11,16). Similarly, Nagoya University Graduate School of Medicine and Nagoya City University Graduate School of Medical Sciences in Japan also contributed by performing molecular analysis of Iraqi childhood AL using next-generation sequencing (NGS). Through such collaborative studies, Al-Kzayer et al. approved the compatibility of the genetic results obtained by NGS regarding B- or T-ALL subtyping with the immunophenotyping results of AL by FCM analyses performed in Iraq (24). Likewise, for the current study cohort, we were also planning to bring the samples for genetic study in Japan, but the pandemic hindered the implementation of our plan.

Currently, utilizing additional markers, such as the NG2 antigen in the ALL panel to facilitate the prompt identification of NG2-positive B-ALL cases harboring KMT2A rearrangements, and using CD300e and CD37 in the AML panel to evaluate the maturation profile of monocytes, are steps toward further improvement (37,38). Similarly, the use of CD58 and CD99 is being considered to aid in the detection of MRD for B- and T-ALL, respectively (39). Meanwhile, hematopathologists at the FCM unit in Mosul are currently undergoing a training course to detect MRD and enhance their work.

Undoubtedly, internationally recognized centers in pediatric oncology are required to support centers in developing countries, either through NPOs or directly, to share knowledge and experiences. Additionally, cooperation in the academic field through conducting research studies together also benefits all parties (7,22,31,40,41). Fortunately, there are good examples of collaboration with Iraq carried out by professional pediatric oncologists from Sapienza University in Italy and St. Jude Children’s Research Hospital in the USA, with the pediatric oncology team working at the Children’s Welfare Teaching Hospital in Baghdad, Iraq. The results have been impressive in improving the diagnosis, management, and care of patients with cancer over the years, ultimately improving their outcomes (31,42,43).

More attention is required to support the health system in Iraq, particularly in terms of infrastructure and human resources. Furthermore, additional steps are crucial for improving the diagnosis and management of hematological malignancies, including the use of cytogenetics and molecular biology methods (9,40).

The main limitation of the current study was the unavailability of cytogenetic studies for the cases due to the shortage of facilities to perform them in Mosul and most of the oncology centers in Iraq.

Finally, we believe that this report will serve as a reference base for future studies from Mosul, as it discloses for the first time the results and outcomes of a cohort of ALL cases from Mosul City; thus, comparison will be possible with further improvement steps.

Conclusions

JCF played a crucial role in providing Mosul City and its surrounding areas with the first FCM unit, which was made possible through a fund from the Japanese government. The project made a breakthrough in AL diagnosis and established one of the essential and supposed routine steps represented by ALL immunophenotyping. Moreover, the Directorate of Health in Mosul has become responsible for meeting the FCM unit’s needs from their local budgets. The HR group represented a significant portion of ALL cases. Although the outcome was satisfactory for the SR group, the survival rate for the HR group was dismal. Obviously, suboptimal facilities related to the shortage of diagnostic and therapeutic resources have impacted the outcome of childhood ALL treatment in Mosul as well as in other places in Iraq. Indeed, further efforts are needed to scale up diagnostic and therapeutic capabilities to improve the outcome of ALL in Mosul City.

Acknowledgments

The authors are grateful to Mrs. Sadako Kamiya, the director of the Japan Chernobyl Foundation (JCF), for her outstanding support throughout this project. We also would like to thank Mr. Takenori Kato for his efforts. We are indebted to Dr. Ahmed Talib Ibrahem, the director of Ibn Al-Atheer Hospital for Children in Mosul, Iraq, for his cooperation. We also appreciate the collaboration of colleagues from Mosul, Dr. Alaa Adrees Al-Dhannoon, and Dr. Shaima Mohammad. We express our thanks to the biologist, Ms. Sarab Yahya Yousif, and the medical laboratory technician, Mr. Ali Omer Ali, from the Flow Cytometry Unit, Hematopathology Department, Ibn Al-Atheer Hospital, for their efforts and collaboration in managing the data. Finally, Iraqi doctors appreciate the significant role of JCF in establishing the first flow cytometry unit in Mosul City.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-24/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-24/dss

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Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-24/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work and ensure that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The scientific and ethics committees at the Directorate of Health in Mosul and the Ministry of Health in Iraq approved the project (IRB Nos. #5909 and #49562), respectively. Informed consent was obtained from the patients’ parents or legal guardians.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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