|Year : 2017 | Volume
| Issue : 3 | Page : 81-87
Cluster of differentiation 97 as a biomarker for the detection of minimal residual disease in common acute lymphoblastic leukemia
Laila M Sherif1, Mervat M Azab2, Ghada M Al-Akad2, Marwa Zakaria1, Maha Atfy2, Sara M Sorour2
1 Department of Pediatric, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
|Date of Submission||25-Mar-2017|
|Date of Acceptance||22-Apr-2017|
|Date of Web Publication||9-Nov-2017|
Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, 44519
Source of Support: None, Conflict of Interest: None
Background Acute lymphoblastic leukemia (ALL) is a biologically heterogeneous disorder. Clinical parameters, immunophenotype, cytogenetic factors, and minimal residual disease (MRD) are among currently used factors in risk stratification and therapy determination of ALL patients. MRD is gaining importance nowadays for therapy efficacy, follow-up, and relapse risk estimation. Recent studies have highlighted potential markers that may improve the sensitivity of MRD detection by flow cytometry. Cluster of differentiation (CD) 97 is one of the markers that show overexpression in pediatric ALL. In this study, we aimed to assess the value of CD97 as a biomarker for MRD detection in pediatric ALL.
Patients and methods This cohort study was conducted on 30 newly diagnosed patients with B-ALL. There were 16 male and 14 female patients with a mean age of 8.38±4.21. Twenty patients were in the low-risk group and 10 patients were in the high-risk group and were treated according to modified CCG 1991. A panel of monoclonal antibodies was used, with special emphasis on CD10, CD19, CD34, and CD97 at diagnosis and at day 14 postinduction of chemotherapy for MRD detection.
Results The percentage of CD19/CD97, CD34/CD97, and CD10/CD97 at day 0 was 57.15±21.74, 57.73±21.20, and 57.87±20.77, whereas at day 14 it was 6.09±2.50, 10.67±8.89, and 5.97±2.44, respectively (P<0.001). CD97 was expressed in 81.5% of patients at diagnosis and was not detected at day 14 (P<0.001). One patient had blast counts more than 5% by light microscopy, whereas 29 patients had MRD more than 0.1 by flow cytometry at day 14 (P<0.001).
Conclusion CD97 can be used for MRD tracing in pediatric ALL.
Keywords: acute lymphoblastic leukemia, cluster of differentiation 97, minimal residual disease, new markers
|How to cite this article:|
Sherif LM, Azab MM, Al-Akad GM, Zakaria M, Atfy M, Sorour SM. Cluster of differentiation 97 as a biomarker for the detection of minimal residual disease in common acute lymphoblastic leukemia. Egypt J Haematol 2017;42:81-7
|How to cite this URL:|
Sherif LM, Azab MM, Al-Akad GM, Zakaria M, Atfy M, Sorour SM. Cluster of differentiation 97 as a biomarker for the detection of minimal residual disease in common acute lymphoblastic leukemia. Egypt J Haematol [serial online] 2017 [cited 2018 Aug 20];42:81-7. Available from: http://www.ehj.eg.net/text.asp?2017/42/3/81/217877
| Introduction|| |
Acute lymphoblastic leukemia (ALL) is a biologically heterogeneous disorder. Clinical parameters, immunophenotyping, cytogenetic factors, treatment response, and minimal residual disease (MRD) are among the currently used factors in risk stratification and therapy determination of ALL patients . The outcome for childhood ALL is being improved during the past 50 years, with current full-remission rates reaching to 90%, which is attributed to the introduction and gradual intensification of combination chemotherapies along with improvement in prognostic factors . On the other hand, a proportion of children are likely to be either overtreated or undertreated with current therapies. Thus, there is a need for increased cure rates with decreased toxicity by the help of individually tailored therapies, for which monitoring MRD gains vital importance . There are two basic methods for the detection of MRD in childhood ALL − molecular analysis of B and T-cell receptor gene rearrangements and flow cytometry (FCM) analysis of aberrant immune phenotypes − both of which provide similar results at MRD level of 0.01, but this typically requires considerable interpretative expertise . The aim is to identify new markers for ALL-MRD detection − for example, cluster of differentiation (CD) 73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, pre-B-cell leukemia transcription factor 1 (PBX1), cadherin-associated protein 1-α (CTNNα1), integrin β-7 (ITGβ7), CD69, and CD49f, which are differentially expressed in a large proportion of ALL cases . These new markers were claimed to have the capacity to detect one leukemic cell among 105 bone marrow (BM) cells and to increase the sensitivity of MRD detection by flow cytometry . CD97 is one of these markers that show overexpression in pediatric ALL .
| Patients and methods|| |
This cohort study was conducted on 30 patients newly diagnosed with B-lineage ALL (16 male and 14 female) with a mean age of 8.38±4.21, ranging from more than 1 to less than 18 years, who were registered in Pediatric Oncology Unit of Zagazig University Hospital. This study was conducted in accordance with the ethical standards of the Helsinki Declaration of 1964 as revised in 2008 and was approved by our local ethics committee. Informed consent was obtained from all individuals participating in the study or their guardians.
Data abstraction form was designed to capture the appropriate information including age, sex, full clinical examination with especial emphasis on pallor, bleeding tendency, fever, and evidence of organ and central nervous system infiltration. Laboratory tests including complete blood count, liver and kidney function, BM study, and cerebrospinal fluid examination immunophenotyping of BM samples were performed on an FACScan flow cytometer (Becton Dickinson BD, San Diego, California, USA) using acute leukemia panel CD3, CD5, CD7, CD10, CD13, CD14, CD19, CD20, CD22, CD33, CD34, CD64, CD79a, TDT, HLA-DR, and MPO. Cells were considered to be positive for malignancy when more than 20% of cells express these markers, except for TDT and CD34; the cutoff value is 10%.
In addition, a panel of four monoclonal antibodies, CD10FITC, CD19PE, CD34PercP, and CD97APC, were defined at diagnosis and at day 14 postinduction of chemotherapy for tracing of MRD.
One milliliter of PB was aseptically collected on K-ethylene diamine tetra acetic acid for CBC and preparation of Leishman-stained PB smears. One milliliter of BM sample on EDTA was used for immunphenotyping and flow cytometric detection of B-lymphoid markers (CD10, CD19, and CD34) in addition to CD97 as a fourth color. Films were prepared directly from the syringe for Leishman and cytochemical staining. Five hundred microliters of BM sample was used for the detection of MRD at day 14 postinduction using MoAbs (CD10, CD19, CD34, and CD97) defined at diagnosis. Sera were collected for routine liver, kidney, and LDH estimation.
Methodology of minimal residual disease detection
Patients were evaluated at diagnosis; monoclonal antibodies combinations were used to define leukemia-associated immunophenotype (LAIP), which allow the discrimination of leukemic blasts from normal lymphocyte progenitors and rely on qualitative or quantitative differences in antigen expression between leukemic cells and their normal counterparts. This step served to define a leukemia phenotypic fingerprint to be used in follow-up samples. The LAIP present in an individual case has been identified by using multiflorescence colors with a comprehensive panel of combinations of monoclonal antibodies. Therefore, MRD during the course of treatment and follow-up can be assessed by the quantification of the frequencies of these cells by multifluorescence colors.
Analysis of MRD
The rationale for MRD detection is to use sequential gating strategy. ALL cases first tight lympho-population gate applied on SSC versus FSC then CD45 versus SSC and CD19 coexpressing CD34 population, then CD19 coexpressing CD10 then subsequently gating on CD34 and CD97, CD19 and CD97, CD10 and CD97. Leukemic events were defined by dot-plots in a region with estimated number of events from statistics.
All data were collected, tabulated, and statistically analyzed using SPSS 18.0 for windows (SPSS Inc., Chicago, Illinois, USA). Quantitative data were expressed as the mean±SD and median (range), and qualitative data were expressed as absolute frequencies (number) and relative frequencies (percentage). Continuous data were checked for normality by using Shapiro–Wilk test. Independent sample Student’s t-test was used to compare two groups of normally distributed data, whereas Mann–Whitney U-test was used for non-normally distributed data. Paired samples t-test was used to compare two dependent groups of normally distributed data, whereas Wilcoxon signed ranks test was used for non-normally distributed data. Kruskal–Wallis test was used to compare more than two groups of non-normally distributed data. Percentages of categorical variables were compared using χ2-test. Paired categorical data were compared using the McNamara’s test. Spearman’s rank correlation analysis was performed between selected study parameters. All tests were two sided. P value of less than 0.05 was considered statistically significant (S), P value of less than 0.01 was considered highly statistically significant (HS), and P value of at least 0.05 was considered nonstatistically significant (NS).
| Results|| |
Thirty patients diagnosed with ALL were included in the study; there were 16 male and 14 female patients, with an age range of 1–17 years and a mean age of 8.38±4.21 years. Twenty (66.7%) patients were classified as the favorable age group (>1 year to <10 years plus LI morphology) and 10 (33.3%) patients were classified as the unfavorable age group (≥10 years to <1 year plus L2 morphology) based on French, American, British classification (FAB) morphological classifications ([Table 1] and [Table 2]). Regarding clinical presentation of patients, 83.3% presented with fever, 40% presented with pallor, 43.3% presented with purpura, 53.3% presented with lymphadenopathy, 33.3% presented with hepatomegaly, and 66.6%presented with splenomegaly([Table 3]). Unfortunately, three patients died before day 14 of chemotherapy. Mean total leukocyte count, hemoglobin (Hb) level, and platelet counts at day 0 were17.98±16.01, 8.68±1.53, and 52.62±24.04, respectively, and at day 14 they were 2.64±1.38, 10.44±1.27, and 106.25±50.21, respectively ([Table 4]). Mean BM blast by light microscopy was 73.29±15.90 and 1.00±1.64 at days 0 and 14, respectively, and mean BM blast by flow cytometry was 72.11±11.90 and 5.68±7.90 at days 0 and 14, respectively ([Table 5]). CD97 was expressed in 22 (81.5%) patients at day 0 and was absent in all patients at day 14 ([Table 6]).
|Table 1 Demographic data of the studied acute lymphoblastic leukemia patients at diagnosis|
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|Table 2 FAB morphological classification of the studied acute lymphoblastic leukemia patients|
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|Table 3 Clinical data of the studied acute lymphoblastic leukemia patients at diagnosis|
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|Table 4 Comparison between laboratory findings at days 0 and at day 14 after therapy of the studied acute lymphoblastic leukemia patients|
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|Table 5 Comparison between blast count at day 0 and at day 14 after therapy of the studied acute lymphoblastic leukemia patients|
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|Table 6 Comparison between cluster of differentiation 97 at day 0 and at day 14 after therapy of the studied acute lymphoblastic leukemia patients|
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Our results showed that mean multiparameter flow cytometry of CD19/CD97 was 57.15±21.74 and 6.09±2.50 at days 0 and 14, respectively; also, mean florescence intensity (MFI) of CD19/CD97 was 91.41±52.67 and 53.13±30.20 at days 0 and 14, respectively. Mean multiparameter flow cytometry of CD34/CD97 was 57.73±21.20 and 10.67±8.89 at days 0 and 14, respectively. In addition, MFI of CD34/CD97 was 91.95±52.8 and 54.07±31.14 at days 0 and 14, respectively. Mean multiparameter flow cytometry of CD10/CD97 was 57.87±20.77and 5.97±2.44 at days 0 and 14, respectively. In addition, MFI of CD10/CD97 was 88.31±50.98 and 53.13±30.20 at days 0 and 14, respectively ([Table 7]). There were 16 (53%) patients with BM blast less than 1% by light microscopy, but the flow cytometry blast was 2.95±6.76, with a range of 0.03–28, and 11 (47%) cases with BM blast 1–5 by light microscopy, whereas by flow cytometry BM blast was 9.66±8.02, with a range of 0.43–23 ([Table 8]).
|Table 7 Comparison between immunophenotyping at day 0 and at day 14 after therapy of the studied acute lymphoblastic leukemia patients|
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|Table 8 Relationship between blasts in bone marrow and blasts count by FCM at day 14 after therapy in studied acute lymphoblastic leukemia patients|
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Our results revealed that 81.48% of patients had MRD level of at least 0.1, 18.51% had MRD level of at least 0.01 to less than 0.1, and 0% had an MRD level less than 0.01 at day 14 postinduction ([Table 9]).
|Table 9 Relationship between IPT/FCM and minimal residual disease at day 14 after therapy in studied acute lymphoblastic leukemia patients|
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| Discussion|| |
MRD is a powerful predictor of the overall response to treatment in childhood ALL .
MRD analysis has been introduced into many treatment protocols for risk assignment and selection of therapeutic regimens . In many ALL protocols, it often begins during induction chemotherapy (day 8, 15, and 19) for the prediction of tumor-relapse-free survival .
MRD detection by multiparameter flow cytometry is based on discrimination of leukemic cells from normal, healthy cells by expressions of aberrant antigens called LAIPs, which were represented by markers normally expressed during lymphohematopoiesis but found in abnormal combinations in leukemic cells .
LAIPs are identified in more than 95% of ALL cases. Thus, multiparameter flow cytometry can be used to monitor 90–95% of MRD in ALL cases during therapeutic management .
In a proportion of patients, leukemic cells express only one set of markers, and have an increased risk of false-negative MRD results because of immunophenotypic shifts. Therefore, the availability of additional markers should allow MRD studies in patients whose ALL cells currently lack suitable leukemia-associated immunophenotype and minimize the risk of false-negative results .
In this study, we evaluate the role of CD97 as a new marker of MRD in childhood C-ALL in combination with CD10, CD19, and CD34.
In the present study, the age of ALL patients ranged between 1 and 17 years, with a mean±SD of 8.38±4.21 years and a median of 8 years; 20 (66.7%) of them were in the favorable age group (> 1 to <10), whereas 10 (33.3%) were in the unfavorable age group (<1 to ≥10). Regarding sex, 16 (53.3%) patients were male and 14 (46.7%) were female.
Our results are in agreement with those of Shalaby et al.  who studied 53 patients of newly diagnosed ALL, with age ranging from 6 months to 17 years; 68.5% were in the favorable age group, between 1 and 10 years, whereas 35% were in the unfavorable age group (<1, ≥10). Regarding sex, 31 (58%) were male and 22 were female (42%) .
Mean total leukocyte count, Hb level, and platelet counts of patients at day 0 were17.98±16.01, 8.68±1.53, and 52.62±24.04, respectively, and at day 14 they were 2.64±1.38, 10.44±1.27, and 106.25±50.21, respectively, with a P value of less than 0.001.
Unfortunately, three (10%) patients died before day 14 postinduction chemotherapy.
On analyzing the relationship of mortality with baseline laboratory findings, no significant association was detected except for total leucocytic count (TLC), which was at least 50 000 as it is considered a prognostic factor and a cutoff point in risk stratification of ALL .
In our study, mean count of BM blasts by light microscopy was 73.29±15.90 with a range of 39–100%, whereas at day 14 postinduction therapy it was 1.00±1.64 with a range of 0–5% and a P value of less than 0.001.
By flow cytometry, the mean±SD of blast in BM at day 0 was 72.11±11.90 with a range of 43–90% and at day 14 postinduction therapy it was 5.68±7.90 with a range of 0.03–28% and a P value of less than 0.001. There were 16 (53%) patients with BM blast less than 1% by light microscopy, but with flow cytometry the BM blast was 2.95±6.76 with a range of 0.03–28 and 11 (47%) cases with BM blast 1–5 by light microscopy, whereas by flow cytometry the mean BM blasts was 9.66±8.02 with a range of 0.43–23 and a P value of 0.008. The difference between blast count at day 14 by morphology and immunophenotyping may be because of miscounting some of the blast cells as hematogones by morphology that proved to be blast cells by immunophenotyping.
In pediatric B-ALL, increased expression of CD10, CD19, and CD58 and decreased expression of CD38 and CD45 are commonly found in association with uniform expression of CD34 and TdT; also, asynchronous expression of CD20 are additional abnormalities that can be found .
In our study, we assessed MRD using CD97 in combination with CD10, CD19, and CD34 and we found that CD97, CD10, CD19, and CD34 were overexpressed in 25 (83.3%) patients at diagnosis, whereas five (16.7%) patients were negative for CD97, which allows us to use this combination for further assessment of MRD at day 14.
In addition, Coustan-Smith et al.  reported in their study that 22 of the 30 markers (CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNα1, ITGβ7, CD69, CD49f) were differentially expressed in about 80% of ALL cases, which can be used to minimize false-negative results and improve sensitivity of immunophenotype MRD studies.
In addition, Djokic et al.  reported that CD86, CD97, and CD123 were overexpressed in hyperdiploid (51–65 chromosome) ALL cases.
Our results revealed that mean multiparameter flow cytometry of CD19/CD97 was 57.15±21.74 and 6.09±2.50 at days 0 and 14, respectively, with a P value of less than 0.001. Mean multiparameter flow cytometry of CD34/CD97 was 57.73±21.20 and 10.67±8.89 at days 0 and 14, respectively, with a P value of less than 0.001. Mean multiparameter flow cytometry of CD10/CD97 was 57.87±20.77 and 5.97±2.44 at days 0 and 14, respectively, with a P value of less than 0.001. Similarly, Victoria et al. found that mean multiparameter flow cytometry of CD34/CD97% in ALL cases was 65.2±32.1% .
According to Coustan-Smith et al. , who stated in their study that markers with MFI more than 10 were overexpressed and markers with MFI less than 10 in ALL are under expressed in ALL patients, and some of these markers such as CD97 and CD99 and CD102 appeared to be overexpressed in a much larger proportion of cases.
Our results revealed that MFI of CD19/CD97 was 91.41±52.67 and 53.13±30.20 at days 0 and 14, respectively, with a P value of less than 0.001, and MFI of CD34/CD97 was 91.95±52.88 and 54.07±31.14 at days 0 and 14, respectively, with a P value of less than 0.001. In addition, MFI of CD10/CD97 was 88.31±50.98 and 53.13±30.20 at days 0 and 14, respectively, with a P value of less than 0.001, which revealed that MFI of CD97 remained high and fluctuating during therapy.
Schrappe et al.  found that MRD less than 0.01% at the end of induction had a favorable outcome, whereas patients with MRD of at least 0.1% at this time point had a high relapse hazard.
Assessment of MRD in our ALL patients at day 14 postinduction showed that 0% of patients had negative MRD (<0.01), 18.51% had positive MRD (≥0.01 to <0.1), and 81.48% of patients had MRD level of at least 0.1.
In agreement with our results, Van der Velden et al.  studied MRD in 99 infants with ALL enrolled in the Interfant-99 protocol and found that all patients classified as ‘high-risk’ because of MRD of at least 0.01% at the end of induction and/or consolidation (26%) relapsed.
| Conclusion|| |
A primary challenge in pediatric ALL is to prospectively identify those children with higher-risk disease − that is to identify MRD. The discovery of new markers of ALL should help widen the applicability of MRD testing and possibly allow reliable MRD studies. CD97 expression is a good marker of leukemic cells and can be used for MRD.
Laila M. Sherif, Mervat M. Azab, Ghada M. Al-Akad, Marwa Zakaria, Maha Atfy and Sara M Sorour contributed to medical practices. Laila M. Sherif, Mervat M. Azab, Ghada M. Al-Akad, Marwa Zakariar contributed to concept, design and analysis or interpretation. Laila M. Sherif, Mervat M. Azab, Ghada M. Al-Akad, Marwa Zakaria, Sara M. Sorour contributed to data collection or processing. Laila M. Sherif, Mervat M. Azab, Ghada M. Al-Akad contributed to literature search. Laila M. Sherif, Marwa Zakaria contributed to writing.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Tekgündüz E, Demir M, Akpinar S. Acute lymphoblastic leukemia; prognostic factors’ perspective. Int J Hematol Oncol
Schrappe M. Minimal residual disease: optimal methods, timing, and clinical relevance for an individual patient. Hematology Am Soc Hematol Educ Program
Irving J, Jesson J, Virgo P, Case M, Minto L, Eyre L et al.
Establishment and validation of a standard protocol for the detection of minimal residual disease in B lineage childhood acute lymphoblastic leukemia by flow cytometry in a multi-center setting. Haematologica
Campana D. Role of minimal residual disease monitoring in adult and pediatric acute lymphoblastic leukemia. Hematol Oncol Clin North Am
Coustan-Smith E, Song G, Clark C, Key L, Liu P, Mehrpooya M et al.
New markers for minimal residual disease detection in acute lymphoblastic leukemia. Blood
Solly F, Angelot F, Garand R, Ferrand C, Seille’s E, Schillinger F et al.
CD304 is preferentially expressed on a subset of B-lineage acute lymphoblastic leukemia and represents a novel marker for minimal residual disease detection by flow cytometry. Cytometry A
Conter V, Bartram CR, Valsecchi MG, Schrauder A, Panzer-Grümayer R, Möricke A et al.
Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of the AIEOPBFM ALL 2000 study. Blood
Pui CH, Campana D, Pei D, Bowman WP, Sandlund JT, Kaste SC et al.
Treating childhood acute lymphoblastic leukemia without cranial irradiation. N Engl J Med
Campana D. Minimal residual disease in acute lymphoblastic leukemia. Hematology Am Soc Hematol Educ Program
Muzzafar T, Medeiros LJ, Wang SA, Brahmandam A, Thomas DA, Jorgensen JL. Aberrant underexpression of CD81 in precursor B-cell acute lymphoblastic leukemia: utility in detection of minimal residual disease by flow cytometry. Am J Clin Pathol
Gaipa G, Basso G, Biondi A, Campana D. Detection of minimalresidual disease in pediatric acute lymphoblastic leukemia. Cytometry B Clin Cytom
Shalaby R, Ashaat N, El waha N, El-Hamid MA, El-Wakeel SH. BCL-2 expression and chromosomal abnormalities in childhood acute lymphoblastic leukemia. Acad J Cancer Res
Pui CH, Pei D, Sandlund JT, Ribeiro RC, Rubnitz JE, Raimondi SC et al.
Long-term results of St. Jude total therapy studies 11, 12, 13A, 13B, and 14 for childhood acute lymphoblastic leukemia. Leukemia
:371–382. doi: 10.1038/leu.2009.252.
Patkar N, Alex AA, Bargavi B, Ahmed R, Abraham A, George B et al.
Standardizing minimal residual disease by flow cytometry for precursor B lineage acute lymphoblastic leukemia in a developing country. Cytometry Part B
Djokic M, Bjorklund E, Blennow E, Mazur J, Soderhall S, Porwit A. Overexpression of CD123 correlates with the hyperdiploid genotype in acute lymphoblastic leukemia. Haematologica
Schrappe M, Valsecchi MG, Bartram CR, Schrauder A, Panzer-Grumayer R, Moricke A et al.
Late MRD response determines relapse risk overall and in subsets of childhood T-cell ALL: results of the AIEOP-BFM-ALL 2000 study. Blood
Van der Velden V, Corral L, Valsecchi MG, Jansen MW, De Lorenzo P, Cazzaniga G et al.
Prognostic significance of minimal residual disease in infants with acute lymphoblastic leukemia treated within the Interfant-99 protocol. Leukemia
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]