R.Z. Yusuf ( Department of Pathology, The Aga Khan University Hospital, Karachi. )
S. Pervez ( Department of Pathology, The Aga Khan University Hospital, Karachi. )
S.A. Aziz ( Department of Pathology, The Aga Khan University Hospital, Karachi. )
M. Khurshid ( Department of Pathology, The Aga Khan University Hospital, Karachi. )
Objective: To collect demographic data for childhood (less than 15 years) leukemias in Karachi, describe the accuracy of the cell surface markers routinely used in the flow cytometric analysis of leukemic cells and arrive at an ideal panel of antibodies for analyzing leukemic samiples.
Materials and Methods: Data from 62 consecutive cases of childhood leukemias referred to the Department of Pathology, Aga Khan University Hospital. (AKUH) between January 1995 and December 1998 was analyzed using Epi Info Version 6. Flow cytometry on all samples was performed using standard protocols.
Results: The mean age of patients was 8.2 years and 49 (79%) were males. Fifty (81%) had acute lymphoblastic leukemias of which 50% were CD1O positive and 24% CD10 negative Pre-B cell leukemias.
Among all Pre B cell All 98% were positive for CD19, 96% for CD22, 89% for HLA-DR and 67% for CD10. Of the 10 AML cases, 100% were positive for CD33, 90% for CD13, 80% for CD19 and 70% for HLA-DR.
Conclusion: The mean age in this study population was significantly higher and percentage of CD10 positive Pre-B All is lower than that in the West. Both these factors might be responsible for the poorer prognosis of these patients. It is not possible to specify a minimum or maximum panel of antibodies that should be used for phenotyping all cases of childhood leukemias. A certain degree or redundancy is essential in any panel of antibodies used for flow cytometry of leukemias (JPMA 51:133:2001).
Leukernias are the most common cancers in children less than IS years of age in Pakistan1. Leukemias also comprise the majority (40%) of the childhood malignancies in the West including United States2, Therefore, they make up the majority of pediatric malignancies in several parts of the world. Most of these are fortunately curable3. The epidemiology and biology of these tumors, unlike the incidence varies from region to region4,5. This makes it essential to ascertain the epidemiology and biology of these tumors in our population, as has been done elsewhere6,7. A search of the past five years of literature reveals very limited statistics relevant to Pakistan, in this regard8. To arrive at these statistics was one of the objectives of this study.
Flow cytometric is highly applicable to the detection and classification of leukemias because of the ease with which single cell suspensions can be made. Composite immunophenotypic analysis is essential for classifying leukemias once the disease is detected by traditional means9. Ongoing efforts to standardize protocols are essential to ensure widespread applicability, together with improved understanding of the attributes and limitations of this technology.
Flow cytometric analysis of leukemic samples was started routinely at The Aga K.han University Hospital, Department of Pathology in 1995 since then there has been continuous evaluation and revision of the panel of antibodies used to analyze leukemic cells. This study also looks at the accuracy of the cell surface markers that have been used routinely for analysis of leukemic samples, since 1995 to arrive at an ideal panel of antibodies for analyzing leukemic samples.
Materials and Methods
A study question was formulated and a list of all the records of leukemias referred to The Department of Pathology, between January 1995 and December 1998 was generated. Patients in the desired age group (less than 15 years) were selected and relevant information from their records was documented on specially designed data entry forms. The data was then entered in Epi. Info Version 6.1. Analysis was performed using the same program. Student’s t test and Chi square test of significancies were performed to determine the presence of a significant difference between mean age and the percentage of CD10 positive preB acute lymphoblastic leukemias between this study population and Western literature. Alpha value was chosen to be 0.05.
Flow cytometry was performed on all the peripheral blood and bone marrow samples by centrifuging mononuclear cells from them. These were then stained with florescence labeled antibodies (obtained from Becton Dickinson, U.S.A.) and run on FACSCAN using cell quest software. The same software was used to analyze the flow cytometry data10.
CD19, CD20, CD22, CD10, CD3, CD5, CD7, CD13, CD33 and HLA-DR were tested on all the cases11. CD2 was not included in the panel because of the presence of CD3, CD5 and CD7, which are all T cell markers as its inclusien would have added no new information. CD14. CD45, Anti-Kappa, Anti-Lambda. lgG, 1gM and CD61 were markers that were tested on selected cases. Positivity of a clone for a specific cell surface marker was defined as more than 30% of the cells staining with the antibody for the respective marker.
The mean age in this study was 8.2 yearswith a standard deviation of 3.9 years. Peak incidence in the 5-9 years age group in males and the 10-14 year age group in females1. In the West the peak age of incidence is 4 years 12 which is significantly less (p<0.05) than that seen in our population. This could be attributed to the late presentation of these children in our setting.
It is established that ALL is more common in males than females, in the pediatric population12,13. In accordance, the majority of our patients were males. Eighty four percent of our cases were those of ALL, the remainder being AML, which is similar to the results reported elsewhere13,14.
A comparison of the immunologic phenotypes of ALL in our population and in the West are shown in Table 2.
The frequencies of gross categories are similar but there is a paucity of CD10 positive Pre-B ALL (p<0.05) in our series as compared to the West and Far East15-18. This decrease in CD10 positive Pre-B ALL, (CD 10 being a good prognostic indicator15,17,19 could account for the poorer prognosis of leukemias in our population20.
There was no marked excess of CD10 positive cells in the bone marrow samples as compared to the peripheral blood samples. This corroborates evidence that significant homing mechanisms do not seem to be actively involved in localizing CD10 positive cells to the bone marrow21.
Since 1995, flow cytometry has routinely been performed on leukemic samples at the Department of Pathology, AKUFI.
The following is an analysis of the accuracy of markers commonly used for flow cytometric analysis of leukemias at AKUH. The denominator for this analysis is 60 cases (excluding the two cases which were reported as inconclusive). The terms “specificiy” and “sensitivity” are not used in the strict statistical sense. Accuracy of various markers was calculated by working out the percentage of cells which tested positive for the markers which were expected biologically, to be present on these cells. This method of analysis has been used before, successfully8.
CDI9 was detected on 92% of B-Cell ALL and 80% of AML cases. As shown earlier, CD19 is not very specific to B Cell leukemias10,22. CD20 was positive in 44% of cases of B Cell ALL and was not expressed on any non B Cell malignancy. Our study confirms that CD20 is less often expressed on acute B lineage leukemias than CD19 but is more “specific” for B cell malignancies10,23. CD22 was found on 90% of the B cell malignancies and on none of the non B cell malignancies. A highly “sensitive” and “specific” marker, CD22’ s usefulness to detect acute B-lineage leukemias is decreased by the fact that it is often dim and is more strongly expressed in the cytoplasm of cells than on their surface10,24. 67% (31/46) of the cases of Pre B Cell ALL were CD10 positive. This is lower than the percentage of CD10 positive Pre-B cell ALL reported in the West and this point has been discussed above. CD10 was not detected on any case of T cell ALL (as has been reported in literature15. This could be due to the small number of cases of T cell ALL in our study. We hypothesize that it could also be due to the high socio-economic status of the patients reporting to the Aga Khan University Hospital since T-ALL is more common in the low socio-economic group. However our study does not have the power to test this hypothesis.
CD3 was detected on 66.7% of the cases of T cell ALL and 7% of the cases of non T cell malignancies. It is hence a “sensitive” and “specific” marker for detecting T cell ALL23. Its usefulness is limited by the fact that it is uncommonly expressed on the surface of 1-lineage ALL. It is almost always present in the cytoplasm of these cells10,25. CD5 was found on all 3 cases of T cell leukemias and 12.3% of non T cell malignancies. Our study corroborates evidence that CD5 is specific for T cells and occurs in a great majority of cases, the drawback once again being that it is dimly expressed10,26. CD7 was seen on 4/46 cases of Pre-B cell ALL. This was seen in samples of peripheral blood for leukemia analysis, which normally has 80% T lymphocytes. This pattern is most likely reactivity to normal circulating lymphocytes. CD7 was also found on all cses of T cell ALL and on 14% of cases of non T cell ALL. It is reported in literature however, that CD7 is found in cases of AML too and is not as “specific for T cell ALL as our study suggests10,27.
CD13 was found on 90% of cases of AML and 8% of non AMI. cases. CD33 was found on all cases of AML and on 2% of non AML cases. Both these markers are hence valuable for diagnosis of AML. Since both can occasionally be expressed on B or T lineage ALL they should be interpreted as strongly suggestive of myeloid leukemia only when expressed in the absence of lymphoid associated antigens10,26. Our study confirms previous reports that HLA-DR is found in most cases of AML and B lineage ALL10,28.
In conclusion, the epidemiology of acute leukemias in our study population is similar to that reported in the West. This has also been shown in another Pakistani studies29,30. Late age at presentation and an excess of CD10 positive cases may lead to poor outcome.
Regarding flow cytometry, antibodies clearly vary in their degree of lineage specificity. Also, many leukemias lack one or more antigens expected to be present on normal cells of a particular lineage9,10,31,32. It is hence recommended, that a certain degree of redundancy be built into a panel for leukemia phenotyping.
Where possible it is recommended that lineage assignment be confirmed with an antibody from a second CD cluster which is known to react principally with the suspected cell lineage. However, failure to detect the confirmatory marker does not exclude the suspected lineage since leukemias commonly exhibit loss of various antigens. It is hence not possible to specify a minimum or maximum paneJ that should be used for phenotyping all cases of leukemias10,14,33.
1.Bhurgi Y, Bhurgi A, Rahim A. et al. Comparbility and Quality Control at the Karachi Cancer Registry Karachi. Karachi Cancer Registry Technical Report Number 4, 1999.
2.Kaplinsky C. Zairov R. Childhood B-Cell non-Hodgkin’s lymphoma in Israel. Leuk, Lymphoina, 1994:14:49-53.
3.Bleyer WA. What can be learned about childhood cancer front “Cancer Statistics Review 1997-1988* Cancer, 1993:71 (Suppl)32,29-36.
4.Roush SW, Krischcr JP, Pollock NC et al. The incidence of pediatric cancer in Florida, 1981 to 1986. Cancer, 1992:69:2212-9.
5.Roguin A, Linn S. Dale J. et al. Patterns of childhood solid tumor incidence in northern Israel, 1973-1990. Pediatr. Hernatol. Oncol., 1997: 4:525-37.
6.Paredes Aguilera R, Roinero Guzman I., Lopez Santiago N. et al. Immunophenotyping of acute lymphoblastic leukemia in Mexican children Sangre (Bare), 1999:44:188-94.
7.Menon BS, Dasgupta A, Jackson N. immunophenotyping pediatric leukemias in Kelanian, Malaysia Pediatr Hematol Oncol., 1998:15 175-8.
8.Pervez S. Khurshid M. Classification and Immunophenotyping of Acute Leukemias: A Prospective Study. J. Pak. Med. Assoc., 1997,47:103-106.
9.Deque RE Flow cytornetric analysis of lymphomas and acute leukemias. Ann. N. Y. Acad. Sci., 1993:677:309-25.
10.Landary L, Auer R. Bach B. Clinical Application of Flow Cytometry: Quality assurance and immunophenotyping of peripheral blood lymphocytes. NCCLS Document H42-T 1992; 12.23-30.
11.Raimondi R, Pellizzari G, Rodeghiero F. Single step immunophenotyping of acute leukemias plot classitiable by standard morphology and cytochemistry: a practical approach. Haematologica, 1 993: 78(6 supplement 2): 66-72.
12.Rudolf AM, Kainci RK, Sagan P. Rudolf’s fundamentals of Pediatrics. First Edition, Appleton and Lange. (Connecticut), 1994, pp. 139-443.
13.Fajardo-Gutierrcz A, Navarrete-Martinez A, Reynoso-(iarcia M et al. Incidence of malignant neoplasins in children attending Social Security Hospitals in Mexico City Med. Pediatr. Oncol., 1997.29 208-12.
14.Klujber V. Koos R, Schuler D, et al. lmmunophenotvping of childhood leukemias-results of the first 100 bone marrow tests. Orv. Hetil., 1996:137 1139-42.
15.De Salvo L. Plurnacher Z. Rios M em al. CD10(CALLA)-negative acute lymphoblastic leukemia. A group with a bad prognosis. Investigation Clinics. 1992;33:147-52.
16.Consolini R, Legitimo A. Rondelli R et al. Clinical relevance of CD10 expression in childhood ALL. The Italian Association for Pediatric Hematology and Oncology (AIEOP). Haematologica. 1998:83:967-73.
17.Lenorinand B, Bene MC. Lesesve iF et at. PreBI (CD10-) acute lymphoblastic leukemia; immunophenotypic and genomic characteristics, clinical features and outcome in 38 adults and 26 children. The Group dEtude Immunologique des Leucemies. Lcuk Lymphoma. 1998:28:329-42.
18,Rivera Luna R, Cardenas Cardos R, Leal Leal C et al. B-lineage acute lvmphoblastic leukemia of childhood. An institutional experience. Arch. Med. Res., 1997:28.233-9.
19.Cap J, Babusikova 0. Kaiserova E, et al. Expression of CD10, CD19 and CD34 markers in bone marrow samples of children with precursor B-Cell acute lymphobiastic leukein ía in ci inical and hematological remission. Neoplasma, 1998:45:231-6.
20.Aziz Z. Zahid M. Mahmood R. et al. Modified BFM protocol for childhood acute lymphoblastic leukemia: a retrospective analysis Med. Pediatr Oncol.. 1 997;28:48-53.
21.Makrynikola V. Bianchi A, Bradstock K. et al. Migration ol acute lymphoblastic leukemia cells into human bone marrow stroina. Leukemia. 1994; 8: 1734-43.
22.Tisone JA, Bohman JE. Thcil KS. Em al. Aberrant expression of CD19 as a marker of monoeytic lineage in acute myelogenous leukemia. Am. J Clin Pathol., 1997;107:283-91.
23.Chuang SS. Li CY. Useful panel of antibodies lot the classification of acute leukemia by immunohistochemical methods in hone marrow trephine biopsy apecimens. Am. i. Clin. Pathol.. 1997;107:410-8.
24.Sartor M, Bradstock K. l)etection of intracellular lymphoid differentation antigens by flow cytometry in acute Iyiaphoblastic leukemia Cytometry, 1994;18:1 19-22.
25.Pizzolo G, Vincenzi C, Nadali G. et al. Detection of membrane and intracellular antigens by flow cytometrv following ORTUO Permea Fix fixation. Leukemia, 1 994;8:672-6.
26.Kuerbitz SJ, civin Cl, Knscher JP. ci al. Expression of myeloid-assocmated and Iyinphoid associated cell-surface antigens in acute myeloid leukemia of childhood: A Pediatric Oncology group study, J. Clin. Oncol., 1992:10:1419 29.
27.Osada H, Erni N, Ueda R. et ai. Genuine CD7 expression in acute leukemia and lymphoblastic lymphoma. Leuk. Res., 1990:14:869-77.
28.Wang L, Zhang M. Somig S. Dynamic analysis of cell surface markers in acute leukeinias unclassified with immunologic criteria Chung. Hua. Nei. Ko. Tsa. Chili.. 1996;35:595-7.
29.Channa J, Shamsi 1, Hashmi K, ci al. Role of lmmunophenotyping in the diagnosis of Acute Leukemia. JCPSP., 2000; 10:158-60.
30.Naeem S, Hayee A. Acute lymphoblastic leukemia-a study of lmmunotvpes. J. Pak. Med. Assoc., 1992.42(4):83-6.
31.Orfao A, Ciudad J, Cronzalez M et at. Flow cytomctrv in the diagnosis of cancer. Scand. J. Clin. Lab. Invest. Suppl., 1995:221 45-52.
32.Pui CH, Behm FG, Crist WM. Clinical and biologic relevance of immunologic marker studies in childhood leukemnias. Blood, 1993:82 343-62.
33.Drexler FIG, Thiel E, Ludwig WD. Acute myeloid leukemias expressing lymphoid associated antigens: diagnostic incidence and prognostic siginifmcance. Leukemia, 1993;7:489-98.