Immunophenotypic Profile of Acute Leukemia: Critical Analysis and Insights Gained at a Tertiary Care Center in India
Authors: Gujral Sumeet , Badrinath Y. , Kumar Ashok , Subramanian Pg , Raje G , Jain H , Pais A , Kadam Ps amre , Banavali Sd , Arora B. , Kumar P , Menon Vg hari , Kurkure Pa , Parikh Pm , Mahadik S , Chogule Ab , Shinde Sc , Nair Cn 
Published: April 2020

Abstract
The purpose of this retrospective study, the largest series in our country, was to illustrate the spectrum of subtypes and the immunophenotypic features of cases referred as acute leukemia (AL). Morphological diagnosis was correlated with cytochemical stains, immunophenotyping, and molecular genetic studies including fluorescent in-situ hybridization (FISH) and polymerase chain reaction (PCR). Two thousand five hundred and eleven consecutive new referral cases of Acute Leukemia (AL) between January 1, 2003 and December 31, 2006 were evaluated based on WHO classification. It included 1464 cases (58.3%) of Acute Lymphoblastic Leukemia (ALL), 962 cases (38.3%) of Acute Myeloid Leukemia (AML), 45 cases (1.8%) of Chronic Myelogenous Leukemia in blast crisis (CMLBC), 37 cases (1.5%) of biphenotypic acute leukemia (BAL), 1 case of Triphenotypic AL and 2 cases of Acute Undifferentiated leukemia (AUL). Common subtypes of ALL were B-cell ALL (76%) which comprised of intermediate stage/CALLA positive (73%), early precursor/Pro BALL (3%). T-cell ALL constituted 24% (348 cases) of ALL. Common subtypes of AML included AMLM2 (27%), AMLM5 (15%), AMLM0 (12%), AMLM1 (12%), APML (11%), AML t(8;21) (9%). CMLBC included myeloid blast crisis (40 cases), lymphoid blast crisis (2 cases), and biphenotypic leukemia (3 cases). CD13 was most sensitive and CD117 most specific for determining myeloid lineage. An immunoprofile of CD34+, HLADR+, CD14+ and CD33− is unlikely to be APML. CD117 is not a sensitive marker for APML and shows negative to weak continuous expression on promyelocytes. CD19 was expressed in 45% of FAB AMLM2 with t(8;21)(q22;q22). At least five cases of hepatosplenic gamma delta T-cell lymphomas were misdiagnosed as T-ALL based on a minimal selective panel where blasts expressed surface CD3. These cases were correctly diagnosed at relapse. Overall incidence of AML in adults is low (53% only). T-ALL is common in adolescent males. We recommend at least ten antibodies mainly CD13, CD33, CD117, CD10, CD19, HLADR, CD7, CD5 (or CD2), CD45 and CD34 as a primary minimal panel for a case of AL. Tdt is invaluable in work-up of pediatric T-cell neoplasm, to differentiate T-ALL from hepatosplenic gamma delta T cell lymphoma. Only 8% cases required additional markers (including intracytoplasmic markers) in the secondary panel for a lineage assignment. cCD22 is more sensitive than cCD79a for B-cell lineage assignment. Drawbacks of this study included referral bias, no clinical correlation, and three color immunophenotyping with FSC/SSC gating. Our approach for immunophenotyping was semi directed, a primary minimal panel followed by additional antibodies, as and when required. Cytoplasmic lineage specific markers (used in additional panel) for a lineage assignment were required only in 8% cases (n=199). We used only 14–20 antibodies per case (mean = 16), a number much less then many other laboratories. WHO classification has made it mandatory to do FISH/PCR to diagnose various leukemias which otherwise could be diagnosed with reasonable accuracy based on morphology, cytochemistry and flow cytometry like CML, APML, AMLM4Eo etc. Significant number of our FAB AMLM2 and AMLM4E0 cases did not show associated translocations. Few patients were partially treated by referring physician (steroids, blood transfusion, heavy metals etc), a category not addressed in WHO classification. Cytogenetics made a major difference in therapeutic decision in 0.8% cases only. Though flow cytometry is extremely popular amongst hematopathology laboratories, cytogenetics is available at few selected centers in countries with limited resources.