ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 10, No. 3 Copyright © 1980, Institute for Clinical Science, Inc. Cell Surface Markers in Acute Lymphoblastic Leukemia* f

G. BENNETT HUMPHREY, M.D., REBECCA BLACKSTOCK, Ph .D., AND JANICE FILLER, M.S.

University of Oklahoma, Health Sciences Center, Oklahoma City, OK 73126

ABSTRACT

During the last nine years, two important methodologies have been used to characterize the cell surfaces of normal lymphocytes and malignant lym­ phoblasts. Normal mature T-cells have a receptor for sheep erythrocytes (E+) while mature B-cells bear membrane-bound immunoglobulin molecules (slg+). These two findings can be used to divide acute lymphoblastic leukemia of childhood into three major groups; B-cell leukemia (slg+ E -), which is rare (approximately 2 percent) and has the poorest prognosis, T-cell leukemia (slg~, E +) which is more common (10 percent) but also has a poor prognosis and null cell leukemia (slg~, E~) which is the most common (85 percent) and has the best prognosis. By the use of additional immunological methods, subgroups within T-cell leukemia and null cell leukemia have also been proposed. One of the most valuable of these additional methods is the detection of surface antigens. Three of the more commonly detected antigens currently being evaluated are (1) common leukemia antigen (cALL), (2) a normal B Lymphocyte antigen the la antigen (la) which is not generally expressed on most T lympho­ cytes and (3) a normal T lymphocyte antigen (T) not expressed on B lympho­ cytes. Within null cell leukemia, the most commonly identified and proba­ bly the largest subgroup is Ia+, cALL+, T”, E _, slg-. In another but smaller subgroup within null cell leukemia, the lymphoblasts contain cytoplasmic immunoglobulin but do not express surface immunoglobulins or E recep­ tors. This subgroup is designated pre B-cell leukemia. Subgroups with T-cell leukemia have also been suggested. These include T+ E~, T+ E+, in which the rosettes are thermolabile and T+ E+, in which the rosettes are thermostable. Whether qr not there are any prognostic differences in these three subgroups remains to be determined.

Introduction homogeneous entity. However, a wide range of clinical responses to therapy ob­ Acute lymphoblastic leukemia (ALL) in served over the past two decades has children is morphologically a relatively suggested that ALL is a heterogeneous * This investigation was supported by Grant disease. Early extensive evaluation of Number CA11233, awarded by The National Cancer clinical and laboratory data identified a Institute, DHEW. number of prognostic factors such as age, f Address reprint requests To: Southwest Oncol­ ogy Group, Operations Office, Suite 100, 3500 Rain­ initial white count, presence or absence of bow Blvd., Kansas City, KS 66103. a mediastinal mass.23 While these prog- 169 0091-7370/80/0500-0169 $01.80 © Institute for Clinical Science, Inc. 1 7 0 HUMPHREY, BLACKSTOCK AND FILLER

nostic factors have been of value, the immunoglobulin. A precursor cell of the search for surface markers as correlates of mature B-cell has been demonstrated to major groups within ALL has dominated have cytoplasmic immunoglobulin (clg) leukemia research for the past nine years but not surface immunoglobulins (slg) and is the principle topic of this review. and is designated a pre-B-cell. A mature In the early 1970’s, several laboratories lymphocyte has receptors for sheep eryth­ reported that normal human peripheral rocytes (E). For human cells, receptor for blood T-lymphocytes spontaneously complement and the Fc portion of IgG formed rosettes with sheep erythrocytes. were initially thought to be limited to During the same period of time, the dem­ B-lymphocytes. Subsequent research has onstration of surface immunoglobulin on shown that these two receptors can also peripheral B-cells became a common lab­ reside on subpopulations of some T-cells oratory procedure. These two discoveries and are therefore not used independently were applied to bone marrow lympho­ to identify either T or B-cells. blasts obtained from children with leukemia and have resulted in the iden­ The Major Groups Within ALL tification of three major groups within this disease: T-cell leukemia, B-cell leukemia For the purpose of discussion, ALL will and null cell leukemia. be divided into three major groups. T-cell, Other methodologies have also been null cell and B-cell leukemia. T-cell applied to ALL cells to identify further leukemia will be limited to those cases subgroups based on cytogenetics, cell cul­ that are E-receptor positive. No distinc­ ture growth characteristics, etc. Subtle tion will be made in this section as to morphological differences based on light thermolabile and thermostable subgroups microscopy have been used to charac­ of E-receptor positive cases. B cell disease terize blast cells. An international will be limited to those cases that are sur­ cooperative committee proposed the face immunoglobulin positive. In some of French-American-British (FAB) classifi­ the early literature, the presence of a Fc cation of ALL which subdivides ALL into receptor or complement receptor was three morphological types: Lj, L2 and L3. used to identify cases designated B-cell These morphological groups have been disease or B-cell leukemia. As both of related to surface marker studies and have these receptors can be found on both been shown recently to be of prognostic T-cells as well as B-cells, these markers significance.38 will not be accepted as characterizing The application of human lymphocyte B-cell disease for this discussion. differentiation and subpopulations to the Null cell disease will include those immunological diagnosis of other disor­ cases that do not express either the ders has been the subject of recent re­ E-receptor or have detectable surface views which are recommended for a more immunoglobulin (slg). In many articles, detailed background study of this impor­ null cell disease is diagnosed by exclusion tant and rapidly developing area of clini­ and is referred to as non-T non-B cell cal and laboratory research.20,46’50 disease.

Surface Markers B-C e l l L e u k e m i a

Cell surface markers have been used to B-cell leukem ia is the rarest o f the major identify functional subdivisions of lym­ or well established groups. This group has phocytes. For example, B-lymphocytes a very poor prognosis, as response to are identified by the presence of surface chemotherapy results in short remis­ CELL SURFACE MARKERS IN ACUTE LYMPHOBLASTIC LEUKEMIA 171 sions.4,10,30,15 T he patients tend to be older a high white cell count, mediastinal mass and a male predominance has been and older patient population, were known suggested.30 Although leukemic presenta­ to be of poor prognostic significance prior tion of Burkitt’s lymphoma is unusual, to the application of markers to ALL. there are a number of characteristics The remission induction rate of T-cell which suggest that B-cell leukemia and leukemia is similar to that of null cell dis­ Burkitt’s lymphoma are closely related or ease with both groups responding well to are even the same disease. For example, a vincristine/prednisone therapy. On 14q+ chromosome has been reported in standard ALL therapies, such as both B-cell leukemia and Burkitt’s lym­ 6-mercaptopurine and methotrexate, a phom a.30,51 The morphology of both is of short median duration of remission has the L3 type and malignant B-cells have been reported by a number of investiga­ been described as “Burkitt cells.” tors. When alkylating agents such as cyc­ lophosphamide or an anthracycline such N u l l C e l l L e u k e m i a as adriamycin is added to the maintenance regimen, longer median durations of re­ Approximately 85 percent of childhood mission are being reported .17,58 ALL is classified as null cell leukemia.

Null cell leukemia has the best prognosis C ytochemical M e t h o d s a s of the major groups. The clinical charac­ R e l a t e d T o M a j o r G r o u p s teristics of this group include those gener­ ally associated with good prognosis such Cytochemical methods have been used as younger age, lack of tumor mass at pres­ for a number of years to classify the entation, and a lower initial white cell leukemias. This has been especially true co u n t.4,10,30,24 T he bone m arrow is thought for the differential diagnosis of acute to be the origin of null cell leukemia. myeloblastic leukemia from acute lym­ These patients respond well to both in­ phoblastic leukemia. Recently, there has duction and remission therapy. This is been a number of papers published that true for both median duration of remission have attempted to correlate surface mar­ and survival.4,10 kers with some enzyme systems in leukemic cells. In table I are summarized some of the T-C e l l L e u k e m i a recent studies in w hich both im m unologi­ T-cell leukemia accounts for approxi­ cal and enzyme markers in leukemia have m ately 10 percent of childhood cases of been studied. Leukemia cells with T-cell ALL.22,30 T-cell leukemia has a poorer surface markers have a markedly reduced prognosis than null cell leukemia, but a 5-nucleotidase activity, while null lym­ better one than B-cell leukemia. T-cell phoblasts have normal levels of this en­ leukemia occurs more frequently in males zym e.44 One of the most widely studied than females and in older children, the enzymes is TdT .13 Elevated levels of TdT median age being 10 to 12 years in studies have been found in children with both limited to children. There is a high fre­ null cell and T-cell leukemia. In general, quency of leukemic masses (76 percent TdT levels are low in B-cell leukemia,13,30 reported in one series)4 of which approx­ but exceptions have been reported .49 imately 40 percent will be a mediastinal While increased levels of adenosine m ass.22 Patients with T-cell leukemia also deaminase (AdA) activity have been re­ present with high initial leukocyte counts, ported in both T-cell and null cell mean values being reported in the leukem ia,52 one report emphasized that neighborhood of 150,000 cells per yu,l. AdA activities did not show any correla­ Many of these clinical characteristics, i.e. tion with immunological markers of pa- 172 HUMPHREY, BLACKSTOCK AND FILLER

T A BL E I thymocytes express a fine localized non­ Enzyme Markers and the Major Group of ALL intense activity (thy-like). In four out of five cases of T-cell leukemia, the acid es­ Enzyme Null Cell T-Cell B-Cell terase was thy-like while in only one out of nine cases of null cell leukemia was _ 5-Nucleotidase Normal Low thy-like reactivity expressed, the remain­ Terminal Increased Increased Low desoxynucleotide ing cases being negative.32 transferase It is our opinion that enzyme markers Adenosine Low Increased Low deaminase and histochemical stains will continue to Hexosaminidase I Increased Low Low Acid phosphatase Low Increased --- be useful in the differentiation of acute Alkaline phosphatase Increased Normal Normal myelocytic leukemia (AML) from ALL. Alkaline Increased Normal Normal phosphodiesterase I However, at present, these enzyme mar­ Acid esterase Low Faint Low kers cannot be substituted for immunolog­ localized activity ical markers in the identification of T, B and null cell disease. tients with ALL, but in fact there was a FAB C lassification, Prognosis wide range of AdA activity in both T-cell and M ajor Groups W ithin ALL and null cell cases.33 The FAB classification defines three Extracts of normal lymphocytes, classes of ALL based upon the amount of thymocytes and granulocytes give differ­ cytoplasm, predominance of nucleoli, the ent isoenzyme patterns of hexosa­ basophilia of the cytoplasm, and other minidase. Increased levels of the characteristics.5 In an analysis of 566 chil­ hexosaminidase component I are found in dren w ith ALL w ho w ere not classified by most cases of acute lymphoblastic surface markers, 83 percent were Li, 15 leukemia, suggesting that this may be of percent were L2 and only 1 percent was diagnostic value in an analysis of this type L3. L2 morphology was associated with a of leukemia.15 T wenty-three out of 27 null significantly higher relapse rate .38 W hen cell cases (defined by antigen studies) FAB morphology is related to null cell demonstrated a pattern of raised leukemias, 75 percent of these cases were hexosaminidase I. Cases of T-cell and Lj and 25 percent were L 2.19 T -c ell B-cell leukemia did not show the raised leukemia can be either the Li or L2 type. hexosaminidase I pattern. The L3 morphology is thought to identify a The last four enzymes listed in table I Burkitt’s type of malignant cell that is sur­ have been used for at least a decade and face immunoglobulin positive. Whether are generally thought of as histochemical the FAB classification is an independent stains rather than enzyme markers. Acid prognostic variable or whether it is more phosphatase, alkaline phosphatase and significant than some of the subgroups alkaline phosphodiesterase I are probably that are currently being defined within of value in distinguishing T-cell disease T-cell and null cell leukemia remains to from null cell disease, but are not reliable be determined. enough to replace immunological markers for the distinction of these two groups.2'31 Surface Antigens on Lymphocytes Four different staining patterns have and Lymphoblasts been recognized for the non-specific acid alphanaphthol acetate esterase or acid es­ The current direction of research in this terase activity. Normal peripheral field relates to the use of antisera to detect T-lymphocytes show a localized, intense surface antigens which are expressed on activity (T-like) while a subpopulation of normal T and B lymphocytes and on CELL SURFACE MARKERS IN ACUTE LYMPHOBLASTIC LEUKEMIA 1 7 3 human leukemia cells. While these anti­ to represent a normal differentiation anti­ sera can be used alone to subclassify ALL, gen of the cell type involved in malignant most laboratories use both antisera and transformation.20 rosetting techniques to identify surface In addition to antigens which may be markers. Before discussing the impact of present on precursors of mature T and both antigen detection and surface recep­ B-lymphocytes, other antigens have been tors on subdivsion within null, B and detected in the common form of ALL T-cell leukemia, a review of surface anti­ which are expressed on mature T and B- gens and corresponding antisera will be lym phocytes.1,3,20,27,28,29,36,56,59,60,62 A set necessary. of B-cell antigens has been referred to as Over the past several years, there have “Ia-like” antigens.36 This terminology has been numerous reports of the detection of come from the fact that these antigens antigens associated with human leukemia closely resemble the la antigens found on cells.36 The antigens detected may be murine B-lymphocytes which are coded truly leukemia specific or may represent by genes of the I-region of the histocom­ differentiation antigens present on vari­ patibility complex (MHC) of mice. The ous precursors of the cells of the lymphoid antigens in man are probably linked to the series.20 Alternatively, the antigens may D-locus of the human MHC. Ia antigens represent fetal antigens which could be have been found on the surface of non-T, present on fetal tissues other than non-B, ALL, CML in blast crisis and lymphoid progenitors.20’35'53 In addition, AML.| 3,20,59 The antigens expressed on antigens detected on mature T and leukemic blasts are apparently identical B-lymphocytes have given valuable in­ to those expressed by mature B-cells and formation which is beginning to allow by a certain T-cell subpopulation.20 Since subclassification of ALL. the antigen can also b e found on im m ature An antigen found on the common form myeloid cells of bone marrow, as well as of acute lymphoblastic leukemias has bone marrow cells of patients with been extensively studied.20,36,35 It is an megaloblastic anemia and myeloid antigen with a molecular weight of about hyperplasia, it has been suggested that Ia 100,000 daltons54 and is found on cells of expression on leukemic blasts may reflect all patients with “common” ALL. Al­ their hematopoietic stem cell origin.20,26 A though the antigen was originally not recent study showed that the presence or detected on blasts cells of T-cell absence of the Ia antigens on the surface leukemia,11,40 more recent evidence has of the blast cell could be useful in the shown weak reactions in 10 percent of subclassification of “null” ALL.1 Other T-cell cases. Cells of Philadelphia investigators29,56 have reported that nor­ chromosome positive CML * in blast crisis mal B-cell antigens can be detected on express the antigen as do AUL f and a few one subgroup of common ALL. It is not lymphomas.45 The antigen has not been possible to determine from these reports if found on the surface of normal, mature the antisera which they have produced is lymphocytes.20,36 The antigen was de­ identical to anti-la antisera or if they have tected on normal fetal bone marrow cells detected other B-cell differentiation and on the cells of regenerating juvenile antigens. bone marrow cells, but it was not detected The B-lymphocyte antigen, BDA (B- on bone marrow cells of adults.26 The ALL cell differentiation antigen),3 is distinct antigen does not cross-react with any from Ia and is present on B-lymphocytes known oncogenic virus, and it is thought in all stages of differentiation. BDA was

* Chronic myelogenous leukemia, f Acute undifferentiated leukemia. { Acute myelogenous leukemia. 174 HUMPHREY, BLACKSTOCK AND FILLER expressed on leukemic blasts of most ALL,37 the data have not yet proven useful (14/18) patients with lymphocytic for subclassification. Certainly this area of leukemia but was not present on investigation deserves to be monitored as leukemias of myelogenous origin. researchers begin to sort out the complex Just as B-cell antigens have been de­ specificities which are detected by their tected on ALL blasts, so have normal anti-viral reagents. T-cell antigens.29,39’41,56,60 An antigen present on thymocytes and leukemia cells Subclassifications Within T-Cell of mice has been called the TL antigen.8 Leukemia and Null Cell Leukemia Antisera having similar reactivities to anti-TL have been produced by injection The preceding discussion of ALL as of rabbits or non-human primates with three major groups was based on only two human thymus cells One such an­ .12,41,56 surface markers, the E-receptor and slg, tiserum detected antigens present on and represents a simple but well recog­ thymocytes and on E-rosette positive ALL nized or “well established” division of blasts.12 The antigen was not found on ALL. These three major groups are now peripheral T-cells or E-rosette negative being divided into subgroups by research blasts. Blasts from other types of leukemia laboratories that are analyzing bone mar­ were not tested for the presence of the row lymphoblasts for cytoplasmic im- “TL-like” antigen. The antigen described munoglobuln, additional surface recep­ by M ohanakum ar and M etzger,41 although tors such as the complement and Fc recep­ TL-like, as determined by its presence on tor, surface antigens as well as the two thymocytes and leukemic blasts, was standard tests (E and slg). At present, present on ALL blasts as well as CLL§ there is no uniform definition of these blasts, a leukemic cell which is known to subgroups owing to the different possess B-cell markers. It is not possible techniques being employed, different to determine if these twp groups have de­ sources of antisera and the lack of uniform scribed the same or different antigens. application of all available surface mar­ Antigens characteristic of peripheral kers to the cells being analyzed. Despite T-cells can also be found on the surface of these problems, some very interesting leukemic blasts. Leukemic blasts, which subgroups have been identified. are classified as T-cell leukemia on the A few examples of subgroups currently basis of their ability to form rosettes with being identified are listed in table II. It is sheep erythrocytes, express these anti­ important to note that in table II the short­ gens as do some ALL cells which do not hand designation has been used as de­ possess the sRBC receptor. Among the scribed in the original research article. “null” cell ALL, those cells which express This takes into account minor differences the T-lymphocyte associated antigen in technique or different sources of anti­ (TLAA) do not express la and vice versa .1 sera. Using a strict definition of B-cell A final possible source of leukemia as­ leukemia which requires the presence of sociated antigens which may someday surface immunoglobulins, no major sub­ prove useful for classification of leukemic groups have been identified within this blasts is those antigens which are as­ major grouping. Since B-cell leukemia is sociated with C-type ribonucleic acid vir­ rare and extensive evaluations for a com­ uses. Although there is currently some in­ plement receptor, Fc receptor, la antigen formation regarding the presence of such and of the different subclasses of immu­ antigens on the surface of blasts cells in noglobulins have not been widely applied to this disease category, it is not surprising § Chronic lymphocytic leukemia. that it remains a single group at this time. CELL SURFACE MARKERS IN ACUTE LYMPHOBLASTIC LEUKEMIA 17 5

T A B L E II

Major Groups Within ALL

ALL' Ia+ E" HuTLA" SMIg+ ALL1- la E- HuTLA- Smlg“ ALL“ Ia“ E+ HuTLA+ Smlg“ (B-ALL) (common ALL) (Thy-ALL) ALL- Ia+ E” HuTLA" Smlg“ ALL“ Ia+ E + HuTLA+ Smlg" (unclassified) ("true T-cell leukemia") Ia+ HTL“ HTL+ la“ Anderson1 E" TLAA” Ia+ (III) E + TLAA+ Ia“ (I) E “ TLAA+ la" (II) K a p l e n ^ SIg+ Anti T Anti B+ Slg Anti T+ Anti B E+ Slg- Anti T+ Anti B" Slg“ Anti T“ Anti B+ r3 7 A peripheral blood E ° //H < 1 0 % ' ’' eTS (Humphrey) peripheral blood E 37/1* 10-40%+ EiL peripheral blood E 37/ 4 > 40%+ Crist, Vogler E" slg clg“ slg" clg” Fc+/- C 3+/- (null) E+ slg“ clg" F c +/- C 3 + / - (SWOG) 57 slg“ clg+ Fc+/_ C 3+/“ (pre B) E" HTLA- SMIg+ Fc+/" EAC+/“ HTLA“ SMIg” Fc“ EAC“ E+ HTLA+ SMIg" HTLA“ SMIg“ Fc+ EAC+ E~ T“ sig+ la CALL T" Slg“ Ia+ cALL+ E+ Tag+/“ Slg“ Ia" c a l l " T" Slg Ia+/“ (undifferentiated) Smlg+ (group VI) Anti cALL+ (group I) Anti CALL- Anti T“ E- (group V) Anti CALL" Anti T E (group III) Anti cALL+ Anti T + (group II) E~ MLC+ GCR++ E + MLC“ GCR“ E“ MLC“ GCR+ SWOG (Pullen) 1,3 E+/5 E +^7 clg slg Tp T la L/L cL C 3 Fcp (peripheral blood £ 37/ 1* < 10%: 10-40% > 40%)

Greaves and coworkers have identified lowup of 28 months. However, for the two subgroups for both null cell and T-cell HTL positive cases, disease free survival leukemia using antisera which detect was only 15 months with all patients hav­ common leukemia antigen (cALL), Ia, and ing relapsed by 24 months. While these a T-antigen (HUTLA). Within null cell two antisera have been used to define null leukemia, most cases were identified as cell leukemia or T-cell leukemia rather common ALL by the presence of both than subgroups, their observations are in­ cALL and Ia positivity.20 A smaller sub­ cluded in this portion of the review be­ group listed as unclassified expressed cause of the length of followup. only the Ia antigen. For T-cell leukemia, Anderson and coworkers using antisera four cases of THY ALL were identified by to detect human T-cell associated antigen the presence of both an E-receptor and (TLAA) and Ia antigens as well as the positivity for HUTLA. One case de­ E-receptor have identified two subgroups scribed as true T-cell leukemia also ex­ within T-cell leukemia. Their group 1 (E pressed the Ia antigen. positive T positive Ia negative E + T + Ia-) Sallen and coworkers have followed a was more common (11 cases) than group 2 large cohort of children for up to four years (E negative T positive Ia negative E~ T+ using an antiserum against a human la”) which included only four cases. T-leukemia antigen (HTL) and the Ia an­ There were 11 cases of null cell leukemia tig en .47 The two antisera were used to (their group 3) which were E negative T identify either null cell leukemia or T-cell negative Ia positive (E_ T_ Ia+). These leukemia. The disease free survival of the investigators believe that both E+ T+ Ia- 76 patients identified as Ia positive was 80 and E~ T+ la- cases respond poorly to percent at four years with a median fol- therapy. This approach suggests that a 176 HUMPHREY, BLACKSTOCK AND FILLER portion of null cell leukemia defined by significant proportion of children with E-receptor and slg methods alone is actu­ bad clinical prognostic signs, such as a ally T-cell leukemia, further suggesting high white cell count or age greater than that antisera may be of more value than ten years. Conversely, for those children E-receptor in detecting T-cell leukemia. in group III with greater than 40 percent E As mentioned in the section on surface positive cells in their peripheral blood, antigens, Kaplan and coworkers have there were none who presented with both generated two antisera against a B-cell a high white cell count and an age greater and T-cell line.29 Within cases of null cell than ten (p 0.001).25 The group with 10 to leukemia, Kaplan identified two males, 40 percent E positive cells in their each of whom had high white counts and a peripheral blood was intermediate be­ mediastinal mass that were T-antigen pos­ tween the first and third groups with re­ itive but did not express the E-receptor. gard to clinical prognostic features. The remaining nine cases within the null Whether or not this observation is an in­ cell group reacted with the B antiserum. dependent prognostic variable remains to In 1974, the Pediatric Division of The be determined. Southwest Oncology Group (SWOG) in­ Vogler and Crist, working indepen­ itiated a group-wide protocol22 requiring dently and in collaboration with the bone marrow blasts to be evaluated for E Pediatric Division of SWOG, have iden­ rosettes at both 37° C and 4° C. A year later, tified the best characterized subgroup the protocol was amended and evaluation within the null cell leukemias.14,57 T heir of peripheral blood at both 37° C and 4°C observation was preceded by extensive was added. In contrast to the earlier report work with animal and human fetal tissue. that all cases of T-cell leukemia would Investigations into lymphocyte ontogeny form stable rosettes at both 37°C and 4°C have suggested the existence of a slg" in approximately one-third of the cases, clg+ B-cell precursor. These pre B-cells the T lymphoblasts were not thermosta­ contain small amounts of intracytoplasmic ble (Ere) but were thermolabile (Ert) at IgM and are initially found in the fetal the higher temperature.21 This observa­ liver and later in the bone marrow. tion has been confirmed by other inves­ Vogler first described four cases of pre tigators.55 There is some suggestion that B-cell leukemia out of a sample of 22 cases this difference in thermal stability may of ALL, suggesting that this subgroup have prognostic significance. might constitute a significant percentage The median duration of remission for of the null cell group.57 These leukemic the Eri subgroup was longer than the Ers pre B-cells also express the common group; however, this difference has not leukemia antigen and a “B-cell” antigen. yet reached statistical significance .21 Of particular interest was the finding of Within the null cell group, the number of two of the four cases that had Fc receptors circulating peripheral blood normal T and one case that had complement recep­ lymphocytes may have prognostic signifi­ tor. Complement receptor and Fc receptor cance. The peripheral blood of children are not found on normal pre B-cells, with less than 20 percent E positive cells suggesting that leukemic transformation in their bone marrow or having null cell occurred beyond the pre B-cell stage or leukemia was analyzed for E rosette for­ that the transformation induced the ex­ mation at both 37° C and 4°C, resulting in pression of these receptors. The existence the identification of three groups. of this subgroup has been confirmed by In group I, children had less than 10 French investigators who reported six percent E positive cells in their cases out of a sample of 50 children .9 In peripheral blood. There was a statistically four of these cases, the disease had CELL SURFACE MARKERS IN ACUTE LYMPHOBLASTIC LEUKEMIA 177

tumoral presentation. These cells also ex­ While adverse clinical features such as pressed the Ia-like and common ALL high white count, central nervous system antigens. leukemia, or mediastinal mass were not The clinical and laboratory characteris­ common features of the undifferentiated tics of pre B-cell leukemia in childhood leukemia, six of their 12 patients were have recently been summarized by Crist, early treatment failures whereas only Vogler, et al.14 Thirty five out of 189 cases, eight out of 55 children expressing both or 18 percent, were pre B-cell leukemia. the la and the CLL antigens were early Tumoral presentation was not a common treatment failures. From their experience, feature and the distribution of patients these authors concluded that a common with regard to hepatosplenomegaly, lymph- leukemia typing pattern of IA+ cALL+ adenopathy, extramedullary disease and represented a favorable prognostic bone marrow involvement was simi­ group.7 lar to null cell and T-cell leukemia. Sex, Netzel and coworkers have divided age and race distribution, as well as the ALL into six groups (I-VI) in a recent initial diagnostic white count, hemoglo­ study of 67 patients.42 In attempting to fit bin and platelet count values, were also their six groups into B, null and T-cell the same for pre B-cell and null cell dis­ leukemia, group number VI clearly falls ease. The initial response to therapy was into B-cell leukemia, two groups within excellent. The median duration of remis­ null cell leukemias (I and V), two within sion for this group has not yet been T-cell leukemia (III and IV) and one reached; the prognostic significance of group (II) that was intermediate between the pre B-cell subgroup is yet to be deter­ T-cell and null cell leukemia. This clas­ m ined. sification was based on the use of two anti­ Esbar and coworkers have made a very sera, one directed against a common interesting observation with the regard to leukemia antigen (anti cALL+) and the the prognostic significance of comple­ second directed against a normal T-cell antigen (anti T). ment and Fc receptors.16 Null cell cases The most common subgroup within that express neither of these two markers null cell leukemia was group I (38 per­ had a significantly longer median dura­ cent) expressing the common leukemia tion of remission than those cases that ex­ antigen (anti cALL+). The smaller group V pressed either the complement or Fc re­ (6 percent) expressed neither the common ceptor markers. This is of particular inter­ leukemia antigen nor the T-cell antigen. est considering Crist and Vogler’s obser­ Group (II) which accounted for 32 percent vation that T-cell, null cell and pre B-cell of the patient sample was intermediate subgroups in each case express these re­ between T-cell and null cell leukemia be­ ceptors in some, but not all, cases. cause these cells expressed both the Using antisera directed against B-cell T-cell antigen (suggesting T-cell antigen (la) and the common leukemia leukemia classification) and the cALL antigen (cALL), Bowman and coworkers antigen (which could suggest null cell have identified two groups within null leukemia). cell leukemia.7 Fifty-five patients expres­ Within T-cell leukemia, group III (anti sed both the la and the CLL antigen while T+ E+) accounted for 14 percent of the 12 patients expressed either the la or the cases and group IV (anti T+ E~) for only 9 CLL antigen but not both (undifferenti­ percent. The T-antigen positive ated leukemia). For both these sub­ E-receptor negative cases (group IV) de­ groups, all cases were E-receptor nega­ serve special comment as Anderson has tive, T-antigen negative, and slg negative. also reported the existence of this sub­ 178 HUMPHREY, BLACKSTOCK AND FILLER

group. Thiel has recently summarized the with the Pediatric Division of the South­ clinical significance of the E- T+ group west Oncology Group, have recently and reported that it may be significant be­ analyzed 238 cases. Cells from 24 of these cause five out of nine patients within this cases were E+ T+ and 11 cases were E- subgroup died soon after diagnosis.55 T +.34 Thus, 15 percent of the total popula­ Yarbro and coworkers have evaluated tion were classified as T-cell leukemia. two unique properties of lymphoblasts One other current direction of research and have related them to T-cell and null needs to be briefly discussed. Serial cell leukem ia.61 The presence of glucocor­ studies of surface characteristics are cur­ ticoid receptors (GCR) in the cytoplasm rently being evaluated in order to look for and the ability of bone marrow lympho­ possible changes in membrane biology at blasts to act as stimulators in a mixed lym­ relapse. In most cases, the same cell phocyte culture (MLC) have been used to phenotypes have been found; however, evaluate cases of ALL. The T-cell cases exceptions have been reported .6,18 T hese (E+) failed to stimulate in the MLC and changes may be due to selective effects of had low levels of GCR. Most of the null therapy or some other malignant advan­ cell cases (18) stimulated in the MLC and tage that allows a small previously unde­ contained high levels of GRC. There was tected subgroup to become dominant or a small group of patients (9) whose cells more easily recognizable. did not stimulate in the MLC and that had intermediate levels of GCR. These find­ Acknowledgment ings may have therapeutic significance Thanks are extended to Leslie Alexander for secre­ based on other observations.48 tarial assistance in preparing this manuscript. Currently the Pediatric Division of R eferences SWOG is evaluating all newly diagnosed 1. Anderson, J. K., M oore, J. O., F alletta, J. cases using institutional laboratories for M., T erry, W. F., and M etzger, R. S.: Acute some of the less complicated method­ lymphoblastic leukemia: Classification and ologies such as E-receptor, complement characterization with antisera to human T-cell and la antigens. J. Nat. Cancer Inst. 62:2 9 3 - receptor and Fc receptor. Specially pre­ 298, 1979. pared bone marrow samples are shipped 2. A n d r e e w a , P., H u h n , D., T h i e l , E., a n d to two research reference laboratories. RoDT, E.: Comparison of enzymecytochemical findings and immunological marker investiga­ The first laboratory, at Duke University, is tions in acute lymphatic leukemia (ALL). Blut measuring surface antigens such as the 36:299-305, 1978. peripheral T lymphocyte antigen (TP), 3. Ba l c h , C. M ., D o u g h e r t y , P. A., Vo g l e r , L. B., Ades, E. W., and F erro n e, S.: A new B-cell thymus antigen (T), B-cell antigen (la), differentiation antigen (BDA) on normal and leukemia lymphoma antigen (L-L), and leukemic human B lymphocytes that is distinct common leukemia antigen (cALL). The from known DR (la-like) antigens. J. Im m u n o l. 121:2322-2328, 1978. second laboratory, at the University of 4. B e l p o m m e , D ., M a t h e , G ., and D a v ie s , A. J. Alabama, is measuring cytoplasmic im­ S.: Clinical significance and prognostic value of munoglobulin (clg), surface immuno­ the T -B immunological classification of human primary acute lymphoid leukemias. Lancet globulin (slg), and Fc receptors for IgM 1:555-558, 1977. (Fc ju,). While this is a very ambitious un­ 5. Be n n e t t , J. M., C a t o v s k y , D ., D a n ie l , M. T., dertaking, the feasibility for such an ap­ F l a n d r in , G ., G a l t o n , D . A. G ., G r a l n ic k , H. R., and Su l t a n , C .: Proposals for the clas­ proach among institutions has already sification of the acute leukaemia. Brit. J. Haem. been proved and multiple subgroups are 33:451-458, 1976. currently being identified .43 6. Bo r e l l a , L., C a s p e r , J. T., and L a u e r , S. J.: Shifts in expression of cell membrane One of these subgroups, pre B-cell phenotypes in childhood lymphoid malignan­ leukemia, has already been discussed.14,57 cies at relapse. Blood 54:64-71, 1979. Doctors Metzger, Falletta and others, 7. B o w m a n , W. P., M e l v in , S. L., R h o m e s , J. A., M a u e r , A. M ., and N ic k s o n , J.: Patterns of cell working independently at Duke Univer­ membrane markers and clinical prognostic fea­ sity Medical Center and in cooperation tures in acute lymphocytic leukemia (ALL) of CELL SURFACE MARKERS IN ACUTE LYMPHOBLASTIC LEUKEMIA 179

childhood. Proc. Amer. Assoc. Cancer Res. teristics of T-cell leukemia and null cell Amer. Soc. Clin. Oncol. 20:156, 1979. leukemia. Proc. Amer. Assoc. Cancer Res.— 8. B o y s e , E. A., O ld , L. T.,and St o c k e r t , E.: The Amer. Soc. Clin. Oncol. 19:220 (#878), 1978. TL (thymus leukemia) antigen. A review. Im- 23. H u m p h r e y , G. B. and F i l l e r , J.: Treatment of munopathology, 4th ed. Grabar, P. and childhood leukemias. Principles of Cancer Miescher, P. A., eds. International Symposium, Treatment. Carter, S. K., Glatstein, E ., and Basel, Schwabc and Co., pp. 23-40. Livingston, R. B., eds. New York, Blakiston 9. B r o u e t , J. C, P r e u d ’h o m m e , J. L., P e n i t , C., Publications, in press. V a l e n s i, F., R o u g e t , P., and Se l ig m a n n , M.: 24. H u m p h r e y , G. B. and L a n k f o r d , ].: Acute Acute lymphoblastic leukemia with pre-B-cell leukemia: The use of surface markers in clas­ characteristics. Blood 54 :269-273, 1979. sification. Seminars Oncol. 3:243-251, 1976. 10. B r o u e t , J. C. and Se l ig m a n n , M.: The im­ 25. Humphrey, G. B., Richie, E., F a lle tta , J. M ., munological classifications of acute lympho­ C r i s t , W. M., Ragab, A. H., and P u l l e n , J. D.: blastic leukemias. Cancer 42:817-827, 1978. The spectrum of the E receptor on bone marrow 11. B r o w n , G ., C a p p il l a r o , D., and G r e a v e s , blast in acute lymphoblastic leukemia (ALL) of M.: Leukemia associated antigens in man. J. childhood. Submitted to Amer. Soc. Hemat. Nat. Cancer Inst. 55:1281-1289, 1975. 1979. 12. C h e c h ik , B. E ., P e r c y , M. E ., a n d G e l f a n d , 26. J a n o s s y , G ., F r a n c is , G. E ., C a p e l l a r o , D. E. W.: Human thymus-leukemia-associated G o l d s t o n e , A. H ., and G r e a v e s , M . F .: Cell antigen: Isolation and partial characterization. sorter analysis of leukemia-associated antigens J. Nat. Cancer Inst. 60:69-75, 1978. on human myeloid precursors. Nature 13. C o l e m a n , M. S., G r e e n w o o d , M. F., H u t ­ 276:176-178, 1978. t o n , J. J., H o l l a n d , P., L a m p k in , B., Kr i l l , C. 27. J a n o s s y , G ., G o l d s t o n e , A. H., C a p e l l a r o , and Ka s i e l i C, J. E.: Adenosine deam inase, D., G r e a v e s , M. F ., Ku l e n k a m p f f , J., P i p - terminal dexynucleotidyl transferase (TdT), PARD, M ., and W e l s h , K.: Differentiation and cell surface markers in childhood acute linked expression of pp. 28, 33 (Ia-like) struc­ leukemia. B lood52:1125-1131, 1978. tures on human leukaemic cells. Brit. J. 14. C r is t , W ., V o g l e r , L., Sa r r if , A., P u l l e n , J., Haemat. 37:391-402, 1977. B a r t o l u c c i, A., F a l l e t t a , J., H u m p h r e y , G. 28. Ka d in , M. E. and B e l l i n g , R. J.: Immuno- B., v a n E y s , J., a n d C o o p e r , M.: Clinical and fluorescent method for positive identification of laboratory characterization of pre-B cell null-cell type acute lymphocytic leukemias: leukemia in children. Submitted to Amer. Soc. Use of heterologous antiserum. Blood 50:771- H e m a t., 1979. 782, 1977. 15. E llis , R. B., Ra p s o n , N. T., P a t r ic k , A.D., and 29. Ka p l a n , J., R avendranath , Y., and P e t e r ­ G r e a v e s , M. F.: Expression of hexosaminidase s o n , W. D.: T and B lymphocyte antigen- isoenzymes in childhood leukemia. New Eng. positive null cell leukemias. Blood49:371-378, J. Med. 298:476-480, 1978. 1977. 16. E s b e r , E . C., M o v a s s a g h i, N., and L e i k in , S. 30. K e r s e y , J. H., G ajl-Peczalska, K. J., C o c c ia , L .: Surface membrane determinants on child­ P. F ., and N esbit, M. E.: The nature of child­ hood acute lymphocytic leukemia cells: Immu­ hood leukemia and lymphoma. Amer. J. Path. noglobulin, Fc and C3 receptors. Clin. E xp. 90:487, 1978. Immunol. 32:523-530, 1978. 31. Kram ers, M. T. C., Catovsky, D., and F o a , R.: 17. E t t in g e r , R., W o o d s , G. W ., T e r r y , W. F., Cell membrane enzymes. II. Alkaline phos­ and F a l l e t t a , J. M.: A new chemotherapeutic phatase and alkaline phosphodiesterase I in approach to T-cell activated lymphocytic normal and leukaemic lymphocytes. Brit. J. leukemia. Clin. Res., (in press). Haemat. 40:111-118, 1978. 18. F u k u n a g a , Y., Ko g o s h i, T., S h i m i z u , H, 32. Ku l e n k a m p f f , J., J a n o s s y , G ., and G r e a v e s , K o g o , T., Ko b a y a s h i, M., Ya m a m o t o , M., and M . F.: Acid esterase in human lymphoid cells U e d a , Y .: A possible change in the surface and leukaemic blasts: A marker for T lympho­ marker of blast cells in childhood acute cytes. Brit. J. Haemat. 36:231, 1977. leukemia. Jap. J. Clin. Hemato. i9:1095-1102. 33. Liso, V.~ T u r s e , A., Sp e c c h ia , G., T r o c c o l i, 19. G r a l n i c k , H. R., G a l t o n , D. A. G., G., L o r ia , M. P., and B o n o m a , L.: Adenosine C a t o v s k y , D., Su l t a n , C ., and B e n n e t t , J. deaminase activity in acute lymphoblastic M.: Classification of acute leukemia. Ann. leukaemia: Cytochemical, immunological and Intern. Med. 87:740-753, 1977. clinical correlations. Scand. J. H aem at. 20. G r e a v e s , M. and Ja n o s s y , G .: Patterns of gene 21:167-175, 1978. expression and the cellular origins of human 34. M cKnight, M. A., F a l l e t t a , J. M., M etzger, leukaemias. Biochim. Biophys Acta 516:193- R., P u l l e n , J., C r i s t , W., H u m p h r e y , G. B., 230, 1978. and v a n E y s, J.: Clinical and laboratory charac­ 21. H u m p h r e y , G. B., C r is t , W., F a l l e t t a , J., teristics of T-antigen positive acute lymphocy­ R i c h i e , E ., Ra g a b , A., and P u l l e n , J.: The tic leukemia in children. Clin. Res., (in press). implication of thermostable and thermolabile 35. M e t z g e r , R. S. and M ohanakumar, T.: T-cell leukemia in randomized trials (potential Serologic studies of the diagnosis and nosology prognostic significance) SWOG study. Proc. of human leukemia. Amer. J. Clin. Path. Amer. Assoc. Cancer Res.—Amer. Soc. Clin. 68:699-705, 1977. Oncol. 20:289 (#1176), 1979. 36. M e t z g e r , R. S. and M o h a n a k u m a r , T.: 22. H u m p h r e y , G. B., F a l l e t t a , J., C r is t , W., Tumor associated antigens of human leukemia and Ra g a b , A.: Laboratory and clinical charac­ cells. Seminars Hemat. i5:139-157, 1978. 180 HUMPHREY, BLACKSTOCK AND FILLER

37. M e t z g a r , R. S., M o h a n a k u m a r , T ., a n d proliferative disease. Clin. Haemat. 6:355-422, BOLOGNESI, D. P.: Relationships between 1977. membrane antigens of human leukemic cells 51. S l a t e r , R. M., P h i l i p , P ., B a d s b e r g , E., and oncogenic RNA virus structural compo­ B e h r e n d t , H ., H a n s e n , N . E., and v a n nents. J. Exp. Med. 143:47-63, 1976. H e e r d e , P.: A 14q+ chromosome in a B-cell 38. M i l l e r , D. R., L e ik in , S., Al b o , V., and H a m ­ acute lymphocytic leukemia and in a leukemic m o n d , D.: Prognostic significance of lympho­ non-endemic Burkitt lymphoma. Internat. J. blast morphology (FAB Classification) in child­ Cancer. 23:639-647, 1979. hood leukemia (ALL). Proc. Amer. Assoc. 52. Sm y t h , J. F., P o p l a c k , D. G.; H o l im a n , G. J., Cancer Res.—Amer. Soc. Clin. Oncol. 20:345, LEVENTHAL, B. G., and Ya r b r o , G.: Correla­ 1979. tion of adenosine deaminase activity with cell 39. M i l l s , B., Se n , L., and B o r e l l a , L.: Reactivity surface markers in acute lymphoblastic of antihuman thymocyte serum with acute leukemia. J. Clin. Invest. 62:710, 1978. leukemic blasts. J. Immunol. iJ5:1038-1044. 53. Su l l iv a n , A. K., J e r r y , L. M., Ro w d e n , G., 40. M in o w a d a , J., Ja n o s s y , G ., G r e a v e s , M . F ., L e w i s , M . G., P i t z e l e , R., L a w , T ., Ad a m s , L. I s u b o t a , T., and Sa h a i Sr iv a s t a v a , B. I.: Ex­ S., T h i , J. L., and Sh e , M.: Fetal antigens on the pression of an antigen associated with acute surface of human lymphoid cells. J. Exp. Med. lymphoblastic leukemia in human leukemia— i 43:1557-1561, 1976. lymphoma cell lines. J. Natl. Cancer Inst. 54. Su t h e r l a n d , R., Sm a r t , T., N ia u d e t , P., and 60:1269-1277, 1978. G r e a v e s , M.: Acute lymphoblastic leukemia 41. M onhanakumar , T. and M e t z g e r , R. S.: associated antigen II. Isolation and partial Human thymus-leukemia related antigen(s): characterization. Leuk. Res. 2:115-126, 1978. Detection by a nonhuman primate antiserum. 55. T h i e l , E., R o d t , H ., N e t z e l , B., H u h n , D ., Cell. Immunol. 12:30-36, 1974. W u n d is c h , G. F., H a a s , R. J., B e n d e r -G o t z e , 42. N e t z e l , R o d t , H., E d k h a r d , T ., Ra in e r , J. C., and T h ie r f e l d e r , S.: T-Zell-antigen pos­ H., and T h ie r f e l d e r , S.: Use of specific anti­ itive, E-Rosetten negative akute lymphoblas- sera against leukemia-associated antigens in tenleukamic. Blut 36:363-369, 1978. the diagnosis of childhoood acute lymphoblas­ 56. T s u b u t a , T ., M in o w a d a , J., N a k a z a w a , S., tic leukemia. Acta Haemat, 61:177-183, 1979. S in k s , L. F., H a n , T ., H ig b y , R. J ., and 43. PULLEN, J.: Personal communication. P r e s s m a n , D .: Correlation of surface markers 44. Re a m a n , G. H ., L e v in , N ., M u c h m o r e , A., of cells of human lymphatic leukemias with H o l im a n , B., andPOPLACK, D. G .: Diminished disease type. J. Nat. Cancer Inst. 59:845-850, lymphoblast 5-nucleotidase activity in acute 1977. lymphoblastic leukemia with T-cell charac­ 57. V o g l e r , L . B., C r is t , W . M., B o c k m a n , D . E., teristics. New Eng. J. M ed . 300:1374-1375, P e a r l , E. R„ La w t o n , A. R., and C o o p e r , M. 1979. D.: Pre-B-cell leukemia a new phenotype of 45. Ro b e r t s , M., G r e a v e s , M., J a n o s s y , G ., childhood lymphoblastic leukemia. New Eng. Su t h e r l a n d , R., and P a in , C.: Acute lym­ J. Med. 298:872-878, 1978. phoblastic leukemia (ALL) associated antigen I 58. W e i n s t e in , H. J., Va n c e , Z. B., J a f f e , N., expression in different haematopoietic malig­ Bu e l l , D ., C a s s a d y , R. J., and N a t h a n , D . G .: nancies. Leuk. Res. 2:105-114. Improved prognosis for patients with mediasti­ 46. Ross, G. D.: Review: Identification of human nal lymphoblastic lymphoma. Blood 53:687, lymphocyte subpopulations by surface marker 1979. analysis. Blood 53:799-811, 1979. 59. W e r n e t , P., B e t s c h , C ., B a r t h , P., 47. Sa l l a n , S. E., C h e s s , L., F r e i, E. Ill, O ’B r ie n , J a r a m il l o , S., Sc h u n t e r , F., W a l l e r , H. D ., C., N a t h a n , D. G., St r o m in g e r , J. L., and and W il m s , K.: Human la alloantigens as cell S c h l o s s m a n , S. F .: Utility of B- and T-cell- differentiation markers of normal and specific antisera in the classification of human pathologic leukocyte surfaces. Scand. J. Im­ leukemias. Differentiation of Normal and munol. 6:563-574, 1977. Neoplastic Hematopoetic Cells. Clarkson, B., et 60. Ya m a n a k a , N., I is h i i , Y., K o s h ib a , H., M ik u n i, al, eds. New York, Cold Spring Harbor Labora­ C., K o n n o , M ., and KlKUCHI, K.: A study of tory Press, 1978, pp. 479-483. surface markers in acute lymphocytic leukemia 48. Sc h m id t , T. J. and T h o m p s o n , E. B.: Glucocor­ by using anti-T and anti-B lymphocyte sera. ticoid receptor function in leukemic cells. En­ Cancer 42:2641-2647, 1978. docrine Control in Neoplasia. Sharma, R. K. and 61. Ya r b r o , G. S. K., L ip p m a n , M . E., J o h n s o n , G. Criss, W. E., eds. Grand Haven, MI, Raven E., and L e v e n t h a l , B. G.: Glucocorticoid re­ Press, 1978, pp. 263-289. ceptors in subpopulations of childhood acute 49. Sh a w , M. T., D w y e r , J. M., A l l a u d e e n , J. S., lymphocytic leukemia. Cancer Res. 37:2688- and W e it z m a n , H. A.: Terminal deoxyribonu- 2695, 1977. cleotidyl transferase activity in B-cell acute 62. Z ig h e l b o im , J., Bick, A., and D u r a n t e z , A.: lymphocytic leukemia. Blood 51:181, 1978. Recognition by human and rabbit sera of com­ 50. SlEGAL , F. P. and G o o d , R. A.: Human lym­ mon antigens to leukemia blast cells, peripheral phocyte diiferentiation markers and their ap­ B-lymphocytes and monocytes. Cancer Res. plication to immune deficiency and lympho- 37:3656-3662, 1977.