Acute myeloid leukemia with 11q23 translocations - Nature

Leukemia (1998) 12, 317–325  1998 Stockton Press All rights reserved 0887-6924/98 $12.00

Acute myeloid leukemia with 11q23 translocations: myelomonocytic immunophenotype by multiparameter flow cytometry ´ MR Baer1, CC Stewart1, D Lawrence1, DC Arthur2,3, K Mrozek4, MP Strout4, FR Davey5, CA Schiffer6,7 and CD Bloomfield4 1

Roswell Park Cancer Institute, Buffalo, NY; 2University of Minnesota, Minneapolis, MN; 5SUNY Health Science Center at Syracuse, Syracuse, NY; and 6University of Maryland Cancer Center, Baltimore, MD, USA

11q23 translocations (t(11q23)) are recurring cytogenetic abnormalities in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia, involving the same gene, ALL1 (or MLL). Mixed lineage antigen expression has been reported in these leukemias, but its frequency and clinical significance are unknown. We immunophenotyped leukemia cells from 19 adult de novo AML patients with t(11q23) by multiparameter flow cytometry. Translocations included t(6;11)(q27;q23), t(9;11) (p22;q23), t(9;11;19)(p22;q23;q13.3), t(2;11)(11;17)(q37;q11q23; q11), t(11;17)(q23;q25), t(11;19)(q23;p13.1), t(11;19)(q23;p13.3) and t(11;22)(q23;q11). FAB types were M4 and M5. The committed stem cell and myeloid antigens HLADr, CD4dim, CD11b, CD13, CD15, CD32, CD33, CD38 and CD64 were each expressed in 80–100% of cases, and the early stem cell and lymphoid antigens CD34, CD56, CD3, CD2 and CD7 in 42, 39, 16, 5 and 5%, respectively. Antigen expression frequencies did not differ from those in 443 adequately karyotyped M4 and M5 cases without t(11q23). Fifteen patients (79%) attained complete remission (CR); median CR duration and survival were 10.0 and 15.1 months. CR duration and survival did not correlate with antigen expression. In particular, patients with t(9;11) survived longer than those with other t(11q23) (median not reached vs 7.6 months; P = 0.048), but antigen expression did not differ in the two groups. Thus frequencies of lymphoid antigen expression are similar in AML with t(11q23) and in other FAB M4 and M5 cases, treatment outcome does not differ in t(11q23) cases with and without lymphoid antigen expression, and better outcome of patients with t(9;11) compared to other t(11q23) does not correlate with differences in antigen expression. Mixed lineage antigen expression is not a distinctive feature of AML with t(11q23). Keywords: acute myeloid leukemia; 11q23 translocations; ALL-1; immunophenotype

Introduction Chromosomal translocations involving band 11q23 are recurring cytogenetic abnormalities in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).1,2 The same gene, variously called ALL1, MLL, HRX or Htrx1, is rearranged in both AML and ALL with 11q23 abnormalities, suggesting a similar molecular pathogenesis regardless of lineage.3–9 Additionally, mixed lineage antigen expression has been reported in acute leukemias with 11q23 involvement, suggesting that these leukemias may arise in pluripotent stem cells with capacity for both myeloid and lymphoid differentiation.10–16 Moreover, pluripotent stem cell involvement might provide an explanation for adverse treatment outcome. Although expression of lymphoid antigens has been reported

in AML with 11q23 abnormalities, the frequency with which these antigens are expressed is not known and the relationship between their expression and treatment outcome has not been defined. We immunophenotyped leukemia cells from 19 adult patients with de novo AML with 11q23 translocations associated with ALL1 gene rearrangements, using multiparameter flow cytometry (MFC).17 We determined the frequency of lymphoid antigen expression in these cases, and correlated lymphoid antigen expression with treatment outcome.

Patients and methods

Patients Nineteen adult patients with de novo AML with 11q23 chromosome translocations enrolled on a Cancer and Leukemia Group B (CALGB) prospective immunophenotyping study of adult AML (CALGB 8361) had pretreatment marrow and/or blood samples submitted to the Laboratory of Flow Cytometry at Roswell Park Cancer Institute (RPCI) for immunophenotyping by MFC over a 4-year period beginning 1 March 1991, when the laboratory became the central immunophenotyping facility for CALGB. All patients were also enrolled on a CALGB prospective karyotyping study (CALGB 8461) and were treated on CALGB treatment protocols (CALGB 8923, 9022 or 9222) (Refs 18, 19 and unpublished, respectively). Induction therapy in all three treatment protocols consisted of standard-dose cytarabine and daunorubicin. Post-remission therapy in CALGB 9222 consisted of either three courses of high-dose cytarabine (HDAC) (3 g/m2) administered as in CALGB 8525,20 or one course each of HDAC, cyclophosphamide and etoposide, and diaziquone and mitoxantrone, administered as in CALGB 9022.19 Four hundred and forty three adequately karyotyped FAB M4 and M5 de novo AML cases without 11q23 translocations were also studied.

Morphologic studies The diagnosis of AML and the assignment of FAB subtypes were based on standard morphological and cytochemical criteria.21 All cases were centrally reviewed.

Cytogenetic analysis Correspondence: MR Baer, Division of Medicine, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263, USA; Fax: 716 845 8446 Current addresses: 3National Cancer Institute, Bethesda, MD, USA; 4 Ohio State University, Columbus, OH, USA; 7Wayne State University, Detroit, MI, USA Received 10 December 1996; accepted 19 November 1997

Bone marrow samples were processed for cytogenetic analysis by standard techniques, using direct and short-term (24–48 h) unstimulated cultures. Chromosomes were G- or Q-banded. A minimum of 15 cells was analyzed in each case. Karyotypes were designated according to the 1995 International System

AML with 11q23 translocations: myelomonocytic immunophenotype MR Baer et al

318

for Human Cytogenetic Nomenclature (ISCN).22 Two karyotypes from each abnormal clone were centrally reviewed.

Results

Cytogenetic and molecular findings ALL1 gene rearrangement studies DNA extracted from cryopreserved leukemia cells by a standard isolation procedure23 was digested with BamHI and HindIII and then size-separated by the method of Southern.24 Southern blots were hybridized with the 859 base pair BamHI fragment (B859) from a complementary DNA subclone spanning exons 5–11 of the ALL1 gene.25

Multiparameter flow cytometry MFC analysis was performed on bone marrow (14 patients) and blood (five patients) samples as previously described.26 The nine three-antibody panels used for immunophenotyping by MFC included fluorescein isothiocyanate (FL)-, phycoerythrin- and either PerCP- or phycoerythrin/Cy5 (PC)-labeled antibodies to CD45, CD14 and HLADr; CD3, CD4 and CD8; CD15, CD34 and CD56; CD33, CD13 and HLADr; CD16, CD32 and CD64; CD7, CD13 and CD2; CD11b, CD13 and CD33; CD38, CD34 and HLADr; and CD33, CD13 and CD19. The antibodies used to study expression of the lymphoid antigens CD2, CD3, CD7, CD19 and CD56 consisted of PC-labeled G11 (Caltag, South San Francisco, CA, USA), FLlabeled SK7 (Becton Dickinson, San Jose, CA, USA), FL-labeled 4H9 (AMAC, Westbrook, ME, USA), PC-labeled J4-119 (AMAC) and PC-labeled NKH1 (Coulter, Hialeah, FL, USA), respectively. Cells were also labeled with identically conjugated isotype controls for each antibody, also in three-antibody panels. Binding of isotype controls was used to create gates defining specific binding.

Statistical methods Complete remission (CR) and relapse were defined according to the criteria of the National Cancer Institute-sponsored workshop on AML.27 CR duration was measured from the date of attainment of CR to the date of relapse; it was censored on the date that patients were last known to be in continued first CR or the date of bone marrow transplantation in first CR. Survival was measured from the date of entry onto treatment protocols to the date of death; it was censored for patients alive at last follow-up. The median follow-up for the four patients whose CR duration was censored was 22 months and that for the six patients whose survival was censored was 27 months. The Kaplan–Meier method was used to determine the distribution of CR duration and survival. Comparisons of continuous or percentage variables were performed with the Mann–Whitney test. Discrete variables were compared between antigen expression groups defined by expression vs lack of expression of any lymphoid antigen or of CD56 or of CD34 with the Fisher exact test. The logrank test was used to analyze differences in distributions for CR duration and survival between antigen expression groups again defined by expression or lack of expression of any lymphoid antigen or of CD56 or of CD34, except when the Kaplan–Meier curves crossed, in which the case the modified Kolmogorov–Smirnov test was used.28 The level of significance was 0.05 for all analyses, with all tests being two-sided.

The 19 adult de novo AML patients in our series had eight different reciprocal translocations involving 11q23, including t(9;11)(p22;q23) in 10 patients and the variant t(9;11;19) (p22;q23;q13.3) in one, t(6;11)(q27;q23) in two patients, t(11;19)(q23;p13.3) in two patients, and t(2;11)(11;17) (q37;q11q23;q11), t(11;17)(q23;q25), t(11;19)(q23;p13.1) and t(11;22)(q23;q11) in one patient each (Table 1). Translocations involving 11q23 were the sole chromosomal change in at least one clone in 12 patients, one of whom also had a related clone with an additional chromosomal abnormality. In the other seven patients, all karyotypically abnormal cells had at least one other chromosomal change in addition to the 11q23 translocation. The additional chromosomal abnormalities included +8 in three patients, and −Y, i(8)(q10), del(13)(q13q22), +19 and +8,+8,+14 in one patient each. None of these additional chromosomal changes were recognized primary abnormalities with favorable or unfavorable prognostic significance. Nine patients had DNA available for analysis, including four with t(9;11)(p22;q23), one with t(11;19)(q23;p13.3), and the patients with t(2;11)(11;17) (q37;q11q23; q11), t(11;17)(q23;q25), t(11;19)(q23;p13.1) and t(11;22)(q23;q11). ALL1 gene rearrangements were demonstrated by Southern blot analysis in all nine (Table 1).

Immunophenotype Immunophenotypes determined by MFC are shown in Table 1, with antigens displayed in decreasing order of frequency of expression. A consistent myeloid immunophenotype was found by MFC. The committed stem cell/early myeloid antigens CD38, CD4dim and HLADr,29,30 the myeloid antigens CD15, CD13, CD33 and CD11b and the granulocytic and monocytic Fc receptor antigens CD32 and CD64 were each expressed in 80–100% of cases. Thus, the prevalent pattern of myeloid antigen expression was HLADr+CD4dim+ CD11b+CD13+CD15+CD32+CD33+CD38+CD64+. Additional myeloid antigens expressed included the monocytic antigen CD14, present in five cases, and the granulocytic/NK cell Fc receptor antigen CD16, present in three cases, all of which also expressed CD14. In contrast to the consistent pattern of committed stem cell and myeloid antigen expression, expression of early stem cell and lymphoid antigens was variable (Table 1). The early stem cell antigen CD34 was expressed in eight cases (42%), the NK lineage antigen CD56 in seven (39%), the T cell antigen CD3 in three (16%), and the T cell antigen CD2 and T lineage antigen CD7 in one case each (5%). The B cell antigen CD19 was not expressed in any of 15 cases in which it was studied. CD34 was expressed in five of the nine cases in which at least one lymphoid antigen was expressed; CD2, CD7 and/or CD56 were expressed in four additional cases without CD34 expression. Lymphoid antigens were expressed in five of 11 cases with t(9;11) or the variant t(9;11;19) and in all three cases with 19p breakpoints, but were not expressed in either of the two t(6;11) cases (Table 1). There were no associations between CD34 expression and specific translocations.

ND ND R R R R ND ND ND ND ND ND ND R R R R ND R

ALL1 gene

t(6;11)(q27;q23) t(6;11)(q27;q23) t(9;11)(p22;q23) t(9;11)(p22;q23) t(9;11)(p22;q23) t(9;11)(p22;q23) t(9;11)(p22;q23) t(9;11)(p22;q23) t(9;11)(p22;q23) t(9;11)(p22;q23) t(9;11)(p22;q23) t(9;11)(p22;q23) t(9;11;19)(p22;q23;q13.3) t(2;11)(11;17)(q37;q11q23;q11) t(11;17)(q23;q25) t(11;19)(q23;p13.1) t(11;19)(q23;p13.3) t(11;19)(q23;p13.3) t(11;22)(q23;q11)

Translocation M4 M4 M5A M5B M5B M5A M5A M4 M4 M5A M5B M5B M5A M5A M5A M4 M5B M5A M4

+ + + + + + + + + + + + + + + + + + ND

+ + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + +

+ + + + + + + + − + + + + + + + + + +

+ + + + + + + − − + + + + + + + + + +

+ + + + + + + − − + + + + + + + + + +

+ + + + + + − + + + + + + + − + + + +

+ + + + + + − + + + + + + + + + + − +

+ − + + + + − + − + + + + + + + + − +

+ + − − − − − + − − + + − + − − + + −

− − − − + + ND − − + + − − + − − + + −

− − − + + − − − − − + + − − + − − − −

− − − − − − − − − − + + − − + − − − −

− − − − − − − − − − + + − − − − + − −

− − − − − − − − − + − − − − − − − − −

− − − − − − − − − − − − − − − + − − −

− ND ND − − − − ND ND − − − − − − − − − −

FAB type CD38 CD4dim CD15 CD32 CD64 HLADr CD33 CD13 CD11b CD34 CD56 CD14 CD16 CD3 CD2 CD7 CD19

Immunophenotypes determined by MFC in cases of AML with 11q23 chromosome translocations

a Also immunophenotyped at relapse. R, rearranged; ND, not determined.

1 2a 3 4 5a 6 7 8a 9 10 11 12 13 14a 15a 16 17a 18 19a

Patient

Table 1

AML with 11q23 translocations: myelomonocytic immunophenotype MR Baer et al

319

AML with 11q23 translocations: myelomonocytic immunophenotype MR Baer et al

320

Correlation of immunophenotype with FAB type FAB types included M4 in six patients, M5A in eight and M5B in five (Table 1). The HLADr+CD4dim+CD11b+CD13+CD15+ CD32+CD33+CD38+CD64+ immunophenotype was a consistent finding irrespective of FAB type. CD34 was expressed in M4, M5A and M5B AML (three, two and three cases, respectively), but CD56, CD14 and CD16 were only expressed in M5A and M5B cases, CD2 only in M5A and CD3 only in M5B. Expression of CD56 was significantly more frequent in M5 compared to M4 cases (58 vs 0%; P = 0.04).

were no differences in antigen expression between FAB M5 cases with and without 11q23 translocations (Table 3). Thus the HLADr+CD4dim+CD11b+CD13+CD15+CD32+CD33+ CD64+ immunophenotype was a common finding in FAB M4 and M5 AML and was not specific for 11q23 translocation cases. Similarly, expression of CD56 and of CD3 on the surface of leukemia cells was found in FAB M5 AML without 11q23 translocations; it was not specific for 11q23 translocation cases.

Immunophenotypes at relapse Frequencies of antigen-positive leukemia cells Percentages of leukemia cells expressing each antigen in antigen-positive cases are shown in Table 2, with antigens again shown in decreasing order of frequency of expression. The antigens which were expressed in a majority of 11q23 translocation cases, including CD38, CD4dim, CD15, CD32, CD64, HLADr, CD33, CD13 and CD11b, were generally present on most of the leukemia cells. In contrast, the less frequently expressed antigens, including CD34, CD56 and CD3, were often expressed on only a minority of cells.

Comparison of immunophenotypes in AML with and without 11q23 translocations Antigen expression in the 19 AML cases with 11q23 translocations was compared to that in 443 adequately karyotyped CALGB cases without 11q23 translocations immunophenotyped during the same time period (Table 3). The HLADr+ CD4dim+CD11b+CD13+CD15+CD32+CD33+CD64+ immunophenotype was significantly more common in cases with 11q23 translocations than in those without (P ⬍ 0.001), as was expression of CD3 (P = 0.002) and of CD56 (P = 0.004). However, only CD56 expression differed in frequency between the 11q23 translocation cases, all of which were FAB M4 and M5, and the 89 adequately karyotyped FAB M4 and M5 cases without 11q23 translocations (P = 0.045), and there Table 2 Frequencies of antigen-positive (antigen+) leukemia cells in antigen+ cases

Antigen

No. antigen+ cases

CD38 CD4dim CD15 CD32 CD64 HLADr CD33 CD13 CD11b CD34 CD56 CD14 CD16 CD3 CD2 CD7

18a 19 19 18 17 17 17 17 15 8 7* 5 3 3 1 1

a

18 cases studied.

Percentage of antigen+ cells in antigen+ cases: median (range) 89 89 74 88 64 85 77 64 72 44 38 48 19 22

(43–99) (22–99) (20–99) (14–100) (21–96) (47–100) (12–90) (26–99) (11–98) (17–100) (11–94) (17–56) (11–32) (18–40) 48 18

Bone marrow samples from seven patients with 11q23 translocations were also immunophenotyped by MFC at first relapse, and relapse and diagnosis immunophenotypes were compared (Table 4). Myeloid antigen expression changed little at relapse compared to diagnosis. The HLADr+CD4dim+ CD11b+CD13+CD15+CD32+CD33+CD64+ immunophenotype remained present at relapse in all four cases in which it had been present at diagnosis (patients 5, 14, 17 and 19). In contrast, expression of lymphoid antigens changed at relapse in five of the seven patients. CD56 was gained in one patient (patient 15) and was lost in two patients (patients 5 and 14), one of whom also gained CD2. CD2 was acquired in two additional patients (patients 8 and 17), one of whom also gained CD7, and the other of whom also lost CD3. There were no changes in CD34 expression at relapse.

Clinical and laboratory features Clinical presentations are summarized in Table 5. All 19 patients had de novo AML. Age range was 17–63 years, with a median of 38 years. Median pre-treatment white blood cell count was 4.3 × 109/l (range 0.6–111.0 × 109/l). Median percent marrow blasts was 89 (range 41–99). Extramedullary infiltration is also shown in Table 5. The only striking finding was the presence of gingival hypertrophy in seven patients. There was no correlation between age, white blood cell count, percent marrow blasts or gingival hypertrophy and lymphoid or early stem cell antigen expression (Table 6). Therapy and clinical course are also summarized in Table 5. Remission induction therapy consisted of standard-dose cytarabine and daunorubicin18,19 in all 19 patients. Fifteen patients achieved CR (CR rate 79%). The four patients who did not achieve CR included three who died during induction therapy and only one (patient 16) who survived induction therapy and failed to achieve CR due to resistant disease. Of note, patient 16 was also the only patient whose leukemia cells expressed CD7 (Table 1). All but one (patient 11) of the 15 patients who achieved CR received intensive post-CR therapy (Table 5). Intensification regimens included HDAC in five patients, and HDAC, cyclophosphamide and etoposide, and diaziquone and mitoxantrone in eight patients. One patient underwent allogeneic bone marrow transplantation (alloBMT) in first CR. To date, 11 of the 15 patients have relapsed, with CR durations ranging between 1.3 and 26.5 months. The patient who underwent alloBMT in first CR (patient 10 in Table 4) remains in first CR at 14.6 months follow-up. Median CR duration for all patients who achieved CR is 10.0 months, and median survival for all patients is 15.1 months (Figure 1). CR duration and survival did not correlate with patient age, white blood cell counts or presence of gingival hypertrophy,

HLADr+CD4dim+CD11b+CD13+CD15+CD32+CD33+CD64+ CD34+ CD56+ CD14+ CD16+ CD3+ CD2+ CD7+

Immunophenotype

68 42 39 26 16 16 5 5

11q23+ (n = 18–19) 20 58 11 17 5 1 11 18

11q23− (n = 405–443)

All cases (%) 11q23+ (n = 18–19) 68 42 39 26 16 16 5 5

P

⬍0.001 0.24 0.004 0.36 0.07 0.002 0.71 0.22 53 38 16 46 12 4 12 13

11q23− (n = 83–89)

FAB M4 and M5

0.31 0.80 0.045 0.13 0.71 0.10 0.69 0.46

P

77 38 58 38 23 23 8 0

11q23+ (n = 12–13)

52 30 38 63 26 7 11 7

11q23− (n = 26–27)

FAB M5

0.18 0.72 0.31 0.19 1.00 0.31 1.00 1.00

P

Table 3 Frequency of the prevalent myeloid immunophenotype and of stem cell and lymphoid antigen expression in adequately karyotyped CALGB AML cases with (11q23+) and without (11q23−) 11q23 translocations

AML with 11q23 translocations: myelomonocytic immunophenotype MR Baer et al

321

AML with 11q23 translocations: myelomonocytic immunophenotype MR Baer et al

322

Table 4

Patient 2 5 8 14 15 17 19

Immunophenotype changes at relapse

not relapsed to date (one of whom underwent alloBMT in first CR) all had AML with t(9;11), but the other five patients with this translocation have relapsed. The incidence of lymphoid antigen expression was similar in cases with t(9;11) and with other 11q23 translocations (45 and 50%; P = 1.0).

Immunophenotype changes at relapse Lost CD32 and CD64 Lost CD14 and CD56 Gained HLADr, CD2 and CD7 Lost CD56. Gained CD2 Lost CD14 and CD16 Lost CD3. Gained CD2 None

Discussion We have demonstrated a consistent myeloid immunophenotype in adult AML with 11q23 translocations associated with ALL1 gene rearrangements, with expression of the committed stem cell and myeloid antigens HLADr, CD4dim, CD11b, CD13, CD15, CD32, CD33, CD38 and CD64 in almost all cases. In contrast, expression of lymphoid antigens and of CD34 was variable, and these antigens, when expressed, were generally present on only a minority of leukemia cells. Moreover, 11q23 translocation cases were all FAB M4 or M5, and expression of myeloid, stem cell and lymphoid antigens in

nor did they correlate with expression of any lymphoid antigen, CD56 or CD34 (Table 6). Outcome was better for patients with t(9;11) compared to those with other 11q23 translocations, with significantly longer survival (median not reached vs 7.6 months; P = 0.048). The four patients who have Table 5

Patient

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Clinical data

Reciprocal breakpoint

Age/ Sex

WBC

% BM blasts

6q27 6q27 9p22 9p22 9p22 9p22 9p22 9p22 9p22 9p22 9p22 9p22 9p22 17q11 17q25 19p13.1 19p13.3 19p13.3 22q11

49M 32F 22F 47M 46M 20M 39M 17F 38F 46M 63F 53F 57F 38F 19M 20F 27M 34/F 36M

6.0 16.0 2.6 71.3 111.0 1.1 1.3 25.2 87.2 1.4 1.1 4.3 1.3 10.6 67.0 97.5 3.6 2.6 0.6

99 89 88 95 96 90 63 82 89 90 41 95 89 90 82 77 88 96 58

CRa

EMI

— G — G,L,H,Spl G,L H — — G H H — — Sk,L G G,L — G —

D Y Y D Y Y Y Y Y Y Y Y D Y Y N Y Y Y

Post-CR therapy

— HDAC, VP/Cy, Mitox/AZQ HDAC, VP/Cy, Mitox/AZQ — HDAC, VP/Cy, Mitox/AZQ HDAC × 3 HDAC, VP/Cy, Mitox/AZQ HDAC, VP/Cy, Mitox/AQQ HDAC × 1 alloBMT (AraC 100 mg/m2 × 5 d) × 2 HDAC, VP/Cy, Mitox/AZQ — HDAC × 3 HDAC × 1 — HDAC × 1 HDAC, VP/Cy, Mitox/AZQ HDAC, VP/Cy, Mitox/AZQ

CRD (months)

Survival (months)

— 5.3 40.4+ — 12.5 4.4 22.7+ 56 2.0 14.6+b 26.5 20.3+ — 12.5 1.3 — 2.2 10.0 8.9

0.4 7.7 41.4+ 0.3 32.6+ 5.7 23.4+ 16.4 14.2 15.6+ 31.3+ 21.5+ 1.3 26.2 4.4 7.5 7.6 15.1 20.8

WBC, white blood cell count (× 109/l); EMI, extramedullary infiltration; G, gingivae; H, liver; L, lymph nodes; Sk, skin; Spl, spleen; CR, complete remission; Y, yes; N, no; D, died; CRD, CR duration; AraC, cytarabine; HDAC, high-dose cytarabine; Ida, idarubicin; Dnr, daunorubicin; VP, etoposide; Cy, cyclophosphamide; Mitox, mitoxantrone; AZQ, diazoquinine; allo, allogeneic; auto, autologous; BMT, bone marrow transplantation. a All patients received cytarabine and daunorubicin remission induction therapy. b CRD censored for life table analysis at time of alloBMT in first CR.

Table 6 Comparisons of pretreatment characteristics and treatment outcome in cases with (LAG+) and without (LAG−) expression of lymphoid antigens, CD56 and CD34

Median age (years) Median WBC (×109/l) Median marrow blasts (%) Gingival hypertrophy (%) CR rate (%) CR duration (months) Survival (months)

LAG+ (n = 9)

LAG− (n = 10)

P

CD56+ (n = 7)

CD56− (n = 11)

P

CD34+ (n = 8)

CD34− (n = 11)

P

38 3.6 90 33 89 12.5 26.2

37.0 11.0 88.5 40 70 5.6 10.9

0.74 0.81 0.35 1.00 0.63 0.81 0.21

38 2.6 90 29 100 11.2 26.2

36 16.0 89 45 64 5.6 7.7

0.72 0.26 0.39 0.64 0.11 0.75 0.18

36 5.2 89.5 25 88 10 15.8

38 2.6 89 45 73 8.9 14.2

0.74 0.97 0.48 0.63 0.60 0.96 0.95

AML with 11q23 translocations: myelomonocytic immunophenotype MR Baer et al

Figure 1 CR duration and survival for AML patients with 11q23 translocations.

AML cases with 11q23 translocations did not differ from their expression in FAB M4 and M5 cases without 11q23 translocations. These data refute the frequently repeated statement that leukemias with 11q23 translocations are mixed lineage leukemias, with the implication that they differ in this regard from other acute leukemias. The 11q23 translocations in the cases of AML included in this report involved seven different partner chromosome breakpoint regions. ALL1 gene rearrangements were demonstrated in cases of AML with t(9;11)(p22;q23), t(2;11) (11;17)(q37;q11q23;q11), t(11;17)(q23;q25), t(11;19)(q23; p13.1) and t(11;19)(q23;p13.3), consistent with the previously reported findings of other laboratories.3–9 We also demonstrated rearrangement of ALL1 in a case of AML with t(11;22)(q23;q11). This translocation has been previously shown to involve ALL1 in only one report.31 It has also been shown not to involve bcr on chromosome 22.31,32 A consistent myeloid immunophenotype, HLADr+CD4dim+ CD11b+CD13+CD15+CD32+CD33+CD38+CD64+, was found in our cases of AML with reciprocal translocations of 11q23 associated with ALL1 gene rearrangements, without regard to partner chromosome breakpoint region. Moreover, additional expression of CD34 and of lymphoid antigens also generally did not correlate with partner chromosome breakpoint region. Lack of differences in immunophenotype based on partner chromosome breakpoint region is consistent with the hypothesis that leukemogenesis in AML with 11q23 translocations results from expression of a truncated ALL1 gene, creating a truncated protein consisting of the amino terminus of ALL1, rather than from formation of a fusion protein.33 The HLADr+CD4dim+CD11b+CD13+CD15+CD32+CD33+ CD38+CD64+ immunophenotype was a consistent finding in cases of AML with 11q23 translocations associated with ALL1 rearrangements, but was not specific for these cases, in that the immunophenotype identified in AML with 11q23 translocations did not distinguish them from FAB M4 and M5 cases without 11q23 involvement. AML with 11q23 translocations thus differs from AML with several other relatively common recurring structural cytogenetic abnormalities, including t(8;21)(q22;q22), inv(16)(p13q22) and t(15;17)(q22;q11-12), which have been reported to be associated with specific immunophenotypes.34–39 In addition, the immunophenotype data did not support the hypothesis that acute leukemias with 11q23 translocations arise in early stem cells and undergo biphenotypic differentiation, because neither CD34 nor lymphoid antigens were expressed in seven of 19 cases (37%), and expression of CD34 and of lymphoid antigens was not

more common in 11q23 translocations cases than in M4/M5 cases without 11q23 involvement. Finally, neither myeloid, lymphoid nor early stem cell antigen expression appeared to have prognostic significance in cases of AML with 11q23 translocations. Treatment outcome was generally poor in our de novo AML patients with 11q23 translocations associated with ALL1 rearrangements, with relatively few disease-free survivors. Our series consisted of a relatively young group of adult AML patients who were all treated since 1991 with standard-dose cytarabine and anthracycline remission induction therapy followed by intensive post-remission chemotherapy or, in one case, allogeneic bone marrow transplantation in first CR. The CR rate was 79%, with only one remission induction failure due to resistant disease. Relapses have occurred to date in 10 of 13 patients less than 60 years of age who achieved CR and received intensive post-CR chemotherapy, including all of the patients with translocations other than t(9;11). These results may be less favorable than those in similarly treated AML patients less than 60 years of age, in whom disease-free survivals of 30–45% have been reported,19,20 but multivariate analyses are needed before definitive conclusions can be drawn about the treatment outcome of patients with AML with 11q23 translocations compared to AML patients less than 60 years of age without this cytogenetic abnormality. A single patient, with AML with t(9;11), underwent allogeneic bone marrow transplantation in first CR. He remains in first CR at 14.6 months. Experience with transplantation in first CR in AML patients with 11q23 translocations is very limited at the present time. Longer follow-up and analysis of results in additional patients are needed before definitive recommendations can be made with respect to transplantation. Previous reports of the prognostic significance of 11q23 abnormalities in AML have been inconsistent.40–48 Remission rates have ranged between 33 and 83% in different series.41–48 CR duration and survival have been reported as poor in most series, but as intermediate or favorable in some.40–48 Likely reasons for this inconsistency are analysis of small numbers of patients, inclusion of both children and adults, and lack of uniformity of therapeutic approaches, with many patients receiving less intensive regimens than those in current use. Moreover, previous reports have generally included patients with translocations, deletions and inversions with 11q23 breakpoints, and ALL1 gene rearrangement studies have not been performed. ALL1 appears to be involved in all reciprocal translocations involving 11q23 in AML other than the t(11;17)(q23;q21) seen in FAB M3 disease, but is not involved in all chromosome 11 deletions with 11q23 breakpoints.8,9,49–51 Ours is the first series restricted to patients with 11q23 translocations known to involve ALL1. CR durations were relatively brief in our series, but there are patients who have not relapsed to date, all of whom had the t(9;11). Results of recent pediatric series suggest that children with de novo AML with the t(9;11) have a favorable outcome when treated with etoposide-containing regimens, with a high CR rate and prolonged disease-free survival,52–54 whereas children with other 11q23 abnormalities do poorly.52,54 A superior treatment outcome for adult AML patients with t(9;11) compared to those with other 11q23 translocations has recently been reported by CALGB.55 All of the disease-free survivors in our series were patients with t(9;11). Of note, however, more than half of our patients with t(9;11) have relapsed. Treatment outcome in 11q23 translocation cases did not correlate with immunophenotype. The combination of high

323

AML with 11q23 translocations: myelomonocytic immunophenotype MR Baer et al

324

CR rate and short CR duration might be explained by initially drug-sensitive disease but rapid regrowth of residual disease. Variables such as drug resistance patterns and cell kinetic parameters need to be studied to explain the predominant pattern of treatment response of AML with 11q23 translocations associated with ALL1 rearrangements as well as the exceptions.

Acknowledgements This research for CALGB 8361 and 8461 was supported, in part, by grants from the National Cancer Institute (CA31946) to the Cancer and Leukemia Group B (Richard L Schilsky, Chairman), National Institutes of Health grant CA37027, the Coleman Leukemia Research Fund and by an award from the Lady Tata Memorial Trust. The authors thank Mary Beth Dell for technical assistance and Sally Palmerton, Erin Trikha and Linda Regal for assistance in data management.

13 14

15

16

17 18

References 1 Heim S, Mitelman F. Cancer Cytogenetics 2nd edn. Wiley-Liss: New York, 1995. 2 Mitelman F, Kaneko Y, Berger R. Report of the committee on chromosome changes in neoplasia. In: Cuticchia AJ, Pearson PL (eds). Human Gene Mapping 1993. A Compendium. Johns Hopkins University Press: Baltimore, 1994, pp 773–812. 3 Cimino G, Moir DT, Canaani O, Williams K, Crist WM, Katzav S, Cannizzaro L, Lange B, Nowell PC, Croce CM, Canaani E. Cloning of ALL-1, the locus involved in leukemias with the t(4;11)(q21;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13) chromosome translocations. Cancer Res 1991; 51: 6712–6714. 4 Ziemin-van der Poel S, McCabe NR, Gill HJ, Espinosa R III, Patel Y, Harden A, Rubinelli A, Smith SD, Le Beau MM, Rowley JD, Diaz MO. Identification of a gene, MLL, that spans the breakpoint in 11q23 translocations associated with human leukemias. Proc Natl Acad Sci USA 1991; 88: 10735–10739. 5 Tkachuk DC, Kohler S, Cleary ML. Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias. Cell 1992; 71: 691–700. 6 Djabali M, Selleri L, Parry P, Bower M, Young BD, Evans GA. A trithorax-like gene is interrupted by chromosome 11q23 translocations in acute leukaemias. Nat Genet 1992; 2: 113–118. 7 Cimino G, Nakamura T, Gu Y, Canaani O, Prasad R, Crist WM, Carroll AJ, Baer M, Bloomfield CD, Nowell PC, Croce CM, Canaani E. An altered 11-kilobase transcript in leukemic cell lines with the t(4;11)(q21;q23) chromosome translocation. Cancer Res 1992; 52: 3811–3813. 8 Corral J, Forster A, Thompson S, Lampert F, Kaneko Y, Slater R, Kroes WG, van der Schoot CE, Ludwig W-D, Karpas A, Pocock C, Cotter F, Rabbits TH. Acute leukemias of different lineages have similar MLL gene fusions encoding related chimeric proteins resulting from chromosomal translocation. Proc Natl Acad Sci USA 1993; 90: 8538–8542. 9 Thirman MJ, Gill HJ, Burnett RC, Mbangkollo D, McCabe NR, Kobayashi H, Ziemin-van der Poel S, Kaneko Y, Morgan R, Sandberg AA, Chaganti RSK, Larson RA, Le Beau MM, Diaz MO, Rowley JD. Rearrangement of the MLL gene in acute lymphoblastic and acute myeloid leukemias with 11q23 chromosomal translocations. New Engl J Med 1993; 329: 909–914. 10 Parkin JL, Arthur DC, Abramson CS, McKenna RW, Kersey JH, Heideman RL, Brunning RD. Acute leukemia associated with the t(4;11) chromosome rearrangement: ultrastructural and immunologic characteristics. Blood 1982; 60: 1321–1331. 11 Kaneko Y, Maseki N, Takasaki N, Sakurai M, Hayashi Y, Nakazawa S, Mori T, Sakurai M, Takeda T, Shikano T, Hiyoshi Y. Clinical and hematologic characteristics in acute leukemia with 11q23 translocations. Blood 1986; 67: 484–491. 12 Ludwig W-D, Bartram CR, Ritter J, Raghavachar A, Hiddeman W, Heil G, Harbott J, Seibt-Jung H, Teichmann JV, Riehm H. Ambigu-

19

20

21

22 23 24 25

26

27

28 29 30

ous phenotypes and genotypes in 16 children with acute leukemia as characterized by multiparameter analysis. Blood 1988; 71: 1518–1528. Hayashi Y, Sugita K, Nakazawa S, Abe T, Kojima S, Inaba T, Hanada R, Yamamoto K. Karyotypic patterns in acute mixed lineage leukemia. Leukemia 1990; 4: 121–126. Pui C-H, Frankel LS, Carroll AJ, Raimondi SC, Shuster JJ, Head DR, Crist WM, Land VJ, Pullen DJ, Steuber CP, Behm FG, Borowitz MJ. Clinical characteristics and treatment outcome of childhood acute lymphoblastic leukemia with the t(4;11)(q21;q23): A collaborative study of 40 cases. Blood 1991; 77: 440–447. Pui C-H, Raimondi SC, Head DR, Schell MJ, Rivera GK, Mirro J Jr, Crist WM, Behm FG. Characterization of childhood acute leukemia with multiple myeloid and lymphoid markers at diagnosis and at relapse. Blood 1991; 78: 1327–1337. Cuneo A, Michaux J-L, Ferrant A, Van Hove L, Bosly A, Stul M, Dal Cin P, Vandenberghe E, Cassiman J-J, Negrini M, Piva N, Castoldi G, Van Den Berghe H. Correlation of cytogenetic patterns and clinicobiological features in adult acute myeloid leukemia expressing lymphoid markers. Blood 1992; 79: 720–727. Stewart CC. Clinical applications of flow cytometry: immunologic methods for measuring cell membrane and cytoplasmic antigens. Cancer 1992; 69 (Suppl. 1): 1543–1552. Stone RM, Berg DT, George SL, Dodge RK, Paciucci PA, Schulman P, Lee EJ, Moore JO, Powell BL, Schiffer CA. Granulocyte–macrophage colony-stimulating factor after initial chemotherapy for elderly patients with primary acute myelogenous leukemia. New Engl J Med 1995; 332: 1671–1677. Moore JO, Dodge RK, Amrein PC, Kolitz J, Lee EJ, Powell B, Godfrey S, Robert F, Schiffer CA. Granulocyte-colony stimulating factor (Filgrastim) accelerates granulocyte recovery after intensive postremission chemotherapy for acute myeloid leukemia with aziridinyl benzoquinone and mitoxantrone: Cancer and Leukemia Group B Study 9022. Blood 1997; 89: 780–788. Mayer RJ, Davis RB, Schiffer CA, Berg DT, Powell BL, Schulman P, Omura GA, Moore JO, McIntyre OR, Frei E. Intensive postremission chemotherapy in adults with acute myeloid leukemia. New Engl J Med 1994; 331: 896–903. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C. Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French– American–British Cooperative Group. Ann Intern Med 1985; 103: 626–629. ISCN (1995). Mitelman F (ed). An International System for Human Cytogenetic Nomenclature. S Karger: Basel, 1995. Gustincich S, Manfioletti G, Del Sal G, Schneider C, Carninci P. A fast method for high-quality genomic DNA extraction from whole human blood. BioTechniques 1991; 11: 298–302. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975; 98: 503–517. ´ Caligiuri MA, Strout MP, Schichman SA, Mrozek K, Arthur DC, Herzig GP, Baer MR, Schiffer CA, Heinonen K, Knuutila S, Nousiainen T, Ruutu T, Block AW, Schulman P, Pedersen-Bjergaard J, Croce CM, Bloomfield CD. Partial tandem duplication of ALL1 as a recurrent molecular defect in acute myeloid leukemia with trisomy 11. Cancer Res 1996; 56: 1418–1425. Baer MR, Stewart CC, Lawrence D, Arthur DC, Byrd JC, Davey FR, Schiffer CA, Bloomfield CD. Expression of the neural cell adhesion molecule CD56 is associated with short remission duration and survival in acute myeloid leukemia with t(8;21)(q22;q22). Blood 1997; 90: 1643–1648. Cheson BD, Cassileth PA, Head DR, Schiffer CA, Bennett JM, Bloomfield CD, Brunning R, Gale RP, Grever MR, Keating MJ, Swatisky A, Stass S, Weinstein H, Woods WG. Report of the National Cancer Institute-sponsored workshop on definitions of diagnosis and response in acute myeloid leukemia. J Clin Oncol 1990; 8: 813–819. Fleming TR, O’Fallon JR, O’Brien PC. Modified Kolmogorov– Smirnov test procedures with application to arbitrarily right-censored data. Biometrics 1980; 36: 607–625. Louache F, Debili N, Marandin A, Coulombel L, Vainchenker W. Expression of CD4 by human hematopoietic progenitors. Blood 1994; 84: 3344–3355. ¨ Terstappen LWMM, Safford M, Unterhalt M, Konemann S, Zur-

AML with 11q23 translocations: myelomonocytic immunophenotype MR Baer et al

31

32 33 34

35

36

37

38

39

40

41 42

43

44

45 46

¨ lutter K, Piechotka K, Drescher M, Aul C, Buchner T, Hiddemann ¨ W, Wormann B. Flow cytometric characterization of acute myeloid leukemia: IV. Comparison to the differentiation pathway of normal hematopoietic progenitor cells. Leukemia 1992; 6: 993–1000. Marukawa O, Akao Y, Inazawa J, Ariyama T, Abe T, Naoe T, Tanimoto M, Saito H, Otsuki Y, Tsujimoto Y. Molecular cloning of the breakpoint of t(11;22)(q23;q11) chromosome translocation in an adult acute myelomonocytic leukaemia. Br J Haematol 1996; 92: 687–691. Kobayashi H, Miyachi H, Ogawa T, Jimbo M. Translocation t(11;22) (q23;q11) in an adult with acute monoblastic leukemia. Jpn J Med 1990; 29: 527–532. Bernard OA, Berger R. Molecular basis of 11q23 rearrangements in hematopoietic malignant proliferations. Genes Chromosom Cancer 1995; 13: 75–85. Kita K, Nakase K, Miwa H, Masuya M, Nishii K, Morita N, Takakura N, Otsuji A, Shirakawa S, Ueda T, Nasu K, Kyo T, Dohy H, Kamada N. Phenotypical characteristics of acute myelocytic leukemia associated with the t(8;21)(q22;q22) chromosomal abnormality: frequent expression of immature B-cell antigen CD19 together with stem cell antigen CD34. Blood 1992; 80: 470–477. Hurwitz CA, Raimondi SC, Head D, Krance R, Mirro J, Kalwinsky DK, Ayers GD, Behm FG. Distinctive immunophenotypic features of t(8;21)(q22;q22) acute myeloblastic leukemia in children. Blood 1992; 80: 3182–3188. Adriaansen HJ, te Boekhorst PAW, Hagemeijer AM, van der Schoot CE, Delwel HR, van Dongen JJM. Acute myeloid leukemia M4 with bone marrow eosinophilia (M4Eo) and inv(16)(p13q22) exhibits a specific immunophenotype with CD2 expression. Blood 1993; 81: 3043–3051. De Rossi G, Avvisati G, Coluzzi S, Fenu S, LoCoco F, Lopez M, Nanni M, Pasqualetti D, Mandelli F. Immunological definition of acute promyelocytic leukaemia (FAB M3): a study of 39 cases. Eur J Hematol 1990; 45: 168–171. Paietta E, Andersen J, Gallagher R, Bennett J, Yunis J, Cassileth P, Rowe J, Wiernik PH. The immunophenotype of acute promyelocytic leukemia (APL): an ECOG study. Leukemia 1994; 8: 1108–1112. Frankel SR, Li K, Baer MR, Stewart CC, Davey F, Regal L, Lawrence D, Schiffer CA, Bloomfield CD. Multiparameter immunophenotype of acute promyelocytic leukemia (APL). Blood 1994; 84: 309a (Abstr.). Fourth International Workshop on Chromosomes in Leukemia 1982. Clinical significance of chromosomal abnormalities in acute non-lymphoblastic leukemia. Cancer Genet Cytogenet 1984; 11: 332–350. Bloomfield CD, de la Chapelle A. Chromosome abnormalities in acute nonlymphocytic leukemia: clinical and biologic significance. Semin Oncol 1987; 14: 372–383. Berger R, Bernheim A, Ochoa-Noguera ME, Daniel M-T, Valensi F, Sigaux F, Flandrin G, Boiron M. Prognostic significance of chromosomal abnormalities in acute nonlymphocytic leukemia: a study of 343 patients. Cancer Genet Cytogenet 1987; 28: 293– 299. Joventino LP, Stock W, Lane NJ, Daly KM, Mick R, Le Beau MM, Larson RA. Certain HLA antigens are associated with specific morphologic and cytogenetic subsets of acute myeloid leukemia. Leukemia 1995; 9: 433–439. Keating MJ, Smith TL, Kantarjian H, Cork A, Walters R, Trujillo JM, McCredie Kb, Gehan EA, Freireich EJ. Cytogenetic pattern in acute myelogenous leukemia: a major reproducible determinant of outcome. Leukemia 1988; 2: 403–412. ¨ Fenaux P, Preudhomme C, Laı JL, Morel P, Beuscart R, Bauters F. Cytogenetics and their prognostic value in de novo acute myeloid leukaemia: a report on 283 cases. Br J Haematol 1989; 73: 61–67. Schiffer CA, Lee EJ, Tomiyasu T, Wiernik PH, Testa JR. Prognostic

47

48

49

50

51

52

53 54

55

impact of cytogenetic abnormalities in patients with de novo acute nonlymphocytic leukemia. Blood 1989; 73: 263–270. Dastugue N, Payen C, Lafage-Pochitaloff M, Bernard P, Leroux D, Huguet-Rigal F, Stoppa A-M, Marit G, Molina L, Michallet M, Maraninchi D, Attal M, Reiffers J. Prognostic significance of karyotype in de novo adult acute myeloid leukemia. Leukemia 1995; 9: 1491–1498. Cortes J, O’Brien S, Kantarjian H, Cork A, Stass S, Freireich EJ, Keating M, Pierce S, Estey E. Abnormalities in the long arm of chromosome 11 (11q) in patients with de novo and secondary acute myelogenous leukemias and myelodysplastic syndromes. Leukemia 1994; 8: 2174–2178. Gu Y, Alder H, Makamura T, Schichman SA, Prasad R, Canaani O, Saito H, Croce C, Canaani E. Sequence analysis of the breakpoint cluster region in the ALL-1 gene involved in acute leukemia. Cancer Res 1994; 54: 2327–2330. Sanford JP, Sait SNJ, Pan L, Nowak NJ, Gill HJ, Le Beau MM, Diaz MO, Zabel B, Shows TB. Characterization of two 11q23.3–11q24 deletions and mapping of associated anonymous DNA markers. Genes Chromosom Cancer 1993; 7: 67–73. Raimondi SC, Frestedt JL, Pui C-H, Downing JR, Head DR, Kersey JH, Behm FG. Acute lymphoblastic leukemias with deletion of 11q23 or a novel inversion (11)(p13q23) lack MLL gene rearrangements and have favorable clinical features. Blood 1995; 86: 1881–1886. Kalwinsky DK, Raimondi SC, Schell MJ, Mirro J, Santana VM, Behm F, Dahl GV, Williams D. Prognostic importance of cytogenetic subgroups in de novo pediatric acute nonlymphocytic leukemia. J Clin Oncol 1990, 8: 75–83. Sandoval C, Head DR, Mirro J, Behm FG, Ayers GD, Raimondi SC. Translocation t(9;11)(p21;q23) in pediatric de novo and secondary acute myeloblastic leukemia. Leukemia 1992; 6: 513–519. Martinez-Climent JA, Lane NJ, Rubin CM, Morgan E, Johnstone HS, Mick R, Murphy SB, Vardiman JW, Larson RA, Le Beau MM, Rowley JD. Clinical and prognostic significance of chromosomal abnormalities in childhood acute myeloid leukemia de novo. Leukemia 1995; 9: 95–101. ´ Mrozek K, Heinonen K, Lawrence D, Carroll AJ, Koduru PRK, Rao KW, Hutchison RE, Moore JO, Mayer RJ, Schiffer CA, Bloomfield CD. Adult patients with de novo acute myeloid leukemia and t(9;11)(p22;q23) have a superior outcome to patients with other translocations involving band 11q23: a Cancer and Leukemia Group B study. Blood 1997; 90: 4532–4538.

Appendix The following CALGB institutions participated in the study: University of Alabama, Birmingham, AL, supported by CA475450; Bowman-Gray Medical Center, Winston-Salem, NC, supported by CA03927; Duke University Medical Center, Durham, NC, supported by CA47577; Eastern Maine Medical Center, Bangor, ME, supported by CA31946; University of Iowa Hospitals, Iowa City IA, supported by CA 47642; Long Island Jewish Medical Center, New Hyde Park, NY, supported by CA11028; University of Maryland Cancer Center, Baltimore, MD, supported by CA31983; University of Missouri/Ellis Fischel Cancer Center, Columbia, MO, supported by CA12046; University of North Carolina at Chapel Hill, Chapel Hill, NC, supported by CA47559; North Shore University Hospital, Manhasset, NY, supported by CA35279; Parkview Memorial Hospital, Fort Wayne, IN, supported by CA31946; SUNY Health Science Center at Syracuse, Syracuse, NY, supported by CA21060; University of Tennessee, Memphis, Memphis, TN, supported by CA47555.

325