The presence of somatic mutations in immunoglobulin genes ... - Nature

Correspondence

1141

The presence of somatic mutations in immunoglobulin genes of B cell acute lymphoblastic leukemia (ALL-L3) supports assignment as Burkitt’s leukemia– lymphoma rather than B-lineage ALL TO THE EDITOR Surface immunoglobulin (Ig)-positive B cell acute lymphoblastic leukemia (B-ALL) comprises 2–3% of pediatric ALL. All other B-lineage ALL are precursor-B-ALL without surface Ig expression but positive for the nuclear enzyme terminal deoxynucleotidyl transferase (TdT), which is regarded to be an immature lymphoid marker. B-ALL is defined by bone marrow (BM) infiltration of ⬎25% blasts with FAB-L3 morphology and/or ALL with surface expression of Ig heavy and light chains. In the majority of patients (70%) there is evidence for extramedullary tumor mass. When the patients have less than 25% blasts with FAB-L3 morphology in their BM, the diagnosis of stage IV Burkitt’s lymphoma (BL) is made. Immunophenotypically, the lymphoblasts of both disease categories are characterized by expression of monotypic surface Ig, B cell markers (CD19, CD20, CD21 and CD22), and CD10, but lack of TdT expression.1 There are two main subtypes of BL: the sporadic and the endemic subtype. Sporadic BL occurs worldwide, whereas endemic BL is predominantly found in African countries. The sites of disease may vary between the two groups, eg orbital masses and jaw tumors are seen almost exclusively in the endemic subtype.2 In both B-ALL and BL, translocations involving the c-MYC gene are found. Translocation (8;14)(q24;q32) involving the Ig heavy chain gene (IGH) is identified in 苲85% of cases, whereas the remainder of cases generally have one of the two variant translocations t(2;8)(p11;q24) and t(8;22)(q24q11) involving the Ig kappa (IGK) and Ig lambda (IGL) light chain genes, respectively. As a result of these translocations the c-MYC gene is overexpressed. It has been shown that c-MYC translocations are essential, but not sufficient to induce B-ALL or BL.2 According to the latest WHO classification of hematopoietic malignancies, B-ALL with FAB-L3 morphology are regarded to be equivalent to BL in leukemic phase and should be diagnosed likewise.3,4 BALL and BL are considered to be similar in biology, oncogenic event, and treatment response, but comparison of their immunogenotype has not been possible, because the specific characteristics of the Ig heavy chain (IGH) gene rearrangements, somatic hypermutation status, and intraclonal heterogeneity have not been reported for B-ALL so far. We therefore addressed the question whether immunogenotype of B-ALL is similar to that of BL or to that of precursor-B-ALL. For this purpose we analyzed the immunogenotype of 12 B-ALL and five BL (four sporadic and one endemic). Southern blot analysis was performed to determine the configuration of the IGH locus and the presence of clonal EBV genome (Table 1). In only one B-ALL was clonal EBV present. Most sporadic BL are also negative for EBV, whereas EBV is almost invariably present in endemic BL.2 The IGH locus was further studied by PCR-heteroduplex analysis for the presence of complete (VH–DH–JH) and incomplete (DH–JH) rearrangements, followed by sequencing of the clonal rearrangements (Table 1). Remarkably, in nine out of 12 cases (75%) incomplete DH–JH rearrangements were detected on the second allele, which was much higher than in our BL control group where in only one of the five patients an incomplete rearrangement was found. No further literature data are available about incomplete DH–JH rearrangements in BL. The frequency of incomplete rearrangements in precursor-B-ALL is 22% (Szczepan´ski et al, submitted). In all B-ALL patients somatic mutations were found in the VH gene segments. This is in strong contrast to precursor-B-ALL, which do not have somatically mutated V genes (van der Burg et al, unpublished results). The percentages of mutations varied from 0.6 to 6.0% with

Correspondence: JJM van Dongen, Dept of Immunology, Erasmus University Rotterdam/University Hospital Rotterdam, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands; Fax: +31-10-4089456 Received 26 February 2001; accepted 15 March 2001

an average of 2.7 ± 1.8% (Table 1). In patient 12 a stop codon was formed as the result of a somatic mutation, explaining the unusual absence of surface Ig expression in this patient. The presence of somatic mutations suggests that B-ALL originate from a (post)-germinal center B cell. The frequency of somatic mutations in B-ALL is lower as compared to BL: sporadic BL (n = 27) with 4.6 ± 3.1%, endemic BL (n = 12) with 7.9 ± 3.9%, and AIDS-associated BL (n = 8) with 7.5 ± 3.6% (Table 2). However, due to the wide range in frequencies of somatic mutations, the difference between B-ALL and sporadic BL was not statistically significant (P ⬎ 0.05, using ␹2 test). There was no evidence for antigen selection in the B-ALL series, because the ratio between replacement and silent mutations (R/S ratio) was comparable for the frame work (FR) sequences and the complementary determining regions (CDR) (Table 1). In antigen-selected B cells the R/S ratio of the mutations in the CDR sequences is generally higher than the R/S ratio of the mutations in the FR sequences. The presence of ongoing mutations resulting in intraclonal heterogeneity was studied by sequencing six to nine individual clones of the cloned PCR products of the in-frame rearrangements. The frequency of intraclonal variation was not higher than the Taq enzyme error rate of 0.11% (data not shown), ie 0.3 mutations per VH clone. Therefore no clear indication for intraclonal heterogeneity in these cases was observed (Table 1). Intraclonal heterogeneity has been observed in two out of 47 BL cases (see Table 2).5 The presence of somatic mutations indicates that B-ALL do not arise from immature B cells, as the name might suggest, but from a postgerminal center B cell. The moderate frequency of somatic mutations and low frequency of EBV positivity are comparable to sporadic BL (Table 2). The obtained Ig gene data indicate that B-ALL differ significantly from the other types of B-lineage ALL, which all represent precursor-B-ALL. In combination with the known similarities in biology, oncogenic event and treatment response between B-ALL and BL, the immunogenotypic data presented here support the view to replace the term B-ALL and diagnose all B-ALL patients as stage IV BL.3 This is fully in line with the newly recognized WHO disease category Burkitt’s leukemia–lymphoma.4 Analogous to the term T-ALL for T-lineage ALL, the term B-ALL could then be reserved for B-lineage ALL, ie ALL of B cell precursor origin.

Acknowledgements We are grateful to Prof Dr R Benner for his continuous support. We thank the Dutch Childhood Leukemia Study Group (DCLSG) for kindly providing B-ALL cell samples. Board members of the DCLSG are WA Kamps, H van den Berg, JPM Bo¨kkerink, ESJM de Bont, B Granzen, PM Hoogerbrugge, R Pieters, JA Rammeloo, T Re´ve`sz, AJP Veerman and M van Weel-Sipman. M van der Burg1 BH Barendregt1 ER van Wering2 AW Langerak1 T Szczepan´ski1,3 JJM van Dongen1

1 Department of Immunology, Erasmus University Rotterdam/University Hospital Rotterdam, Rotterdam; 2Dutch Childhood Leukemia Study Group, The Hague, The Netherlands; 3 Dept of Pediatric Hematology and Chemotherapy, Silesian Medical Academy, Zabrze, Poland

References 1 Sandlund JT, Magrath IT. B-cell acute lymphoblastic leukemia and Burkitt lymphoma. In: Pui CH (eds). Childhood Leukemias. Cambridge University Press: Cambridge, 1999, pp 313–321. 2 Sandlund JT, Downing JR, Crist WM. Non-Hodgkin’s lymphoma in childhood. N Engl J Med 1996; 334: 1238–1248. Leukemia

Leukemia

5170

5190

5263

5333

5364

6058 6194 6441

5269

4694

4930

6137

1

2

3

4

5

6 7 8

9

10

11

12

t(8;14)

t(8;22)

IgM/Ig␭

—

t(8;14)

IgM/Ig␬

t(8;14)

IgM/Ig␭

t(8;14)

t(8;14)

IgM/Ig␭

IgM/Ig␭

ND

IgM/Ig␬

t(8;14) t(8;14) t(8;14)

t(8;14)

IgM/Ig␬

IgM/ND ND IgM/Ig␭

t(8;14)

NDa

Surface Ig C-MYC expression translocation

60%

90%

95%

60%

ND ND 50%

85%

30%

50%

ND

60%

Tumor load in BM

R/R/g

R/R/g

R/R

R/R/g

R/R/Rg R/g R/R/g

R/R/g

R/R/g

R/R/g

R/R/g

R/R/ga

SBa of IGH locus

VH5-51-DH6-25-JH6bg (out)

+

—

—

—

NI NI VH3-9-DH5-12-JH4b (in) VH4-39-DH3-22-JH4b (in) VH4-34-DH3-22-JH4b (in) VH4-59-DH4-17-JH4b (in)

VH4-30-DH1-1-JH1 (in)

NIa

VH4-34-DH1-14-JH4b (in)

VH1-69-DH3-3-JH5b (in) VH3-48-DH6-13-JH6c (in)

1st allele (frame)

DH3-3-JH6

DH2-8-JH4

NI

DH3-10-JH6

DH 3-9-JH6c DH5-5-JH4 DH2-2-JH6

DH3-22-JH4b

VH3-30DH 2-21JH4b (out)

VH1-18-DH5-24-JH6b (out)

DH3-22-JH5b

DH1-JH

2nd allele (frame)

PCR HDa analysis and sequencing of IGH locus

— — —

—

—

—

—

—

EBV

2.1 (6/279)

— — 2.1 (5/234) 1.3 (3/243) 3.4 (11/321) 3.0 (8/267)

0.9 (3/318)

—

4.5 (7/155)

0.6 (2/318) 6.0 (15/261)

%

4e (4:1)

NI NI n.d.d (3:0) n.d.d (1:0) 1e (1:1) n.d.d (3:0)

n.d.d (1:0)

NI

n.d.d (2:0)

0 (0:1) 3e (3:1)

R/S ratiob CDR

2 (2:1)

NI NI 4 (4:1) n.d. (2:0) 2 (6:3) 0.25 (1:4)

0 (0:1)

NI

0.7 (2:3)

n.d. (1:0) 1.7 (5:3)

R/S ratioc FR

Somatic mutations in 1st allele

ND

0 (6)

ND

0 (6)

— — 0.25 (4)

0.17 (6)

ND

ND

0.25 (8)

0.33 (6)

Intraclonal heterogeneity (No. of clones)f

a ND, not determined; NI, not identified; −, negative; SB, Southern blotting; R, rearranged allele; g, remaining germline band, mainly derived from non-leukemic cells with germline IGH genes; HD, heteroduplex analysis. b R/S ratio, ratio of replacement mutations and silent mutations in the complementary determining regions (CDR) with the number of respective mutations between brackets. c R/S ratio of mutations in the frame work regions (FR). d No calculation for antigen selection, because one of the R/S ratios was not defined (n.d.) (S = 0). e No evidence for antigen selection, because no significant difference in R/S ratio of CDR and FR (P ⬎ 0.05 in ␹2 test). f Mean number of intraclonal mutation differences per clone with the number of sequenced clones between brackets. g As a result of somatic mutation a stop codon was formed in the VH region (AAA (Lys) into TAA (stop)) explaining the absence of surface Ig expression in this patient.

Patient code

Immunogenotypic characteristics of the 12 B-ALL samples

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No.

Table 1

Correspondence

Correspondence

Table 2

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Characteristics of IGH gene rearrangements in B-ALL, precursor B-ALL, and the three types of BL

Disease category Number of casesa Somatic mutations in VH (range) Antigen selection in VHd Intraclonal heterogeneity

B-ALL

Precursor-B-ALL

Sporadic BL

9 2.7 ± 1.8% (0.6–6.0%) 0/9 0/9

50b 0% — 0/50 0/50

27 (15 cell lines)c 4.6 ± 3.1% (0.3–13%) 1/27 1/27

Endemic BL 12 (9 cell lines)c 7.9 ± 3.9% (1.4–15%) 0/12 1/12

AIDS associated BL 8 (5 cell lines)c 7.5 ± 3.6% (3.1–13.3%) 1/8 0/8

a The nine B-ALL cases as well as four sporadic BL and one endemic BL were analyzed in this study, whereas the other BL cases were reviewed by Chapman et al.5 b Van der Burg et al, unpublished results. c Cell lines were included, since Chapman et al6 demonstrated that somatic mutations of V-genes do not occur in vitro. d Absence or presence of antigen selection was deduced from the R/S ratios of the CDR and FR sequences.

3 Dayton VD, Arthur DC, Gajl-Peczalska KJ, Brunning R. L3 acute lymphoblastic leukemia. Comparison with small noncleaved cell lymphoma involving the bone marrow. Am J Clin Pathol 1994; 101: 130–139. 4 Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman J, Lister TA, Bloomfield CD. The World Health Organization classification of hematological malignancies report of the Clinical Advisory Committee Meeting, Airlie House, Virginia, November 1997. Mod Pathol 2000; 13: 193–207.

5 Chapman CJ, Wright D, Stevenson FK. Insight into Burkitt’s lymphoma from immunoglobulin variable region gene analysis. Leuk Lymphoma 1998; 30: 257–267. 6 Chapman CJ, Mockridge CI, Rowe M, Rickinson AB, Stevenson FK. Analysis of VH genes used by neoplastic B cells in endemic Burkitt’s lymphoma shows somatic hypermutation and intraclonal heterogeneity. Blood 1995; 85: 2176–2181.

The growth of highly proliferative acute lymphoblastic leukemia may be independent of stroma and/or angiogenesis TO THE EDITOR Lymphoproliferative diseases, like acute leukemia (AL) in children, have potential to invade locally and infiltrate a variety of different tissues other than bone marrow, ie central nervous system or testis. The mechanisms of tumoral invasion involved in solid tumors are related to angiogenesis, endothelial adhesion and cell migration. Recent studies have hypothesized similar mechanisms for hemopathies, especially AL.1 It has been suggested that angiogenesis could participate in AL cell proliferation, and therefore the secretion of modulators of angiogenesis such as bFGF has been considered as a possible indicator of the leukemic proliferation. The importance of angiogenic factors is not fully established. These angiogenic cytokines seem to be produced by leukemic and/or bone marrow (BM) microenvironmental cells.2 Recently, Veiga et al2 have shown a contact-dependent interaction between acute lymphoblastic leukaemia (ALL) cells of patients and BM endothelium in presence of angiogenic cytokines. This interaction leads to the survival of leukemic cells by maintaining the expression of anti-apoptotic genes. A recent study showed that high levels of cellular VEGF are significantly correlated with shorter survival in adult acute myeloblastic leukemia (AML) patients with high WBC at diagnosis (P ⬍ 0.04), whereas no association was found with the plasma levels of bFGF.3 However, no correlation between angiogenesis and the prognosis of ALL has been described yet. In a series of children with ALL, we found a correlation between usual poor prognostic features and the interactions between leukemic cells and BM microenvironment. Twenty-three newly diagnosed proB and pre-B-lineage ALL were studied for the urinary secretion of angiogenic cytokines (bFGF, VEGF). Diagnosis was established on BM aspirates and classification was made according to the usual immunophenotyping and cytogenetics. All children were treated according to the French protocol FRALLE 93.4 The urinary bFGF and VEGF levels

Correspondence: P Schneider, Faculte´ de Me´decine-Pharmacie, 22 boulevard Gambetta, 76183 Ronen Cedex, France; Fax: 02 32 88 86 92 Received 5 September 2000; accepted 15 February 2001

(pg/mmol creatinine/24 h) were determined by an ELISA method using the Quantikine FGF immunoassay (R&D Systems, Minneapolis, MN, USA) performed on a 24-h urine collection. For 11 patients, apoptosis of the leukemic cells was studied in the presence and the absence of an adherent monolayer of human embryonic fibroblast line (MRC5, Biomerieux, March-L’Etoile, France). Fibroblasts were cultured to confluence in RPMI 1640 and 15% FCS before the addition of leukemic cells in stem span serum-free medium (Stem Technologies, Meylan, France) (1 × 106 ALL cells/ml/well). Cocultures were carried out in triplicate, in 24-well plates, at 37°C with 5% CO2. The percentage of apoptotic cells was determined by flow cytometry (EPICS XL-MCL, Coulter, Margency) after 4 days of culture using the annexin V binding assay (Immunotech, Coulter Company, Marseilles, France). The median urinary bFGF levels were significantly elevated in children with leukemia (15 months to 15 years old, median: 8) compared to the levels of 19 healthy controls under the age of puberty (3 to 13 years old, median: 10). At diagnosis the median values were significantly different (respectively 460 pg/mmol creatinine and 30 pg/mmol creatinine, P ⬍ 0.01) (Figure 1a). A wide range was observed among our 23 patients at diagnosis, with bFGF levels from less than 0.25 to 29 837 pg/mmol creatinine. The normal value was 333 pg/ mmol creatinine of bFGF (mean + 2 s.d. of the control group). A large overlap between patients and controls was observed and 12 patients had normal values of bFGF. Eleven of them had very high risk leukemias, with adverse prognosis factors: five had a very high WBC at diagnosis (⬎50 × 109/l), three a conserved hemoglobin level (⬎10 g/dl), one positive myeloid antigens (CD13, CD33), and two a resistance to initial treatment (Table 1). Most of these prognosis factors are associated with a high proliferation rate. Among the 11 patients with an elevated level of bFGF (⬎333 pg/mmol creatinine), five patients had poor prognosis risk factors. These five patients did not have any special characteristics except elevated WBC or normal hemoglobin level. In the whole series there is a significant relationship between normal bFGF levels and high risk factors of ALL (Fisher exact test: P = 0.027). When considering the leukemic burden, no correlation was found between the WBC at presentation, the presence of a spleno- and/or hepatomegaly and the level of bFGF (data not shown). Leukemia