Letters to the Editor 2064 study is the first to present the mRNA expression of Cernunnos/ 6Department of Otolaryngology, Faculty of Otolaryngology, XLF, which was newly identified as an NHEJ member in 2006,6,7 College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan and as well as of other NHEJ components including Artemis, XRCC4 7 and DNA ligase IV in pediatric ALL. We showed that Graduate Institute of Medical Genetics, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan overexpressions of NHEJ were closely associated with E-mail: [email protected] malignant leukemic cells, since elevations of NHEJ mRNA transcripts were found only in fresh ALL and in relapsed cases, but not in thalassemia, complete remission or peripheral blood References of normal individuals. Elevations of NHEJ mRNAs could also be found in malignant cell lines K562 and BJAB, but not in oral 1 Khanna KK, Jackson SP. DNA double-strand breaks: signaling, cancer. Therefore, we propose that upregulation of NHEJ mRNA repair and the cancer connection. Nat Genet 2001; 27: 247–254. transcripts is a characteristic of pediatric ALL. This feature may 2 Brady N, Gaymes TJ, Cheung M, Mufti GJ, Rassool FV. Increased be valuable for proper prognosis or for providing strategies for error-prone NHEJ activity in myeloid leukemias is associated with DNA damage at sites that recruit key nonhomologous end-joining treatment of this malignancy. . Cancer Res 2003; 63: 1798–1805. 3 Gaymes TJ, Mufti GJ, Rassool FV. Myeloid leukemias have increased activity of the nonhomologous end-joining pathway and Acknowledgements concomitant DNA misrepair that is dependent on the Ku70/86 heterodimer. Cancer Res 2002; 62: 2791–2797. This work was supported by Kaohsiung Medical University 4 Rassool FV. DNA double strand breaks (DSB) and non-homologous Pediatric Cancer Funds (QP094001 to SSC) and Kaohsiung end joining (NHEJ) pathways in human leukemia. Cancer Lett 2003; Medical University Cancer Funds (QC094004 to CSL). 193: 1–9. 5 Greaves MF, Wiemels J. Origins of translocations in S-S Chiou1,2, J-L Huang3, Y-S Tsai4, T-F Chen4, K-W Lee5,6, childhood leukaemia. Nat Rev Cancer 2003; 3: 639–649. S-HH Juo4,7, Y-J Jong2,4, C-H Hung1,2, T-T Chang1 and 6 Ahnesorg P, Smith P, Jackson SP. XLF interacts with the XRCC4– C-S Lin4 DNA ligase IV complex to promote DNA nonhomologous end- 1Department of Pediatrics, Kaohsiung Medical University joining. Cell 2006; 124: 301–313. Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan; 7 Buck D, Malivert L, de Chasseval R, Barraud A, Fondaneche MC, 2 Sanal O et al. Cernunnos, a novel nonhomologous end-joining Department of Pediatrics, Faculty of Pediatrics, College of factor, is mutated in human immunodeficiency with microcephaly. Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 3 Cell 2006; 124: 287–299. Department of Bioscience Technology, College of Health 8 Hosoi Y, Watanabe T, Nakagawa K, Matsumoto Y, Enomoto A, Science, Chang Jung Christian University, Tainan, Taiwan; 4 Morita A et al. Up-regulation of DNA-dependent Graduate Institute of Medicine, College of Medicine, kinase activity and Sp1 in colorectal cancer. Int J Oncol 2004; 25: Kaohsiung Medical University, Kaohsiung, Taiwan; 461–468. 5 Department of Otolaryngology, Kaohsiung Medical 9 Livak KJ, Schmittgen TD. Analysis of relative expression University Hospital, Kaohsiung Medical University, data using real-time quantitative PCR and the 2ÀDDCT method. Kaohsiung, Taiwan; Methods 2001; 25: 402–408.

Conventional cytogenetics of nodular lymphocyte-predominant Hodgkin’s lymphoma

Leukemia (2007) 21, 2064–2067; doi:10.1038/sj.leu.2404736; node biopsy was used for morphologic, immunologic, mole- published online 10 May 2007 cular and cytogenetic studies, including molecular cytogenetics. After an expert review, patients suspected to have atypical diffuse large B-cell lymphoma or a CHL were excluded and 13 The World Health Organization classification of the tumors of patients with an ascertained NLPHL diagnosis were retained and hematopoietic and lymphoid tissues distinguishes nodular are reported here. Clinical and pathological characteristics are lymphocyte-predominant Hodgkin’s lymphoma (NLPHL) from summarized in Table 1. Median age was 32 years (range 6–57). classical Hodgkin’s lymphoma (CHL) on morphological, clinical All patients had localized peripheral lymphadenopathy (stage I: and biological differences.1 NLPHL accounts for approximately 7 or stage II: 6). None had extra-nodal involvement or B 5% of all Hodgkin’s lymphomas and usually presents as an early symptoms. Five were treated at diagnosis or later, and eight stage disease, with slow progression and a favorable outcome.2 patients did not receive any treatment and remained in stable Response to conventional therapy, when indicated, is excellent. disease. Median follow-up for this series is 59.8 months. Transformation to diffuse large B-cell lymphoma has been In all cases, the morphologic, cytologic and immunopheno- reported in approximately 3–5% of cases. Tumor cells, known typic features were consistent with the diagnosis of NLPHL. L&H as ‘popcorn’ or L&H cells, express B-cell antigens like CD20 and cells expressed B-cell markers: CD79a, CD20, Oct2, Bob1 and CD45, usually do not express CD15 and CD30 and probably are Bcl6. Rosetting with CD57 was not constant, as reported in of centroblastic origin. Cytogenetic characteristics of NLPHL literature. Mum1 was expressed in 50% of cases, whereas CD30 have been rarely reported, and were mainly obtained using and CD15 were not expressed. Two cases were identified as molecular cytogenetic methods like fluorescent in situ hybridi- germinal center progressive transformation with features of early zation (FISH) or comparative genomic hybridization (CGH).3,4 NLPHL. These two cases also presented typical NLPHL areas. Between 1979 and 2004, 22 patients referred to the Centre Good quality metaphases were obtained in all 13 patients and Henri Becquerel had a diagnosis of NLPHL. A single lymph clonal abnormalities were identified in 12/13 (Figure 1).

Leukemia Letters to the Editor 2065 Table 1 Clinical data, histology and immunohistology

Sex/age Clinical stage CD20 CD79a CD57 CD15 ema Bob1 Oct2 Bcl6 Mum1 CD30 Histology at diagnosis

1 M/19 IIAa + ++À NA + + + ÀÀFpt, early poppema 2 F/21 IIAa + ++À NA + + + ÀÀTypical 3 M/32 IIAa + NA + ÀÀ++ + ÀÀFpt, early poppema 4 F/57 IIAa + +r+À NA + + + ÀÀTypical 5 M/45 Iaa + +r+À NA + + + ÀÀTypical 6 M/35 IIAa + +r+À NA + + + + À Typical 7 F/17 Iaa + ++À NA + + + ÀÀDiffuse pattern 8 M/30 Iaa + +r+À + + + NA +weak À Typical 9 F/6 Iaa + +weak + À +++ + + À Typical 10 F/10 Iaa + +weak + À + + + +weak +weak À Typical 11 M/38 Iaa + ++À + + + + +weak À Typical 12 F/41 IIAa + +r+ÀÀ+ À ++À Typical, fpt 13 M/41 Iaa + ++À NA + + + ÀÀTypical Abbreviations: F, female; Fpt, germinal center progressive transformation; M, male; NA, not available; r+, rosetting with cd57.

a b

add(6) der(3)

1 23 45

3

6 789101112

13 14 15 16 17 18 14 add(6) add(14)

19 20 21 22 x y

3 c d

BCL6-cen IgH cen BCL6-tel IgH-tel

Figure 1 Representative metaphase from patient 11. (a) R banding, (b) R-banded karyotype, (c) FISH with LSI BCL6 probe, (d) FISH with LSI IgH probe.

Karyotypes are reported in Table 2. All were complex but one,  3q27 rearrangements in eight patients, associating more than three numerical and/or structural  loss of chromosome 4 or 4q28 rearrangements resulting in a abnormalities. Nine karyotypes were in the diploid range loss of the 4q28–q32 region in six patients, (46–49 ), one was hypodiploid and one tetraploid.  loss of chromosome 7 or 7q23–q33 rearrangements in five One patient had both a diploid and a tetraploid component. As patients, in CHL,5 the number of abnormal metaphases was usually low  loss or partial deletion of chromosome 13 in three patients as compared to the number of normal mitoses. and Recurrent chromosomal defects included  14q32 rearrangements in six patients.

 a gain or a partial gain of the long arm of chromosome 1 in 10 Chromosome 9 imbalances were present in seven patients, as patients, either as an isochromosome for the long arm (7/10) gain (one patient), loss (one patient), add(9)(p24) (two patients), (Figure 1) or as a translocated supernumerary long arm (3/10), add(9)(q22) (one patient) and 9q33–34 rearrangement (two patients).

Leukemia Letters to the Editor 2066 Table 2 Cytogenetic, FISH and molecular results of 13 patients with LPHD

Patient Conventional cytogenetics 3q27 BCL6 IgH rearrangement, abnormality rearrangement FISH/molecular (FISH) 3

1 46,XY,der(4)t(1;4)(q21;q28),t(3;14)(q27;q32) [3]/46,XY [33] + NT NT 2 48,XX,i(1)(q10),t(3;5)(q27;q31),add(7)(q31),+add(8)(p23),+9 [3]/46,XX [16] + + NT 3 46,XY,i(1)(q10),del(4)(p13) [3]/94,o4n4,XXYY,i(1)(q10),+8,+8,t(9;14)(q22;q32) ÀÀt(9;14) [6]/46,XY [1] 4 49,X,der(X)t(X;7)(q21;q13),add(2)(p23),add(3)(q27),+5,del(6)(q16q23),À7, + + NT/t(3;14) add(9)(p24),À10,À13,+mar[4]/46,XX [13] 5 92,o4n4,XXXX,add(1)(p36),add(1)(p12),i(1)(q10),À2,add(3)(p21)x2,À4, ÀÀÀ del(4)(q28),del(6)(q12q27)x2,+8,À9,À10,add(10)(q25),+12,add(13)(p11), add(13)(q31),t(14;22)(q32;q11),+16,À19,+21 [8]/46,XY [5] 646À48,XY,t(3;5)(q27;q31),+5,+8 [cp3]/46,XY [13] + À NT 7 46,XX,i(1)(q10),del(2)(q31q34),add(3)(p21),del(4)(q28),À5,del(6)(q12q22),À7, ÀÀNT add(7)(p21),add(9)(q34),add(12)(q24),del(14)(q11q22),add(16)(q23), add(19)(q13),+mar [3]/46,XX [15] 8 46,X,ÀY,del(4)(q28),+8,add(9)(p24),der(14)t(1;14)(q11;p11), ÀÀNT der(21)t(1;21)(q11;p11) [3]/46,XY [13] 9 41,X,ÀX,i(1)(q10),À2,der(3)t(2;3)(q23;q27),À4,À10,add(14)(q32), + + inv(14) der(16)t(16;17)(q23;q11),À17,add(19)(p13),+r [4]/46,XX [14] 10 46,X,ÀX,i(1)(q10),add(2)(q12),t(3;17)(q27;q21),add(4)(p13)(q28), +NTNT add(14)(q32),i(18)(q10),+mar [5]/46,XX [7] 11 46,XY,i(1)(q10),dup(2)(p12p16),t(3;6)(q27;q22),del(7)(q22),À14,+mar [3]/46, + + +/t(3;14;6) XY,i(1)(q10),dup(2)(p12p16),t(3;6)(q27;q22),del(7)(q22),i(7)(q10),add(14)(q32) [2]/46,XY [15] 12 47,X,ÀX,t(3;9)(q27;q33),add(5)(q31),add(7)(p21),add(7)(q31),der(13)t(1;13) +NTNT (q24;q34),À14,add(21)(q22),+mar [2]/46,XX [15] 13 46,XY [18] À NT NT Abbreviations: LPHD, lymphocyte-predominant Hodgkin’s disease; NT, not tested.

The partner region involved in 3q27 rearrangements could be proliferation rate and the complexity of results), the cytogenetic identified in seven cases as 5q31 in two patients and 2q23, pattern of these patients is rather well characterized, comprised 6q22, 9q22, 14q32 and 17q21 in one patient each. Conven- of hyperdiploid metaphases, mainly in the triploid range, tional cytogenetics failed to identify the translocated material in without any of the abnormalities usually encountered in B-cell one case. or T-cell non-Hodgkin’s lymphoma. This pattern was confirmed FISH analysis could be performed on previously R-banded by molecular cytogenetics studies, including FICTION. metaphases in nine patients. Among these, five displayed a 3q27 To date, for the same reasons the cytogenetic characterization rearrangement and the involvement of BCL6 was confirmed by of NLPHL remains partial, limited to some CGH data,4 FISH in four (patients 2, 4, 9 and 11). However, whereas the to a partial analysis using interphase FISH with probes translocation of patient 9 was interpreted as a t(2;3)(q23;q27), exploring genes usually involved in B-cell lymphoma3 and to FISH showed that the telomeric part of BCL6 was translocated conventional cytogenetics data from a few number of patients on a small marker chromosome, suggesting a three-way with NLPHL or transformed lymphoma, reported together with translocation. In the fifth case (patient 6), the rearrangement extensive FISH and molecular analysis.6 was telomeric to the BCL6 gene. Normal signals were found on Our study, where the only significant imbalances involved all abnormal metaphases in 4/4 patients without 3q27 rearran- chromosomes 1, 4, 7, 9 and 13, does not confirm the results gements, confirming the presence of four copies of chromosome obtained using CGH by Franke.4 3 in patients 5 and in the tetraploid clone of patient 3. Search for In the present series, a gain of chromosome 1q was an implication of the IgH in four patients with 14q32 unexpectedly found in 10 of 12 patients, mainly represented rearrangements confirmed the involvement of the IgH gene in by an isochromosome for chromosome 1 long arm. This three cases. In patient 5, the 14q32 rearrangement found using abnormality is not frequent in other subtypes of B-cell conventional cytogenetics that is t(14;22)(q32;q11) did not lymphomas, present in 9/23 Burkitt’s lymphomas, 20/330 involve the IgH nor the Ig l gene. An attempt to identify the IgH diffuse large cell lymphomas, 17/303 follicular lymphomas partner at 9q22 in patient 3 showed that the corresponding and 12/144 CHL with cytogenetic abnormalities studied at breakpoint was telomeric to RP11-346B7. diagnosis (C Bastard, unpublished data). Unexpected BCL6-IgH translocations were identified in A chromosome 4q rearrangement resulting in a loss of the patient 4 who expressed fusion transcripts joining Im to BCL6 4q28–qter region was present in six patients (46%). Such 4q exon 2 and BCL6 exon1 to Cm, and in patient 11, who expressed deletions are unusual in NHL, whereas they are more frequent a fusion transcript joining the BCL6 first exon to Ce but not the in CHL.5 reciprocal Ie/BCL6-exon2 transcript, indicating a complex As reported by Wlodarska et al.,3,6 we confirm the frequent translocation t(3;14;6)(q27;q32;q22). The genomic breakpoint involvement of BCL6 and immunoglobulin heavy chain genes. of the derivative chromosome 14 was also cloned in this patient, As underlined by these authors, 3q27 and 14q32 chromosomal confirming the transcription data. defects, as detected by conventional cytogenetics, are not Despite the difficulty of conventional cytogenetic studies in present in CHL. On the other hand, a recent report by Martin CHL, (due to the low number of malignant cells, their low Subero et al.7 demonstrates that such rearrangement can be

Leukemia Letters to the Editor 2067 found by FISH in a notable proportion of CHL patients. A Stamatoullas1, J-M Picquenot1, C Dumesnil2, P Ruminy1, Therefore, these authors conclude that the identification of IgH D Penther1, P Bertrand1, M-N Courel1, C Maisonneuve1, and BCL6 rearrangements cannot be used to differentiate CHL A Franc¸ois3, P Gaulard4, H Tilly1 and C Bastard1 from NLPHL. Whereas we agree with this conclusion when such 1Groupe d’e´tude des prolife´rations lymphoı¨des, Centre Henri defects are detected by FISH alone, we think that the results of a Becquerel, Rouen, France; 2Service d’He´matologie Pe´diatrique, CHU Rouen, France; conventional cytogenetic analysis, which shows a chromosomal 3 pattern globally different from that of CHL (mainly diploid De´partement d’Anatomo-Pathologie, CHU Rouen, France and context, iso 1q chromosome or 1q gain, higher frequency of 4De´partement d’Anatomo-Pathologie, Hoˆpital Henri Mondor, 3q27 and 14q32 rearrangements), provides useful arguments Cre´teil, France that can contribute to distinguishing CHL from NLPHL. E-mail: [email protected] To the best of our knowledge, the BCL6 partner regions that we identified here have not been previously reported, with the exception of the 14q32 region. In one of two patients carrying a References t(3;5)(q27;q31), a FISH control using a break-apart probe demonstrated that the BCL6 gene was probably not involved, 1 Jaffe ES, Harris NL, Stein H, Vardiman JW (eds) World Health as the break occurred telomeric to BCL6. In this case, FISH Organization classification of tumours.Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. IARC Press: analysis did not suggest an ABR breakpoint (no faint signal from Lyon, 2001, 238–253. the commercial break-apart telomeric probe). 2 Diehl V, Sextro M, Franklin J, Hansmann ML, Harris N, Jaffe E et al. Using interphase FISH, Renne´ and colleagues8 studied the Clinical presentation, course, and prognostic factors in lymphocyte- involvement of immunoglobulin genes loci in 24 patients with predominant Hodgkin’s disease and lymphocyte-rich classical NLPHL and found a disruption of the IgH locus in five patients. Hodgkin’s disease: report from the European Task Force on Using triple-color FISH, they identified a BCL6-IgH translocation Lymphoma Project on Lymphocyte-Predominant Hodgkin’s Disease. J Clin Oncol 1999; 17: 776–783. in all five cases and conclude that this event was recurrent in 3 Wlodarska I, Nooyen P, Maes B, Martin-Subero JI, Siebert R, NLPHL. In our series, a single t(3;14)(q27;q32) was identified by Pauwels P et al. Frequent occurrence of BCL6 rearrangements conventional cytogenetics, whereas in two cases the same in nodular lymphocyte predominance Hodgkin lymphoma translocation could only be identified by FISH and/or but not in classical Hodgkin lymphoma. Blood 2003; 101: molecular methods, the corresponding derivative chromosomes 706–710. 3 being interpreted as add(3)(q27) and t(3;6)(q27;q22), respec- 4 Franke S, Wlodarska I, Maes B, Vandenberghe P, Delabie J, Hagemeijer A et al. Lymphocyte predominance Hodgkin disease is tively. Two other rearrangements disrupting the IgH genes characterized by recurrent genomic imbalances. Blood 2001; 97: involved genes located in the 9q22 and 14q23 regions, 1845–1853. respectively, indicating that BCL6 is not the only partner of 5 Tilly H, Bastard C, Delastre T, Duval C, Bizet M, Lenormand B. IgH translocations. Cytogenetic studies in untreated Hodgkin’s disease. Blood 1991; 77: Despite a long follow-up, none of our patients transformed to 1298–1304. diffuse large B-cell lymphoma. 6 Wlodarska I, Stul M, Wolf-Peeters C, Hagemeijer A. Heterogeneity of BCL6 rearrangements in nodular lymphocyte predominant This study reporting the results of conventional cytogenetic Hodgkin’s lymphoma. Haematologica 2004; 89: 965–972. investigations performed in a series of 13 NPHL patients 7 Martin-Subero JI, Klapper W, Sotnikova A, Callet-Bauchu E, demonstrates that in such patients, conventional karyotypic Harder L, Bastard C et al. Chromosomal breakpoints affecting analysis is feasible, confirms the clonal nature of the tumor and immunoglobulin loci are recurrent in Hodgkin and Reed–Sternberg provides additional informations when compared to molecular cells of classical Hodgkin lymphoma. Cancer Res 2006; 66: cytogenetic methods. The identification of 3q27 and 14q32 10332–10338. 8 Renne C, Martin-Subero JI, Hansmann ML, Siebert R. Molecular rearrangements in a mainly diploid context confirms previously cytogenetic analyses of immunoglobulin loci in nodular lymphocyte published results and demonstrates that NLPHL patients share predominant Hodgkin’s lymphoma reveal a recurrent IGH-BCL6 genetic defects with NHL patients rather than with CHL patients. juxtaposition. J Mol Diagn 2005; 7: 352–356.

HS1 complexes with cytoskeleton adapters in normal and malignant chronic lymphocytic leukemia B cells

Leukemia (2007) 21, 2067–2070; doi:10.1038/sj.leu.2404744; remained elusive. Previous studies were not unequivocal, as published online 17 May 2007 they suggested various possibilities including actin-binding activity, involvement in apoptosis and a nuclear function. Likewise, the subcellular localization is unclear, as HS1 was Hematopoietic cell-specific Lyn substrate 1 (HS1, HCLS1, reported to be inside the nucleus, in the cytoplasm and in the LckBP1) is a 75-kDa intracellular protein expressed in cells of lipid rafts of B-lymphocytes. The analysis of the amino-acid lymphohemopoietic origin. It was originally identified in sequence showed that HS1 has a modular structure and is B-lymphocytes as a major substrate of B-cell receptor (BCR)- structurally related to cortactin, a cortical actin-associated induced phosphorylation after antigen (Ag) stimulation. Geneti- protein that modulates the dynamics of the actin cytoskeleton. cally modified mice confirmed that in both B and T-lymphocytes This structural relationship is underscored by the existence of a HS1 is involved in the processes of Ag-receptor-induced clonal putative actin-binding domain at the N-terminus of HS1 expansion and deletion.1 However, the specific role of HS1 molecule. Accordingly, it has been shown recently that in

Leukemia