Extra copies of ALK gene locus is a recurrent genetic aberration and favorable prognostic factor in both ALK-positive and ALK-negative anaplastic large cell lymphomas

Leukemia Research 36 (2012) 1141–1146

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Extra copies of ALK gene locus is a recurrent genetic aberration and favorable prognostic factor in both ALK-positive and ALK-negative anaplastic large cell lymphomas Ran Yu a,b , Gang Chen c , Chunju Zhou d , Zifen Gao e , Yunfei Shi e , Yan Shi f , Xiaoge Zhou g , Jianlan Xie g , Hongxiang Liu h , Liping Gong a,∗ a

Department of Pathology, Basic Medical College, Capital Medical University, Beijing, China Department of Basic Science, Weifang Nursing Vocational College, Weifang, China c Department of Pathology, Fujian Provincial Tumor Hospital, Fuzhou, China d Department of Pathology, Beijing Children’s Hospital, Capital Medical University, Beijing, China e Department of Pathology, Peking University Healthy Science Center, Beijing, China f Department of Pathology, The Second Affiliated Hospital of the Harbin Medical University, Harbin, China g Department of Pathology, Beijing Friendship Hospital, Capital Medical University, Beijing, China h Molecular Malignancy Laboratory and Department of Histopathology, Addenbrooke’s Hospital, University of Cambridge, Cambridge, UK b

a r t i c l e

i n f o

Article history: Received 19 March 2012 Received in revised form 1 June 2012 Accepted 3 June 2012 Available online 20 June 2012 Keywords: Anaplastic large cell lymphoma (ALCL) Anaplastic lymphoma kinase (ALK) Molecular genetics Fluorescence in situ hybridization (FISH)

a b s t r a c t Systemic anaplastic large cell lymphoma (ALCL) is subtyped into ALK-positive ALCL and ALK-negative ALCL based on the presence or absence of ALK protein expression. ALK-positive ALCL is characterized by t(2;5)(p23;q35)/NPM-ALK or variant ALK-involved translocations, while little is known about the genetic changes in ALK-negative ALCL. We investigated the structural and numerical aberrations of the ALK gene using interphase fluorescence in situ hybridization (FISH) in 81 cases with ALCL and analyzed their association with clinical outcome of the patients. ALK gene rearrangement was found in 47 of 50 (94%) ALK-positive ALCLs but in none of 31 ALK-negative ALCLs. Extra copies of the ALK gene locus, representing mainly extra copies of chromosome 2, were seen in 19 ALK-positive (38%) and 15 ALK-negative (48%) cases (P = 0.357). In 55 cases with follow-up information, the mean survival time of the 38 ALK positive cases (58 months) was significantly longer than that of 17 ALK-negative cases (22.5 months) (P = 0.038). Interestingly, the cases with extra copies of ALK had a significantly longer mean survival time than those without (64.4 months vs 35.3 months) (P = 0.023) and this difference was observed in both ALK-positive (72.3 vs 45.9 months) and ALK-negative (34.7 vs 9.9 months) cases. Multivariate analysis showed that both ALK protein expression and extra copies of ALK gene were independent predictors for better survival (P = 0.008). Our results suggest that the extra copies of ALK gene locus are a frequent genetic aberration in both ALK-positive and ALK-negative ALCL and is a favorable prognostic marker for the patients. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Systemic anaplastic large cell lymphoma (ALCL) is a biologically and clinically heterogeneous type of mature T-cell lymphoma characterized by consistent expression of CD30. In the current World Health Organization (WHO) Classification of Tumours of Hematopoietic and Lymphoid Tissues [1], systemic ALCL is divided into two different entities, i.e. anaplastic lymphoma kinase (ALK)positive ALCL and ALK-negative ALCL based on whether or not

∗ Corresponding author at: Department of Pathology, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, China. Tel.: +86 10 83911432; fax: +86 10 83911699. E-mail address: [email protected] (L. Gong). 0145-2126/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2012.06.005

the tumor cells express ALK. ALK-positive ALCL is usually found in children and young adults and has better prognosis, whereas the ALK-negative ALCL occurs in patients of a wide range of ages and shows a more aggressive behavior [2–6]. ALK-positive ALCL is genetically distinctive, with the tumor cells almost invariably carrying ALK-involved translocations [1]. The most frequent translocation is t(2;5)(p23;q35) which fuses the ALK gene on chromosome 2p23 with the nucleophosmin (NPM) gene on chromosome 5q35, resulting in expression of a chimeric NPM-ALK protein with oncogenic potential due to its constitutive tyrosine kinase activity [7,8]. The variant ALK-associated translocations involve other partner genes including TPM3 in 1q25, ATIC in 2q35, TFG in 3q21 and MSN in Xq11et al, all resulting in ALK protein expression [9]. However, the genetic feature of ALK-negative ALCL cases, which lack ALK gene translocation, is largely unknown.

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In a previous study of 28 ALCL cases, we observed extra copies of ALK gene in a substantial number of ALK-positive (3/19) and ALK-negative (7/9) cases using interphase fluorescence in situ hybridization (FISH). Other workers [10,11] also detected extra copies of ALK in ALCL. However, it remained to confirm whether the gain of extra copies of ALK is a recurrent genetic feature of ALCL, especially ALK-negative ALCL, and whether this genomic aberration has any clinical implications. To address these questions, we investigated the structural and numerical changes of ALK gene locus by FISH in a large series of ALK-positive and ALK-negative ALCL cases and analyzed their association with clinical outcome of the patients. 2. Materials and methods 2.1. Tissue specimens Formalin-fixed and paraffin-embedded (FFPE) specimens from 81 patients with systemic ALCL were retrieved from Department of Pathology of Beijing Friendship Hospital (N = 25), Beijing Children’s Hospital (N = 25), Fujian Provincial Cancer Hospital (N = 21), Beijing University Healthy Science Center (N = 6) and the Second Affiliated Hospital of Harbin Medical University (N = 4) in China. The disease was diagnosed between 1997 and 2009. All cases were reviewed by two specialist hematopathologists and appropriate immunohistochemistry was carried out to confirm diagnosis according to the WHO Classification of Tumours of Hematopoietic and Lymphoid Tissues [1]. The immunohistochemical studies were performed on 4 ␮m thick paraffin sections using the EnVision method. The antibodies used included those against the CD20, CD3, CD2, CD45RO, CD30, ALK, EMA, GranzymeB and Ki-67. All stainings were performed with appropriate positive and negative controls. The stainings were evaluated under at least 10 high-power fields of tumor areas. The use of archival paraffin-embedded tissues for research was approved by the local ethics committee of each authors’ institution.

determine statistical significances. The rates of survival were estimated by the method of Kaplan and Meier and compared by log-rank tests. Multivariate analysis was performed on possible prognostic factors using the Cox model. The two-sided P values less than 0.05 were considered to be statistically significant.

3. Results 3.1. Clinical–pathological features Among the 81 patients with ALCL, 46 were male and 35 were female (the male to female ratio 1.3:1). The ages ranged from 1 to 77 years with a mean of 28.2 and a median of 26. Morphologically, “common pattern” as described in the WHO Classification of Tumours of Hematopoietic and Lymphoid Tissues [1] was the feature of the tumor in the majority of cases. The characteristic hallmark cells with eccentric, horseshoe or kidney-shaped nuclei were observed in all cases (Fig. 1A). The tumor cells expressed CD30 in all of the cases studied and the staining was seen mainly on the surface membrane and in the Golgi apparatus. Fifty of 81 (50/81, 61.7%) cases expressed ALK protein, and its subcellular distribution varied, with 24 (48%) cases showing a nuclear and cytoplasmic staining (Fig. 1B), 2 cases showing diffuse cytoplasmic staining only (Fig. 1C), 3 cases showing nuclear staining only and 2 cases showing membranous staining only. In the remaining 9 ALK positive cases, the ALK staining pattern could not be determined. The mean age was significantly younger in ALK-positive cases (19.4 years, ranged from 1 to 61 years) than in ALK-negative cases (42.4 years, ranged from 3 to 77 years) (P = 0.000).

2.2. Interphase FISH

3.2. Interphase FISH ALK dual color break apart rearrangement probe was used to detect ALK gene rearrangements and copy number changes. This probe contains two differently labeled probes on opposite sides of the breakpoint of the ALK gene. An approximately 250 kb probe for the telomeric side of the ALK breakpoint is labeled with SpectrumOrange. The centromeric probe is approximately 300 kb and labeled with SpectrumGreen. When hybridized with the ALK dual color break apart rearrangement probe, the 2p23 ALK region in its native state will be seen as two immediately adjacent or fused orange/green (yellow) signals. However, if a t(2;5) or other chromosome rearrangement at the 2p23 ALK breakpoint region has occurred, one orange and one green signal will be seen, while the native ALK region will remain as an orange/green fusion signal. Chromosome 2 enumeration probe (CEP2), which is a SpectrumOrange labeled probe specific for the alpha satellite (centromeric) region 2p11.1–q11.1, was used to determine copy number of chromosome 2 where the ALK gene is located. Chromosome 10 enumeration probe (CEP10), which is a SpectrumGreen labeled probe specific for the alpha satellite (centromeric) region 10p11.1–q11.1, was used as an additional control for evaluation of copy numbers of chromosomes since chromosome 10 aneuploidy has been reported to be rare in this type of lymphoma [11]. In a normal cell, the expected pattern for a nucleus hybridized with the CEP 2 probe or CEP10 is a two orange or two green signals pattern. In an abnormal cell, for example trisomy 2 or trisomy 10, the expected pattern will be a three orange or three green signals pattern. All the probes were purchased from Vysis, Abbott Laboratories, USA. Interphase FISH was performed on 4 ␮m thick paraffin sections of the specimens as previously described [12] with some modifications. Briefly, the tissue sections were deparaffinized and pressure-cooked in 1 mM EDTA buffer for 3 min. The sections were then digested in 0.1% of pepsin solution at 37 ◦ C for 20 min and 2–5 ␮l probe was applied onto the appropriate tissue areas. The slides were incubated at 80 ◦ C for 25 min and at 45 ◦ C for two days, followed by washing in gradient SSC solutions and counter-staining with anti-fade solution containing DAPI (Vector Labs, USA). The FISH signals were examined independently by two investigators using a fluorescence microscope (Olympus BX51, Japan). Slides with known structural or numerical abnormality for the above probes were used as the positive controls, and a case of reactive hyperplasia of tonsil served as the negative control. In each case, around 100–200 nuclei from at least 5–8 areas were examined. Nuclei with apparent overlapping or truncation were excluded from analysis. The cut-off value was established on 5 paraffin slides of the benign lymph nodes and tonsils, and was calculated as the mean plus 3 standard deviations of nuclei counted. All the cut-off values of the probes were less than 5%. 2.3. Statistical analysis Data analysis was performed using the Statistical Package for the Social Sciences software for Windows (SPSS version 11.5). The 2 and t tests were used to

The FISH study was successful in all of the ALCL cases studied, and the results are summarized in Table 1. 3.2.1. ALK gene Forty-seven of 50 ALK-positive cases (94%) showed ALK (2p23) gene break apart (Fig. 1D), indicating the presence of ALK gene involved translocations. The remaining 3 ALK-positive cases showed neither break apart nor copy number change at the ALK locus. Interestingly, among the 47 cases positive for ALK break apart, 19 cases (40.4%) also showed extra copies (up to 8 copies) of both the rearranged and non-rearranged ALK gene locus (Fig. 1E). In contrast, ALK gene break apart was not detected in any of 31 ALK-negative cases. However, two to eight extra copies of ALK gene locus were detected in 15 cases (48.4%) (Fig. 1F). The incidence of extra copies of ALK gene in ALK-positive cases (19/50, 38%) and ALK-negative cases (15/31, 48.4%) was not significantly different (P = 0.357). In 20 cases (11 ALK-positive and 9 ALK-negative) showing break apart and/or copy number changes at ALK locus, the FISH signal patterns were not uniform in tumor cells of the same case, suggesting heterogeneity of tumor cell populations in these cases (Fig. 1E and F). 3.2.2. Chromosome 2 and chromosome 10 To investigate whether the above described copy number changes were restricted to ALK gene locus or involving the centromere of chromosome 2 as well, 12 ALK-positive and 11 ALKnegative cases with extra copies of ALK were examined with CEP2 probe. Meanwhile, CEP10 probe was used as an additional reference to estimate the polyploidy change of genome. As shown in Table 2, concurrent extra copies of chromosome 2 and chromosome 10 were seen in 9 ALK-positive (75%) and 10 ALK-negative cases (91%), and the copy number of ALK (Fig. 1G), CEP2 (Fig. 1H) and CEP10 (Fig. 1I) were identical in each of these cases, suggesting polyploidy of the respective chromosomes in these cases. One

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Fig. 1. Representative cases showing typical morphology, ALK protein expression and genetic features of ALCL. (A) Hallmark cells of ALCL (HE ×400). (B) ALK protein expression showing both nuclear and cytoplasmic staining pattern (IHC ×400). (C) ALK protein expression showing cytoplasmic staining pattern. (IHC ×400). (D) Interphase FISH with ALK Dual Color Break Apart Rearrangement Probe shows nuclei with break apart of the ALK locus, indicating ALK involved translocation. Signal pattern: 1 red-and-green fused signal (indicating 1 copy of normal ALK gene) and 1 separate red and green signals (indicating 1 copy of rearrange ALK genes). (E) Interphase FISH with ALK Dual Color Break Apart Rearrangement Probe shows nuclei with both break apart and extra copies of the ALK locus. Signal pattern: 2 fusion signals (indicating 2 copies of normal ALK genes), 2 red and 2 green signals (indicating 2 copies of translocated ALK genes) in one cell, and 3 fusion signals (indicating 3 copies of normal ALK genes), 1 red and 1 green signals (indicating 1 copy of translocated ALK gene) in another cell. (F) Interphase FISH with ALK Dual Color Break Apart Rearrangement Probe shows nuclei with multiple copies of non-rearranged ALK gene. Signal pattern: multiple fusion signals (indicating extra copies of normal ALK genes). (G) Interphase FISH with ALK Dual Color Break Apart Rearrangement Probe shows nuclei with three copies of ALK gene. Signal pattern: 3 fusions. (H) Interphase FISH with CEP2 (D2Z1) Spectrum orange probe shows nuclei with three copies of chromosome 2. Signal pattern: 3 orange signals (the same case with three copies of ALK). (I) Interphase FISH with CEP10 Spectrum Green probe shows nuclei with three copies of chromosome 10. Signal pattern: 3 green signals (the same case with three copies of ALK). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Table 1 FISH results of ALK gene in ALK+ and ALK− ALCL. Diagnosis

Total no. cases

Cases with ALK break apart (%)

Cases with extra copies ALK (%)

Cases with neither break apart nor copy number change of ALK (%)

ALK+ ALK−

50 31

47 (94) 0 (0)

19 (38) 15 (48.4)

3 (6) 16 (51.6)

Total

81

47 (58.2)

34 (42)

19 (23.5)

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Table 2 FISH results of CEP2 and CEP10 in ALCL cases with extra copies of ALK gene. Total no. cases

ALK+ with extra copy of ALK (N = 12)

ALK− with extra copy of ALK (N = 11)

Extra copy of CEP2

Normal copy of CEP2

Extra copy of CEP2

Normal copy of CEP2

Extra copy of CEP10 Normal copy of CEP10

19 4

9 (75%) 0 (0%)

0 (0%) 3 (25%)

10(90.9%) 1 (9.1%)

0 (0%) 0 (0%)

Total

23

9 (39.1%)

3 (13.4%)

11 (47.8%)

0 (0%)

Fig. 2. Overall survival of ALCL. (A) ALK-positive ALCL (n = 38) vs ALK-negative ALCL (n = 17). (B) ALCL cases with 2 copies of ALK gene (n = 33) vs ALCL cases with extra copy of ALK gene (n = 22) regardless of ALK positivity. (C) ALK-positive ALCL with 2 copy of ALK gene (n = 25) vs ALK-positive ALCL with extra-copy of ALK gene (n = 13). (D) ALK-negative ALCL with 2 copy of ALK gene (n = 8) vs ALK-negative ALCL with extra copy of ALK gene (n = 9). (E) ALK-positive ALCL with extra copies of ALK gene (n = 12) vs ALK-negative ALCL without extra copies of ALK gene (n = 7).

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ALK-negative case with extra copies of chromosome 2 showed no copy number change of chromosome 10. The other 3 ALK-positive cases with extra copies of ALK gene did not show any numeric aberrations of chromosome 2 or 10, suggesting that copy number change in these cases were restricted to ALK locus or part or whole arm of 2p. 3.3. Survival Fifty-five cases had follow-up information available, with 35 survived and 20 died at the time of writing up of the study. The patients typically received multiagent chemotherapy and in most cases an anthracycline-based regime was used. The survival time ranged from 0 to 78 months with a mean of 49.3 (one-year cumulative survival 66.3%). The mean survival time of 38 ALK-positive cases (58 months) was significantly longer than that of 17 ALKnegative cases (22.5 months) (P = 0.038) (Fig. 2A). Interestingly, 22 cases (12 ALK+ and 10 ALK−) with extra copies of ALK had a significantly longer mean survival time than 33 cases (26 ALK+ and 7 ALK−) without extra copies of ALK (64.4 vs 35.3 months) (P = 0.023) (Fig. 2B), and this difference was observed in both ALK positive (72.3 vs 45.9 months) and ALK negative (34.7 vs 9.9 months) cases although the difference was not statistically significant (P = 0.062 and 0.070, respectively) (Fig. 2C and D). However, the mean survival time of ALK-positive cases with extra copies of ALK gene (12 cases, 71.8 months) was significantly longer than that of ALK-negative cases without extra copies of ALK gene (7 cases, 10.6 months) (P = 0.02, Fig. 2E). Multivariate analysis showed that ALK protein expression, multiple copy of ALK gene and age were independent predictors of the survival (P = 0.009) 4. Discussion In the 2008 edition of WHO classification [1], systemic ALCL is stratified into ALK-positive ALCL and ALK-negative ALCL according to the presence or absence of ALK protein expression. The present study, consistent with previous studies [2–6], showed that ALKpositive ALCL cases are much younger and have a more favorable outcome when compared to ALK-negative ALCL cases. Genetically, ALK-positive ALCL is characterized by carrying t(2;5)(p23;q35) or variant ALK translocations [1] whereas little is known about the molecular genetic changes in ALK-negative ALCL. In the present study, we confirmed the previous findings [13] that the great majority of ALK-positive ALCLs, but not ALK-negative ALCL, harbor an ALK gene-involved translocation. The negative finding of ALK gene rearrangement in 3 ALK-positive cases might be due to the unusual breakpoints of ALK (not detectable by FISH), such as inv(2)(p23;q35), or false ALK positivity by immunohistochemistry as previously reported by Perkins et al. [14]. In addition, we showed that a high proportion of both ALK-positive (19/50, 38.0%) and ALKnegative (15/31, 48.4%) cases carried extra copies of the ALK gene locus. Previously, Kansal et al. [11] detected extra copies of ALK gene in all of their 5 ALK-negative cases, but not in any of 6 ALKpositive cases. Our finding of the recurrent copy number gain of ALK gene locus in both ALK-positive and ALK-negative ALCLs is therefore novel. Further investigations using CEP2 and CEP 10 probes confirmed that the extra copies of ALK gene represent the extra copies of chromosome 2 in the majority of cases (20/23, 87%) and in all but one such case there were also extra copies of chromosome 10 detected (19/20, 95%), indicating the presence of polyploid genome in these cases. In only a minority of our cases, the copy number gain was restricted to ALK gene locus or part or whole arm of 2p. Interestingly, we observed in some cases with extra copies of ALK gene that the changes of ALK gene, both rearranged and non-rearranged, varied in different cells of the same case, consistent with the

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presence of heterogeneous neoplastic clones in the same tumor. Such heterogeneity of the genomic aberration may account for the clonal evolution and the morphological heterogenicity during the tumor progression. More significantly, we demonstrated for the first time that the presence of extra copies of ALK gene is an independent prognostic factor for ALCLs in our case series. The patients with extra copies of ALK gene had a significantly longer mean survival time than those without and the outcome of ALK-positive cases with extra copies of ALK gene was much better than that of ALK-negative cases without extra copies of ALK gene. This finding suggests that the extra copies of ALK gene locus have a favorable prognostic value in ALCL, making detection of the numerical changes of ALK gene necessary in both ALK-positive and ALK-negative cases. The ALK gene-involved chromosomal abnormalities include translocations, amplifications, gain or mutations [15,16]. Whilst ALK translocations have long been recognized as the characteristic genetic abnormalities resulting in activation of ALK in ALCL, pathogenesis of numerical changes of the ALK gene or its associated chromosome is unknown. Our and other study [17] suggest that the ALK gene with low copy number gain appears to remain silent, as none of the ALCL cases with extra copies of ALK but without the ALK gene rearrangement expressed ALK protein. However, gain, amplification and mutations of ALK gene have been thought to be pathogenic in neuroblastoma [16,18–20]. In future, more researches on gain, amplification and mutations of ALK gene in ALCL are needed to uncover the pathogenesis of ALCL, especially the ALK-negative ALCLs. In summary, using simple and easily applicable FISH technique we showed in the present study that copy number gain of ALK gene is a frequent genetic aberration in ALCL including both ALK-positive and ALK-negative cases. Furthermore, the copy number gain of ALK gene locus appeared to define a subgroup of ALCL with much better outcome regardless of the positivity of ALK expression. The rearrangement and copy number change of the ALK gene should therefore be evaluated for both ALK-positive and ALK-negative cases in routine practice. Conflict of interest statement The authors of this manuscript have no conflict to declare. Acknowledgement This work was supported by grants from the Beijng Natural Science Foundation of China (7122025) and the Beijing Medicine Research and Development Fund, China (2007-1030). Contributions: R.Y. and G.Ch. contributed equally to this work. R.Y, G.Ch. performed and designed experiments; Y.-F.Sh., Y.Sh., J.X. performed experiments, analysis and interpretation of data; Ch.Zh., Z.G., X.Zh. reviewed the pathologic diagnoses and interpretation of data; H.L. and L.G. designed experiments and contributed to the writing of the manuscript. References [1] Swerdlow S, Campo E, Harris N, Jaffe E, Pileri S, Stein H, et al. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon, France: IARC; 2008. [2] Shiota M, Nakamura S, Ichinohasama R, Abe M, Akagi T, Takeshita M, et al. Anaplastic large cell lymphomas expressing the novel chimeric protein p80NPM/ALK: a distinct clinicopathologic entity. Blood 1995;86:1954–60. [3] Falini B, Pileri S, Zinzani PL, Carbone A, Zagonel V, Wolf-Peeters C, et al. ALK+ lymphoma: clinico-pathological findings and outcome. Blood 1999;93:2697–706. [4] Gascoyne RD, Aoun P, Wu D, Chhanabhai M, Skinnider BF, Greiner TC, et al. Prognostic significance of anaplastic lymphoma kinase (ALK) protein expression in adults with anaplastic large cell lymphoma. Blood 1999;93:3913–21. [5] Ten BR, Oudejans JJ, Ossenkoppele GJ, Pulford K, Willemze R, Falini B, et al. ALK expression in extranodal anaplastic large cell lymphoma favours systemic

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