- Email: [email protected]
T-cell lymphoma, nasal type, with aggressive and indolent course
Clinicopathologic and molecular features in cutaneous extranodal natural killere/T-cell lymphoma, nasal type, with aggressive and indolent course Isabella Fried, MD,a Monika Artl, MSc,b Carlo Cota, MD,c Hansgeorg M€ uller, MD,d Elvira Bartolo, MD,e f g Sebastiana Boi, MD, Concetta Chiarelli, MD, Esmeralda Vale, MD,h Matthias Schmuth, MD,d Thomas Wiesner, MD,a Michael R. Speicher, MD,b and Lorenzo Cerroni, MDa Graz, Austria; Rome, Trento, and Belluno, Italy; Innsbruck, Austria; and Almada and Lisbon, Portugal Background: Extranodal natural killere/T-cell lymphoma, nasal type (ENKTCL-NT) is a highly aggressive lymphoma and prognosis is usually poor. The genetic background of primary cutaneous cases is poorly understood. Objective: We sought to evaluate the clinicopathologic features of cutaneous ENKTCL-NT, and the prognostic significance of genomic copy number alterations. Methods: Eight cases of cutaneous ENKTCL-NT (5 primary, 2 secondary, 1 no staging performed), including 2 patients with an unusually prolonged course of 5 and 23 years, were investigated using array comparative genomic hybridization. Results: All patients presented with typical clinicopathologic features. Epstein-Barr virus was found in neoplastic cells in all specimens. Copy number alterations were detected in all 8 cases with losses on 6q (37.5% of cases) and 7p (37.5% of cases), and gains on 7q (37.5% of cases) being the most frequent. Complexity of array comparative genomic hybridization profile did not correlate with the course of the disease. However, an increase of copy number alterations was detected in sequential biopsy specimens of 1 long-term survivor. Limitations: This was a small case series retrospective study. Conclusion: Clinicopathologic features of cutaneous ENKTCL-NT are distinctive. Lower number of copy number alterations cannot be used as predictor for prolonged survival in cutaneous ENKTCL-NT. ( J Am Acad Dermatol 2014;70:716-23.) Key words: array comparative genomic hybridization; clinicopathologic features; cutaneous T-cell lymphoma; extranodal natural killere/T-cell lymphoma; nasal type; genomic aberrations; prognosis.
xtranodal natural killer (NK)-/T-cell lymphoma, nasal type (ENKTCL-NT), is a highly aggressive lymphoma usually presenting in the upper respiratory tract, especially the nasal cavity. The skin is the second most affected organ and may also be the primary site of onset. Although in the past prognosis was considered to be almost invariably poor, new treatment modalities have
E
allowed a better course in recent years.1-5 However, without aggressive treatment, only a few patients show a prolonged survival.6-8 Studies on copy number alterations in cutaneous lesions of ENKTCL-NT are very limited. In a report on 4 patients with cutaneous ENKTCL-NT (3 primary and 1 secondary), Berti et al,9 using array comparative genomic hybridization (aCGH),
From the Research Unit Dermatopathology, Department of Dermatology,a and Institute of Human Genetics,b Medical University of Graz; Dermatopathology Unit, San Gallicano Dermatological Institute, Romec; Department of Dermatology, Innsbruck Medical Universityd; Dermatology Department, Hospital Garcia de Orta, Almadae; Department of Pathology, Santa Chiara Hospital, Trentof; Department of Pathology, San Martino Hospital, Bellunog; and Departments of Dermatology and Pathology, Hospital da Luz, Lisbon.h Supported in part by the Jubil€aumsfonds of the Oesterreichische Nationalbank (OeNB 13837).
Conflicts of interest: None declared. Accepted for publication November 20, 2013. Reprint requests: Lorenzo Cerroni, MD, Research Unit Dermatopathology, Department of Dermatology, Medical University of Graz, Auenbruggerplatz 8, 8036 Graz, Austria. E-mail: [email protected]. Published online January 16, 2014. 0190-9622/$36.00 Ó 2013 by the American Academy of Dermatology, Inc. http://dx.doi.org/10.1016/j.jaad.2013.11.028
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described gains of 1q and 7q and loss of 17p in primary Green methodology (both from Life Technologies, cutaneous ENKTCL-NT and gain of 7q and loss of 9p, Darmstadt, Germany) according to the manufac12p, and 12q in secondary cutaneous ENKTCL-NT. turer’s instructions. The cyclin-dependent kinase This high frequency of copy number alterations is inhibitor 2A (CDKN2A) copy number of a given consistent with findings in other NK-cell neoplasms, sample was calculated using the comparative regardless of organ of origin.10-13 However, data for DDCt method15,16 using neurofibromin 2 (NF2) cutaneous ENKTCL-NT remain very limited. As long(22q12.2) as a reference gene and male megapool term survivors are extremely reference DNA (Kreatech rare,7,8 the prognostic signifiDiagnostics, Amsterdam, CAPSULE SUMMARY The Netherlands) as refercance of chromosomal aberence sample. THP1 human rations is unclear. Cutaneous extranodal natural killere/ acute monocytic leukemia We investigated 8 cases of T-cell lymphoma, nasal type, is an cells, which have been cutaneous ENKTCL-NT using aggressive lymphoma with poor previously shown to harbor aCGH, including 2 patients prognosis. Skin is the second most a loss of CDKN2A,17 served as with an unusually prolonged common site of involvement. course. a negative control. Losses on 6q and 7p and gains on 7q are the most frequent genetic aberrations. RESULTS METHODS Rare patients experience a very Clinical data Patients prolonged course. A summary of clinical Eight patients with cutaand molecular characterisResults of array comparative genomic neous ENKTCL-NT were tics is provided in Table I. hybridization do not predict prognosis. included in the study. The There were 5 male and 3 study was approved by the female patients with a mean Ethical Committee of the age of 50 years (median: 53; range: 17-84). All Medical University of Graz, Austria, and was conpatients presented with cutaneous lesions; after ducted in accordance with the declaration of staging investigations, 5 were classified as primary Helsinki. All cases were classified according to cutaneous and 2 as secondary cutaneous ENKTCLdiagnostic criteria published in the 2008 World NT; 1 patient died before staging investigations Health Organization (WHO) classification of tumors were completed. of hematopoietic and lymphoid tissues.1 EpsteinLesions were located on 1 anatomic site in 4 cases Barr virus was positive in tumor cells in all cases. In (face 2, back 1, and arm 1, respectively) and each case a skin biopsy specimen at time of first generalized in the other 4 cases. One of the patients presentation was used for histopathological and with facial involvement showed the typical clinical molecular analyses. In 1 case (case 8), a total of 3 picture of lethal midline granuloma (Fig 1), whereas biopsy specimens over a period of 23 years could be the second presented with a persistent swelling of investigated. Partial data of this case were previously both cheeks. A persistent swelling of 1 cheek published.7 was present also in 1 patient with generalized plaques and tumors (Fig 2). The other 3 patients Array comparative genomic hybridization with generalized lesions had multiple, nonspecific Areas with at least 80% of tumor cells were microplaques and nodules. dissected from 10-m tissue sections. DNA was d
d
d
then purified using a QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany) and aCGH was performed as previously described14 using an oligonucleotide array containing 60,000 probes (Agilent, Santa Clara, CA). As fragmented DNA from FFPE section may result in unspecific cross-hybridization in subtelomeric regions, we excluded these regions to perform our analyses under very stringent conditions. Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction (PCR) was performed in triplicates using genomic DNA on a 7500 Fast Real-time PCR system using SYBR
Abbreviations used: aCGH:
array comparative genomic hybridization ARF: alternate reading frame CDKN2A: cyclin-dependent kinase inhibitor 2A EBER: Epstein-Barr virus encoded RNA-1 ENKTCL-NT: extranodal natural killer-/T-cell lymphoma, nasal type NF2: neurofibromin 2 NK: natural killer p53: tumor suppressor protein 53 PCR: polymerase chain reaction TIA-1: T-cell intracellular antigen-1 WHO: World Health Organization
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Table I. Clinical and molecular characteristics of investigated extranodal natural killere/T-cell lymphoma, nasal type Case no.
Gender
Age, y
Staging
1
M
59
PC
2 3 4 5 6 7
F M M F M M
17 51 25 63 48 84
8 (1989) 8 (2011) 8 (2012)
F
55
Skin lesions at onset
Survival, mo
aCGH profile
Generalized
3, A1
SC PC SC PC ND PC
Localized (face) Localized (face) Generalized Localized (back) Generalized Generalized
12, D1 13, A1 8, D1 64, De 1, D1 13, D1
PC
Solitary (arm) Solitary recurrence at same site Generalized
284, A1
Gain 2q; loss 1q, 3p, 3q, 4p, 4q, 6q, 9p, 12p, 12q, 15q Gain 7q; loss 7p Gain 7q; loss 6q Gain 1q; loss 19 p, 19q Gain 2q Gain 6p; loss 1p Gain 1q, 7q, 8q; loss 4p, 5p, 6q, 7p, 7q, 8p, 9p, 11q, 16p, 17q Gain 6p; loss 5p, 7p Gain 7q; loss 7p, Loss 19p, 19q Gain 1q, 3p, 3q, 4q, 6p, 6q, 7q, 14p, 14q, 16p, 20p, 20q; loss 4q, 6p, 6q, 7p, 9p, 9q, 13q, 19p, 19q
A1, Alive with lymphoma; aCGH, array comparative genomic hybridization; D1, died of lymphoma; De, died without evidence of lymphoma; F, female; M, male; ND, not determined; PC, primary cutaneous; SC, secondary cutaneous.
Fig 1. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Clinical presentation of so-called ‘‘lethal midline granuloma’’ characterized by necrotic lesions on the nose (case 3).
One patient with prolonged survival presented with a solitary tumor on the arm (a photograph was not taken at time of first diagnosis). The tumor recurred on the same arm as a solitary nodule 16 years later (a photograph was not taken), and then recurred again as a solitary tumor at the same location 6 years after successful therapy (Fig 3, A). One year later the patient had progressive disease with multiple plaques and tumors (Fig 3, B). The mean survival after diagnosis was 50 months (median: 12.5; range: 1-284). Four patients died of progressive disease with a mean survival of 8.5 months (median: 10; range: 1-13). Two patients were alive with disease at last follow-up 3 and 13 months after diagnosis, respectively. The last 2 patients had an unusually prolonged course with long survival. One of these patients (case 5) died free
Fig 2. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Persistent swelling of 1 cheek in 1 patient who also had generalized plaques and tumors (case 4).
of disease 64 months after diagnosis. The other (case 8) is alive with progressive skin disease 23 years after first diagnosis. Histology, immunohistology, and in situ hybridization All cases fulfilled histologic criteria for diagnosis of ENKTCL-NT as described in the WHO classification of hematopoietic neoplasms published in 2008.1 Briefly, biopsy specimens were characterized by dense, diffuse infiltrate of small and medium lymphocytes admixed with a minor proportion of larger cells (Fig 4). Large cells predominated in 1 case only. In 7 cases the epidermis was not affected, whereas in 1 case there was subepidermal edema with scattered intraepidermal lymphocytes. A prominent involvement of the subcutaneous tissue was observed in 3 cases. Although some degree of vessel involvement was found often,
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Fig 3. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Case 8. A, Recurrent, localized lesion on the arm of a patient with prolonged survival, arising 22 years after first diagnosis. B, One year later the patient had progressive disease with multiple plaques and tumors.
Fig 4. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Dense, diffuse infiltrates (A) of small and medium atypical lymphocytes (B) (case 1).
typical angiodestructive features were found only in 1 case (Fig 5). In all cases in situ hybridization for Epstein-Barr virus was strongly positive in all tumor cells. Tumor cells expressed a typical phenotypic profile with positivity for CD2 and negativity for other T-cell markers (but positive cytoplasmic reaction for CD33) and positivity for cytotoxic proteins (T-cell
intracellular antigen-1 [TIA-1], granzyme B) and CD56 (Fig 6). aCGH and correlation with prognosis In all 8 cases aCGH copy number alterations were identified. Two aCGH patterns were defined according to the number of chromosomal aberrations: a complex pattern (defined as presence of
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comprised only 85kb and harbors CDKN2A, an important gene for cell cycle G1 control. CDKN2A encodes the inhibitor of cyclin D-dependent kinase, p16INK4a, and the unrelated alternate reading frame (ARF) protein, which acts as stabilizer of the tumor suppressor protein p53. Loss of CDKN2A could be confirmed by quantitative real-time PCR in all 3 cases.
DISCUSSION Fig 5. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Prominent angiodestruction was found in 1 case only (case 3).
[4 aberrations), and a less complex profile (defined as presence of 1-4 aberrations). Losses on 6q (37.5% of cases) and 7p (37.5% of cases), and gains on 7q (37.5% of cases) were the most frequent aberrations (Fig 7). In 2 of 7 patients with only 1 biopsy specimen analyzed (case 1-7), a highly complex profile affecting different chromosomes could be observed, whereas the other 5 demonstrated a less complex profile with a considerably lower number of chromosomal aberrations. Comparison of aCGH pattern with follow-up information failed to reveal a correlation with prognosis (Table I). Analysis of sequential biopsy specimens in a long-term survivor We also performed aCGH analyses of 3 different biopsy specimens from 1989, 2011, and 2012 in 1 long-term survivor (case 8) (Fig 8). The patient presented in 1989 with a localized lesion on the arm that responded well to local radiotherapy. She experienced a recurrence on the same site in 2005 that was successfully treated by systemic chemotherapy (material from this biopsy specimen was not available for the study), and a new recurrence in 2011, again on the same site, treated by local radiotherapy. A less complex profile was observed in the first biopsy specimen from 1989. Only a few additional aberrations could be observed in 2011 as compared with 1989. In 2012, she experienced a new recurrence, this time with progressive skin disease. Several additional aberrations were present in this last biopsy specimen. Interestingly, we detected a newly acquired loss of 9p21 in the sample of 2012 that was not present in the previous biopsy specimens. A loss of the same region was found in 2 other cases in our study, both showing a highly complex aCGH profile. The minimal common deleted region of all 3 cases
Our study provides data on copy number alterations in cutaneous lesions of ENKTCL-NT at time of diagnosis and in follow-up samples of 1 long-term survivor. The clinicopathologic features of our patients are typical of ENKTCL-NT and underline the heterogeneous clinical and histologic features of cutaneous lesions of this rare lymphoma. In particular, ENKTCL-NT should be considered as a differential diagnosis of persistent facial swelling (observed in 2 of our patients). Another suggestive clinical presentation is that of lethal midline granuloma, characterized by necrotic lesions on the central part of the face, particularly the nose. Although angiodestruction is considered as a characteristic histopathologic finding of ENKTCL-NT, cutaneous biopsy specimens in our cases showed this feature in 1 case only, suggesting that angiodestruction may be less common in skin lesions of ENKTCL-NT. Prominent subcutaneous involvement simulating the histopathologic picture of subcutaneous ‘‘panniculitis-like’’ T-cell lymphoma is relatively frequent (3/8 cases in our series), but in contrast to genuine subcutaneous panniculitis-like T-cell lymphoma the infiltrate in ENKTCL-NT clearly involves also the dermis. However, besides evaluation of the morphologic pattern, complete phenotypic analyses are necessary in all cases of malignant lymphoma with prominent involvement of the subcutaneous fat for a precise classification. The genetic aberrations most commonly found in our cohort were losses on 6q and 7p and gains on 7q, which is consistent with previous studies. Especially loss of 6 (q21q25) has been frequently reported in ENKTCL-NT,10,11,13,18 and the transcription factors PRDM1 and FOXO3 have been identified as most likely targets in this locus. In fact, re-expression of both genes suppressed proliferation, and reconstitution of PRDM1 led to G2/M-cell cycle arrest, increased apoptosis, and a strong negative selection pressure in 6q-deleted NK-cell lines, supporting their role as tumor suppressors.19,20 Interestingly, loss of 6q was not present in the first aCGH profile of a long-term survivor but was present in the last, progression-associated profile, suggesting that it is a progression-associated rather than primary
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Fig 6. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Phenotypic and in situ hybridization features with positivity for Epstein-Barr virus encoded RNA-1 (EBER)-1 (case 4) (A); TIA-1 (case 5) (B); and CD56 (case 8) (C).
Fig 7. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Genomic aberrations detected by array comparative genomic hybridization. The ideogram summarizes copy number alterations of 8 cases at time of first diagnosis (2 sequential biopsy specimens from case 8 have not been included in this plot). The green and red bars on the right side of each chromosome demonstrate in what percentage loss ( green bar) or gain (red bar) was found in this genomic region.
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Fig 8. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Genetic aberrations in a long-term survivor (case 8). A less complex profile was present in 1989 with a few additional aberrations in the recurrent lesion of 2011, whereas a more complex profile was found in the biopsy specimen of 2012 corresponding to progressive disease.
genetic event. Aberrations on chromosome 7 have also been repeatedly reported in ENKTCL-NT and in NK-cell leukemia10,11,13 and gain on 7q was also reported in 1 primary and 1 secondary cutaneous case of ENKTCL-NT.9 Deletion of 7p was a primary event in a long-term survivor, suggesting that at least in some cases this genetic aberration may be involved in the primary lymphomagenesis. Interestingly we detected a loss of 9p21 in 3 samples of our cohort (2 at first diagnosis and 1 in the last biopsy specimen of the patient with prolonged course). Quantitative real-time PCR confirmed loss of the tumor suppressor CDKN2A in all 3 samples. Loss of 9p21 has been infrequently reported in ENKTCL-NT in the literature.10,21 As Berti et al9 have detected loss of 9p21 only in their case of secondary cutaneous ENKTCL-NT, but in none of the 3 primary cutaneous cases, they suggested that CDKN2A loss could be useful to distinguish primary cutaneous from secondary cutaneous cases of ENKTCL-NT. However, our data showing CDKN2A loss in 3 of 5 primary cutaneous ENKTCL-NT do not confirm these results. As 9p21 deletions were detected in 3 samples with a highly complex aCGH profile, and were present only in the most recent profile in 1 patient with prolonged disease and sequential biopsy specimens over 23 years, it may be speculated that CDKN2A loss is a late event in lymphomagenesis in ENKTCL-NT. Berti et al9 found also loss of 17p13.1, harboring the tumor suppressor gene 53 (TP53), in 2 of 3 primary cutaneous cases, and proposed a relevant role for TP53 in the
lymphomagenesis of primary cutaneous ENKTCLNT. Loss of 17p13.1 was detected also in a few cases of noncutaneous ENKTCL-NT12 and of aggressive NK-cell leukemia.13 In contrast to these data, we could not detect this aberration in any of our cases, thus not supporting a crucial role of TP53 in the pathogenesis of ENKTCL-NT. In our study complexity of aCGH pattern did not correlate with prognosis. Several of the cases with a less complex profile showed a rapidly progressive course and a short survival. Thus, aCGH profiles with a lower number of genetic alterations cannot be used as predictor for prolonged survival. On the other hand, both patients with prolonged course had a less complex profile at first diagnosis. It may be that some cases showing only a few genetic aberrations require further genetic events for progression to more aggressive disease. In fact, transition from a less complex to a highly complex profile was found in different biopsy specimens from 1 long-term survivor. This patient had relatively stable disease until very recently, and did present with progressive cutaneous disease in the recent past. This suggests that the lymphoma had remained genetically relatively stable for many years, becoming instable and progressive only after 23 years. In summary, our study suggests that complexity of aCGH profiles does not correlate with prognosis. In addition, we showed for the first time to our knowledge aCGH profiles in long-term survivors, including sequential biopsy specimens
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from a patient revealing changes in profile pattern that correlate with more progressive disease. The differences between the aCGH profile at first diagnosis and the last, progression-associated profile, may help to better characterize the genetic aspects of ENKTCL-NT, and may be useful for a more precise characterization of primary genetic events and progressionassociated aberrations. REFERENCES 1. Chan JKC, Quintanilla-Martinez L, Ferry JA, Peh S-C. Extranodal NK/T-cell lymphoma, nasal type. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al, editors. WHO classification of tumors of hematopoietic and lymphoid tissues. Lyon (France): IARC press; 2008. pp. 285-8. 2. Choi YL, Park JH, Namkung JH, Lee JH, Yang JM, Lee ES, et al. Extranodal NK/T-cell lymphoma with cutaneous involvement: ‘nasal’ vs ‘nasal-type’ subgroupsea retrospective study of 18 patients. Br J Dermatol 2009;160:333-7. 3. Bekkenk MW, Jansen PM, Meijer CJ, Willemze R. CD561 hematological neoplasms presenting in the skin: a retrospective analysis of 23 new cases and 130 cases from the literature. Ann Oncol 2004;15:1097-108. 4. Li S, Feng X, Li T, Zhang S, Zuo Z, Lin P, et al. Extranodal NK/T-cell lymphoma, nasal type: a report of 73 cases at MD Anderson Cancer Center. Am J Surg Pathol 2013;37:14-23. 5. Lee J, Cho SG, Chung SM, Ryu MR, Kim SH, Jang HS, et al. Retrospective analysis of treatment outcomes for extranodal NK/T-cell lymphoma (ENKL), nasal type, stage I-IIE: single institute experience of combined modality treatment for early localized nasal extranodal NK/T-cell lymphoma (ENKL). Ann Hematol 2013;92:333-43. 6. Au WY, Kim SJ, Yiu HH, Ngan RK, Loong F, Kim WS, et al. Clinicopathological features and outcome of late relapses of natural killer cell lymphomas 10-29 years after initial remission. Am J Hematol 2010;85:362-3. 7. Zuriel D, Fink-Puches R, Cerroni L. A case of primary cutaneous extranodal natural killer/T-cell lymphoma, nasal type, with a 22-year indolent clinical course. Am J Dermatopathol 2012;34: 194-7. 8. Watabe D, Kanno H, Inoue-Narita T, Onodera H, Izumida W, Kowata S, et al. A case of primary cutaneous natural killer/T-cell lymphoma, nasal type, with indolent clinical course: monoclonal expansion of Epstein-Barr virus genome correlating with the terminal aggressive behavior. Br J Dermatol 2009;160:205-7. 9. Berti E, Recalcati S, Girgenti V, Fanoni D, Venegoni L, Vezzoli P. Cutaneous extranodal NK/T-cell lymphoma: a
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clinicopathologic study of 5 patients with array-based comparative genomic hybridization. Blood 2010;116:165-70. Iqbal J, Kucuk C, Deleeuw RJ, Srivastava G, Tam W, Geng H, et al. Genomic analyses reveal global functional alterations that promote tumor growth and novel tumor suppressor genes in natural killer-cell malignancies. Leukemia 2009;23: 1139-51. Nakashima Y, Tagawa H, Suzuki R, Karnan S, Karube K, Ohshima K, et al. Genome-wide array-based comparative genomic hybridization of natural killer cell lymphoma/leukemia: different genomic alteration patterns of aggressive NK-cell leukemia and extranodal Nk/T-cell lymphoma, nasal type. Genes Chromosomes Cancer 2005;44:247-55. Ko YH, Choi KE, Han JH, Kim JM, Ree HJ. Comparative genomic hybridization study of nasal-type NK/T-cell lymphoma. Cytometry 2001;46:85-91. Siu LL, Wong KF, Chan JK, Kwong YL. Comparative genomic hybridization analysis of natural killer cell lymphoma/leukemia: recognition of consistent patterns of genetic alterations. Am J Pathol 1999;155:1419-25. Wiesner T, Obenauf AC, Cota C, Fried I, Speicher MR, Cerroni L. Alterations of the cell-cycle inhibitors p27(KIP1) and p16(INK4a) are frequent in blastic plasmacytoid dendritic cell neoplasms. J Invest Dermatol 2010;130:1152-7. Aarskog NK, Vedeler CA. Real-time quantitative polymerase chain reaction: a new method that detects both the peripheral myelin protein 22 duplication in Charcot-Marie-Tooth type 1A disease and the peripheral myelin protein 22 deletion in hereditary neuropathy with liability to pressure palsies. Hum Genet 2000;107:494-8. Zebisch A, Wolfler A, Fried I, Wolf O, Lind K, Bodner C, et al. Frequent loss of RAF kinase inhibitor protein expression in acute myeloid leukemia. Leukemia 2012;26:1842-9. Adati N, Huang MC, Suzuki T, Suzuki H, Kojima T. High-resolution analysis of aberrant regions in autosomal chromosomes in human leukemia THP-1 cell line. BMC Res Notes 2009;2:153. Siu LL, Chan V, Chan JK, Wong KF, Liang R, Kwong YL. Consistent patterns of allelic loss in natural killer cell lymphoma. Am J Pathol 2000;157:1803-9. Karube K, Nakagawa M, Tsuzuki S, Takeuchi I, Honma K, Nakashima Y, et al. Identification of FOXO3 and PRDM1 as tumor-suppressor gene candidates in NK-cell neoplasms by genomic and functional analyses. Blood 2011;118:3195-204. Kucuk C, Iqbal J, Hu X, Gaulard P, De Leval L, Srivastava G, et al. PRDM1 is a tumor suppressor gene in natural killer cell malignancies. Proc Natl Acad Sci U S A 2011;108:20119-24. Sakajiri S, Kawamata N, Egashira M, Mori K, Oshimi K. Molecular analysis of tumor suppressor genes, Rb, p53, p16INK4A, p15INK4B and p14ARF in natural killer cell neoplasms. Jpn J Cancer Res 2001;92:1048-56.
xtranodal natural killer (NK)-/T-cell lymphoma, nasal type (ENKTCL-NT), is a highly aggressive lymphoma usually presenting in the upper respiratory tract, especially the nasal cavity. The skin is the second most affected organ and may also be the primary site of onset. Although in the past prognosis was considered to be almost invariably poor, new treatment modalities have
E
allowed a better course in recent years.1-5 However, without aggressive treatment, only a few patients show a prolonged survival.6-8 Studies on copy number alterations in cutaneous lesions of ENKTCL-NT are very limited. In a report on 4 patients with cutaneous ENKTCL-NT (3 primary and 1 secondary), Berti et al,9 using array comparative genomic hybridization (aCGH),
From the Research Unit Dermatopathology, Department of Dermatology,a and Institute of Human Genetics,b Medical University of Graz; Dermatopathology Unit, San Gallicano Dermatological Institute, Romec; Department of Dermatology, Innsbruck Medical Universityd; Dermatology Department, Hospital Garcia de Orta, Almadae; Department of Pathology, Santa Chiara Hospital, Trentof; Department of Pathology, San Martino Hospital, Bellunog; and Departments of Dermatology and Pathology, Hospital da Luz, Lisbon.h Supported in part by the Jubil€aumsfonds of the Oesterreichische Nationalbank (OeNB 13837).
Conflicts of interest: None declared. Accepted for publication November 20, 2013. Reprint requests: Lorenzo Cerroni, MD, Research Unit Dermatopathology, Department of Dermatology, Medical University of Graz, Auenbruggerplatz 8, 8036 Graz, Austria. E-mail: [email protected]. Published online January 16, 2014. 0190-9622/$36.00 Ó 2013 by the American Academy of Dermatology, Inc. http://dx.doi.org/10.1016/j.jaad.2013.11.028
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described gains of 1q and 7q and loss of 17p in primary Green methodology (both from Life Technologies, cutaneous ENKTCL-NT and gain of 7q and loss of 9p, Darmstadt, Germany) according to the manufac12p, and 12q in secondary cutaneous ENKTCL-NT. turer’s instructions. The cyclin-dependent kinase This high frequency of copy number alterations is inhibitor 2A (CDKN2A) copy number of a given consistent with findings in other NK-cell neoplasms, sample was calculated using the comparative regardless of organ of origin.10-13 However, data for DDCt method15,16 using neurofibromin 2 (NF2) cutaneous ENKTCL-NT remain very limited. As long(22q12.2) as a reference gene and male megapool term survivors are extremely reference DNA (Kreatech rare,7,8 the prognostic signifiDiagnostics, Amsterdam, CAPSULE SUMMARY The Netherlands) as refercance of chromosomal aberence sample. THP1 human rations is unclear. Cutaneous extranodal natural killere/ acute monocytic leukemia We investigated 8 cases of T-cell lymphoma, nasal type, is an cells, which have been cutaneous ENKTCL-NT using aggressive lymphoma with poor previously shown to harbor aCGH, including 2 patients prognosis. Skin is the second most a loss of CDKN2A,17 served as with an unusually prolonged common site of involvement. course. a negative control. Losses on 6q and 7p and gains on 7q are the most frequent genetic aberrations. RESULTS METHODS Rare patients experience a very Clinical data Patients prolonged course. A summary of clinical Eight patients with cutaand molecular characterisResults of array comparative genomic neous ENKTCL-NT were tics is provided in Table I. hybridization do not predict prognosis. included in the study. The There were 5 male and 3 study was approved by the female patients with a mean Ethical Committee of the age of 50 years (median: 53; range: 17-84). All Medical University of Graz, Austria, and was conpatients presented with cutaneous lesions; after ducted in accordance with the declaration of staging investigations, 5 were classified as primary Helsinki. All cases were classified according to cutaneous and 2 as secondary cutaneous ENKTCLdiagnostic criteria published in the 2008 World NT; 1 patient died before staging investigations Health Organization (WHO) classification of tumors were completed. of hematopoietic and lymphoid tissues.1 EpsteinLesions were located on 1 anatomic site in 4 cases Barr virus was positive in tumor cells in all cases. In (face 2, back 1, and arm 1, respectively) and each case a skin biopsy specimen at time of first generalized in the other 4 cases. One of the patients presentation was used for histopathological and with facial involvement showed the typical clinical molecular analyses. In 1 case (case 8), a total of 3 picture of lethal midline granuloma (Fig 1), whereas biopsy specimens over a period of 23 years could be the second presented with a persistent swelling of investigated. Partial data of this case were previously both cheeks. A persistent swelling of 1 cheek published.7 was present also in 1 patient with generalized plaques and tumors (Fig 2). The other 3 patients Array comparative genomic hybridization with generalized lesions had multiple, nonspecific Areas with at least 80% of tumor cells were microplaques and nodules. dissected from 10-m tissue sections. DNA was d
d
d
then purified using a QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany) and aCGH was performed as previously described14 using an oligonucleotide array containing 60,000 probes (Agilent, Santa Clara, CA). As fragmented DNA from FFPE section may result in unspecific cross-hybridization in subtelomeric regions, we excluded these regions to perform our analyses under very stringent conditions. Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction (PCR) was performed in triplicates using genomic DNA on a 7500 Fast Real-time PCR system using SYBR
Abbreviations used: aCGH:
array comparative genomic hybridization ARF: alternate reading frame CDKN2A: cyclin-dependent kinase inhibitor 2A EBER: Epstein-Barr virus encoded RNA-1 ENKTCL-NT: extranodal natural killer-/T-cell lymphoma, nasal type NF2: neurofibromin 2 NK: natural killer p53: tumor suppressor protein 53 PCR: polymerase chain reaction TIA-1: T-cell intracellular antigen-1 WHO: World Health Organization
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Table I. Clinical and molecular characteristics of investigated extranodal natural killere/T-cell lymphoma, nasal type Case no.
Gender
Age, y
Staging
1
M
59
PC
2 3 4 5 6 7
F M M F M M
17 51 25 63 48 84
8 (1989) 8 (2011) 8 (2012)
F
55
Skin lesions at onset
Survival, mo
aCGH profile
Generalized
3, A1
SC PC SC PC ND PC
Localized (face) Localized (face) Generalized Localized (back) Generalized Generalized
12, D1 13, A1 8, D1 64, De 1, D1 13, D1
PC
Solitary (arm) Solitary recurrence at same site Generalized
284, A1
Gain 2q; loss 1q, 3p, 3q, 4p, 4q, 6q, 9p, 12p, 12q, 15q Gain 7q; loss 7p Gain 7q; loss 6q Gain 1q; loss 19 p, 19q Gain 2q Gain 6p; loss 1p Gain 1q, 7q, 8q; loss 4p, 5p, 6q, 7p, 7q, 8p, 9p, 11q, 16p, 17q Gain 6p; loss 5p, 7p Gain 7q; loss 7p, Loss 19p, 19q Gain 1q, 3p, 3q, 4q, 6p, 6q, 7q, 14p, 14q, 16p, 20p, 20q; loss 4q, 6p, 6q, 7p, 9p, 9q, 13q, 19p, 19q
A1, Alive with lymphoma; aCGH, array comparative genomic hybridization; D1, died of lymphoma; De, died without evidence of lymphoma; F, female; M, male; ND, not determined; PC, primary cutaneous; SC, secondary cutaneous.
Fig 1. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Clinical presentation of so-called ‘‘lethal midline granuloma’’ characterized by necrotic lesions on the nose (case 3).
One patient with prolonged survival presented with a solitary tumor on the arm (a photograph was not taken at time of first diagnosis). The tumor recurred on the same arm as a solitary nodule 16 years later (a photograph was not taken), and then recurred again as a solitary tumor at the same location 6 years after successful therapy (Fig 3, A). One year later the patient had progressive disease with multiple plaques and tumors (Fig 3, B). The mean survival after diagnosis was 50 months (median: 12.5; range: 1-284). Four patients died of progressive disease with a mean survival of 8.5 months (median: 10; range: 1-13). Two patients were alive with disease at last follow-up 3 and 13 months after diagnosis, respectively. The last 2 patients had an unusually prolonged course with long survival. One of these patients (case 5) died free
Fig 2. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Persistent swelling of 1 cheek in 1 patient who also had generalized plaques and tumors (case 4).
of disease 64 months after diagnosis. The other (case 8) is alive with progressive skin disease 23 years after first diagnosis. Histology, immunohistology, and in situ hybridization All cases fulfilled histologic criteria for diagnosis of ENKTCL-NT as described in the WHO classification of hematopoietic neoplasms published in 2008.1 Briefly, biopsy specimens were characterized by dense, diffuse infiltrate of small and medium lymphocytes admixed with a minor proportion of larger cells (Fig 4). Large cells predominated in 1 case only. In 7 cases the epidermis was not affected, whereas in 1 case there was subepidermal edema with scattered intraepidermal lymphocytes. A prominent involvement of the subcutaneous tissue was observed in 3 cases. Although some degree of vessel involvement was found often,
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Fig 3. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Case 8. A, Recurrent, localized lesion on the arm of a patient with prolonged survival, arising 22 years after first diagnosis. B, One year later the patient had progressive disease with multiple plaques and tumors.
Fig 4. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Dense, diffuse infiltrates (A) of small and medium atypical lymphocytes (B) (case 1).
typical angiodestructive features were found only in 1 case (Fig 5). In all cases in situ hybridization for Epstein-Barr virus was strongly positive in all tumor cells. Tumor cells expressed a typical phenotypic profile with positivity for CD2 and negativity for other T-cell markers (but positive cytoplasmic reaction for CD33) and positivity for cytotoxic proteins (T-cell
intracellular antigen-1 [TIA-1], granzyme B) and CD56 (Fig 6). aCGH and correlation with prognosis In all 8 cases aCGH copy number alterations were identified. Two aCGH patterns were defined according to the number of chromosomal aberrations: a complex pattern (defined as presence of
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comprised only 85kb and harbors CDKN2A, an important gene for cell cycle G1 control. CDKN2A encodes the inhibitor of cyclin D-dependent kinase, p16INK4a, and the unrelated alternate reading frame (ARF) protein, which acts as stabilizer of the tumor suppressor protein p53. Loss of CDKN2A could be confirmed by quantitative real-time PCR in all 3 cases.
DISCUSSION Fig 5. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Prominent angiodestruction was found in 1 case only (case 3).
[4 aberrations), and a less complex profile (defined as presence of 1-4 aberrations). Losses on 6q (37.5% of cases) and 7p (37.5% of cases), and gains on 7q (37.5% of cases) were the most frequent aberrations (Fig 7). In 2 of 7 patients with only 1 biopsy specimen analyzed (case 1-7), a highly complex profile affecting different chromosomes could be observed, whereas the other 5 demonstrated a less complex profile with a considerably lower number of chromosomal aberrations. Comparison of aCGH pattern with follow-up information failed to reveal a correlation with prognosis (Table I). Analysis of sequential biopsy specimens in a long-term survivor We also performed aCGH analyses of 3 different biopsy specimens from 1989, 2011, and 2012 in 1 long-term survivor (case 8) (Fig 8). The patient presented in 1989 with a localized lesion on the arm that responded well to local radiotherapy. She experienced a recurrence on the same site in 2005 that was successfully treated by systemic chemotherapy (material from this biopsy specimen was not available for the study), and a new recurrence in 2011, again on the same site, treated by local radiotherapy. A less complex profile was observed in the first biopsy specimen from 1989. Only a few additional aberrations could be observed in 2011 as compared with 1989. In 2012, she experienced a new recurrence, this time with progressive skin disease. Several additional aberrations were present in this last biopsy specimen. Interestingly, we detected a newly acquired loss of 9p21 in the sample of 2012 that was not present in the previous biopsy specimens. A loss of the same region was found in 2 other cases in our study, both showing a highly complex aCGH profile. The minimal common deleted region of all 3 cases
Our study provides data on copy number alterations in cutaneous lesions of ENKTCL-NT at time of diagnosis and in follow-up samples of 1 long-term survivor. The clinicopathologic features of our patients are typical of ENKTCL-NT and underline the heterogeneous clinical and histologic features of cutaneous lesions of this rare lymphoma. In particular, ENKTCL-NT should be considered as a differential diagnosis of persistent facial swelling (observed in 2 of our patients). Another suggestive clinical presentation is that of lethal midline granuloma, characterized by necrotic lesions on the central part of the face, particularly the nose. Although angiodestruction is considered as a characteristic histopathologic finding of ENKTCL-NT, cutaneous biopsy specimens in our cases showed this feature in 1 case only, suggesting that angiodestruction may be less common in skin lesions of ENKTCL-NT. Prominent subcutaneous involvement simulating the histopathologic picture of subcutaneous ‘‘panniculitis-like’’ T-cell lymphoma is relatively frequent (3/8 cases in our series), but in contrast to genuine subcutaneous panniculitis-like T-cell lymphoma the infiltrate in ENKTCL-NT clearly involves also the dermis. However, besides evaluation of the morphologic pattern, complete phenotypic analyses are necessary in all cases of malignant lymphoma with prominent involvement of the subcutaneous fat for a precise classification. The genetic aberrations most commonly found in our cohort were losses on 6q and 7p and gains on 7q, which is consistent with previous studies. Especially loss of 6 (q21q25) has been frequently reported in ENKTCL-NT,10,11,13,18 and the transcription factors PRDM1 and FOXO3 have been identified as most likely targets in this locus. In fact, re-expression of both genes suppressed proliferation, and reconstitution of PRDM1 led to G2/M-cell cycle arrest, increased apoptosis, and a strong negative selection pressure in 6q-deleted NK-cell lines, supporting their role as tumor suppressors.19,20 Interestingly, loss of 6q was not present in the first aCGH profile of a long-term survivor but was present in the last, progression-associated profile, suggesting that it is a progression-associated rather than primary
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Fig 6. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Phenotypic and in situ hybridization features with positivity for Epstein-Barr virus encoded RNA-1 (EBER)-1 (case 4) (A); TIA-1 (case 5) (B); and CD56 (case 8) (C).
Fig 7. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Genomic aberrations detected by array comparative genomic hybridization. The ideogram summarizes copy number alterations of 8 cases at time of first diagnosis (2 sequential biopsy specimens from case 8 have not been included in this plot). The green and red bars on the right side of each chromosome demonstrate in what percentage loss ( green bar) or gain (red bar) was found in this genomic region.
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Fig 8. Cutaneous extranodal natural killere/T-cell lymphoma, nasal type. Genetic aberrations in a long-term survivor (case 8). A less complex profile was present in 1989 with a few additional aberrations in the recurrent lesion of 2011, whereas a more complex profile was found in the biopsy specimen of 2012 corresponding to progressive disease.
genetic event. Aberrations on chromosome 7 have also been repeatedly reported in ENKTCL-NT and in NK-cell leukemia10,11,13 and gain on 7q was also reported in 1 primary and 1 secondary cutaneous case of ENKTCL-NT.9 Deletion of 7p was a primary event in a long-term survivor, suggesting that at least in some cases this genetic aberration may be involved in the primary lymphomagenesis. Interestingly we detected a loss of 9p21 in 3 samples of our cohort (2 at first diagnosis and 1 in the last biopsy specimen of the patient with prolonged course). Quantitative real-time PCR confirmed loss of the tumor suppressor CDKN2A in all 3 samples. Loss of 9p21 has been infrequently reported in ENKTCL-NT in the literature.10,21 As Berti et al9 have detected loss of 9p21 only in their case of secondary cutaneous ENKTCL-NT, but in none of the 3 primary cutaneous cases, they suggested that CDKN2A loss could be useful to distinguish primary cutaneous from secondary cutaneous cases of ENKTCL-NT. However, our data showing CDKN2A loss in 3 of 5 primary cutaneous ENKTCL-NT do not confirm these results. As 9p21 deletions were detected in 3 samples with a highly complex aCGH profile, and were present only in the most recent profile in 1 patient with prolonged disease and sequential biopsy specimens over 23 years, it may be speculated that CDKN2A loss is a late event in lymphomagenesis in ENKTCL-NT. Berti et al9 found also loss of 17p13.1, harboring the tumor suppressor gene 53 (TP53), in 2 of 3 primary cutaneous cases, and proposed a relevant role for TP53 in the
lymphomagenesis of primary cutaneous ENKTCLNT. Loss of 17p13.1 was detected also in a few cases of noncutaneous ENKTCL-NT12 and of aggressive NK-cell leukemia.13 In contrast to these data, we could not detect this aberration in any of our cases, thus not supporting a crucial role of TP53 in the pathogenesis of ENKTCL-NT. In our study complexity of aCGH pattern did not correlate with prognosis. Several of the cases with a less complex profile showed a rapidly progressive course and a short survival. Thus, aCGH profiles with a lower number of genetic alterations cannot be used as predictor for prolonged survival. On the other hand, both patients with prolonged course had a less complex profile at first diagnosis. It may be that some cases showing only a few genetic aberrations require further genetic events for progression to more aggressive disease. In fact, transition from a less complex to a highly complex profile was found in different biopsy specimens from 1 long-term survivor. This patient had relatively stable disease until very recently, and did present with progressive cutaneous disease in the recent past. This suggests that the lymphoma had remained genetically relatively stable for many years, becoming instable and progressive only after 23 years. In summary, our study suggests that complexity of aCGH profiles does not correlate with prognosis. In addition, we showed for the first time to our knowledge aCGH profiles in long-term survivors, including sequential biopsy specimens
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from a patient revealing changes in profile pattern that correlate with more progressive disease. The differences between the aCGH profile at first diagnosis and the last, progression-associated profile, may help to better characterize the genetic aspects of ENKTCL-NT, and may be useful for a more precise characterization of primary genetic events and progressionassociated aberrations. REFERENCES 1. Chan JKC, Quintanilla-Martinez L, Ferry JA, Peh S-C. Extranodal NK/T-cell lymphoma, nasal type. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al, editors. WHO classification of tumors of hematopoietic and lymphoid tissues. Lyon (France): IARC press; 2008. pp. 285-8. 2. Choi YL, Park JH, Namkung JH, Lee JH, Yang JM, Lee ES, et al. Extranodal NK/T-cell lymphoma with cutaneous involvement: ‘nasal’ vs ‘nasal-type’ subgroupsea retrospective study of 18 patients. Br J Dermatol 2009;160:333-7. 3. Bekkenk MW, Jansen PM, Meijer CJ, Willemze R. CD561 hematological neoplasms presenting in the skin: a retrospective analysis of 23 new cases and 130 cases from the literature. Ann Oncol 2004;15:1097-108. 4. Li S, Feng X, Li T, Zhang S, Zuo Z, Lin P, et al. Extranodal NK/T-cell lymphoma, nasal type: a report of 73 cases at MD Anderson Cancer Center. Am J Surg Pathol 2013;37:14-23. 5. Lee J, Cho SG, Chung SM, Ryu MR, Kim SH, Jang HS, et al. Retrospective analysis of treatment outcomes for extranodal NK/T-cell lymphoma (ENKL), nasal type, stage I-IIE: single institute experience of combined modality treatment for early localized nasal extranodal NK/T-cell lymphoma (ENKL). Ann Hematol 2013;92:333-43. 6. Au WY, Kim SJ, Yiu HH, Ngan RK, Loong F, Kim WS, et al. Clinicopathological features and outcome of late relapses of natural killer cell lymphomas 10-29 years after initial remission. Am J Hematol 2010;85:362-3. 7. Zuriel D, Fink-Puches R, Cerroni L. A case of primary cutaneous extranodal natural killer/T-cell lymphoma, nasal type, with a 22-year indolent clinical course. Am J Dermatopathol 2012;34: 194-7. 8. Watabe D, Kanno H, Inoue-Narita T, Onodera H, Izumida W, Kowata S, et al. A case of primary cutaneous natural killer/T-cell lymphoma, nasal type, with indolent clinical course: monoclonal expansion of Epstein-Barr virus genome correlating with the terminal aggressive behavior. Br J Dermatol 2009;160:205-7. 9. Berti E, Recalcati S, Girgenti V, Fanoni D, Venegoni L, Vezzoli P. Cutaneous extranodal NK/T-cell lymphoma: a
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