- Email: [email protected]
CD8 expression in anaplastic large cell lymphoma (ALCL) correlates with non-common morphologic variants and T-cell antigen expression suggesting biological differences with CD8-negative ALCL
Journal Pre-proof CD8 expression in anaplastic large cell lymphoma (ALCL) correlates with noncommon morphologic variants and T-cell antigen expression suggesting biological differences with CD8-negative ALCL Jing Shen, L. Jeffrey Medeiros, Shaoying Li, Sa A. Wang, Pei Lin, Mahsa Khanlari, Swaminathan P. Iyer, C. Cameron Yin, Guilin Tang, Jeffrey L. Jorgensen, Shimin Hu, Roberto N. Miranda, Jie Xu PII:
S0046-8177(20)30018-6
DOI:
https://doi.org/10.1016/j.humpath.2020.01.005
Reference:
YHUPA 4965
To appear in:
Human Pathology
Received Date: 20 September 2019 Revised Date:
14 January 2020
Accepted Date: 28 January 2020
Please cite this article as: Shen J, Medeiros LJ, Li S, Wang SA, Lin P, Khanlari M, Iyer SP, Yin CC, Tang G, Jorgensen JL, Hu S, Miranda RN, Xu J, CD8 expression in anaplastic large cell lymphoma (ALCL) correlates with non-common morphologic variants and T-cell antigen expression suggesting biological differences with CD8-negative ALCL, Human Pathology, https://doi.org/10.1016/ j.humpath.2020.01.005. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
CD8 expression in anaplastic large cell lymphoma (ALCL) correlates with non-common morphologic variants and T-cell antigen expression suggesting biological differences with CD8-negative ALCL Jing Shen1,2, L. Jeffrey Medeiros1, Shaoying Li1, Sa A. Wang1, Pei Lin1, Mahsa Khanlari1, Swaminathan P. Iyer3, C. Cameron Yin1, Guilin Tang1, Jeffrey L. Jorgensen1, Shimin Hu1, Roberto N. Miranda1, Jie Xu1 1 Department of Hematopathology, 3Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX 2
Department of Hematology, Capital Medical University Beijing Friendship Hospital, Beijing, China
Words of Abstract: 250 Number of Tables: 2 Number of Figures: 5 Running Title: CD8, anaplastic large cell lymphoma Key Words: CD8, anaplastic large cell lymphoma, pathology, prognosis Conflicts of Interest and Source of Funding: The authors have disclosed that they have no relationships with, or financial interest in, any commercial companies pertaining to this article. Part of the data has been presented at the Annual Meeting of United States and Canadian Academy of Pathology in 2019. Correspondence: Jie Xu, MD PhD Department of Hematopathology MD Anderson Cancer Center 1515 Holcombe Blvd, Unit 72 Houston, TX 77030 Phone: 713-794-1220 Fax: 713-792-8438 Email: [email protected]
1
ABSTRACT Anaplastic large cell lymphoma (ALCL) is a T cell neoplasm characterized by uniformly strong CD30 expression and common absence of T cell markers. Most ALCL cases express CD4, but a small subset of ALCL cases has been reported to express CD8. Little is known about the clinicopathologic and prognostic features of CD8+ ALCL. In this study, CD8 was assessed in 158 patients with systemic ALCL: CD8 was positive in 13 of 67 (19%) ALK+ and 13 of 91 (14%) ALK-negative neoplasms. In ALK+ ALCL, the CD8+ subgroup more often showed a non-common morphologic pattern (69% vs 13%, p = 0.0001) and was more often positive for CD2 (100% vs 45%, p = 0.001), CD3 (92% vs 24%, p = 0.0001), and CD7 (100% vs. 39%, p = 0.002), but less frequently positive for CD25 (50% vs. 100%, p = 0.02). ALK+ ALCL patients with CD8+ neoplasms also had a higher relapse rate (82% vs 48%, p = 0.05) and more often underwent stem cell transplant (73% vs 36%, p = 0.04). CD8 expression did not correlate with patient overall survival or progression-free survival regardless of ALK status (all p > 0.05). We conclude that CD8+ ALCL cases appear to be biologically different from the more common CD8-negative ALCL cases. Our data suggest that CD8 positivity in ALK+ ALCL helps to identify a patient subset more prone to relapse or more in need of stem cell transplant during their clinical course, although there was no impact on survival in this cohort.
2
Introduction Systemic anaplastic large cell lymphoma (ALCL) is a T cell neoplasm characterized by lymphoma cells that are usually large and pleomorphic with horseshoe-shaped nuclei and abundant cytoplasm (so-called “hallmark” cells), uniform and strong CD30 expression, a CD4+ cytotoxic immunophenotype, and aberrant loss of one or more T cell antigens. Patients with ALCL frequently have disease involving lymph nodes and extranodal sites, and many patients present with advanced stage disease and B symptoms. ALCL can be further classified into ALK+ and ALK-negative types.[1] ALK+ ALCL is characterized by ALK expression resulting from translocations involving ALK at 2p23, most commonly t(2;5)(p23;q35)/NPM1-ALK. Patients with ALK+ ALCL tend to be younger with a male predominance. These neoplasms are morphologically heterogeneous and five morphologic patterns are recognized in the current World Health Organization (WHO) classification: common, small cell, lymphohistiocytic, Hodgkin-like, and composite patterns.[1] Patients with ALK-negative ALCL lack abnormalities involving ALK. Compared to ALK+ ALCL, patients with ALK-negative ALCL are usually older, less often have extranodal involvement, and more often have an aggressive clinical course and poorer outcome. The long-term overall survival rates are 70-90% in patients with ALK+ ALCL, compared with < 50% in patients with ALK-negative ALCL.[2-11] CD8+ cases of ALCL have been reported, mostly in the form of case reports and small case series.[12, 13] The clinicopathologic features and outcome of patients with CD8+ ALCL are not well studied. One study in the literature using immunofluorescence showed that CD8 expression is associated with a non-common morphologic pattern and poorer outcome in pediatric patients with ALK+ ALCL.[14] We are not aware of a similarly designed study for patients with ALK-negative ALCL.
3
In this study, we assessed CD8 expression in ALK+ and ALK-negative ALCL cases by using flow cytometry immunophenotypic and/or immunohistochemical methods and correlated the results with clinicopathologic features and patient outcome.
4
Materials and Methods Case Selection We searched the database of the Department of Hematopathology at The University of Texas MD Anderson Cancer Center from January 1, 2007 through December 31, 2018 for cases of systemic ALCL that had CD8 results available by flow cytometry and/or immunohistochemistry. The diagnosis and sub-classification of ALCL were based on criteria specified in the 2016 WHO classification.[1] The diagnosis of ALK+ ALCL was confirmed by ALK expression by immunohistochemistry and/or t(2;5)(p23;q35) by conventional cytogenetics or ALK rearrangement by fluorescence in situ hybridization (FISH) analysis. Clinical information was obtained by review of medical records. This study was approved by the institutional review board.
Immunophenotypic Analysis Immunohistochemical studies were performed using formalin-fixed, paraffin-embedded (FFPE) tissue sections, either at the time of diagnosis or retrospectively for this study as described previously.[15] Immunohistochemical analysis was performed on an automated immunostainer (Leica Bond-Max IHC Stainer, San Diego, CA). Tissue sections, 4-µm-thick, were deparaffinized and underwent heat-induced antigen retrieval using the Bond Max Epitope Retrieval 1 solution for 15 minutes. The antibodies used were specific for CD2, CD7, EMA (Leica Biosystems, Newcastle, United Kingdom); CD3, CD20, CD43, CD45 (Dako, Carpinteria, CA, USA); CD4 (Cell Marque, Rocklin, CA, USA); CD5 (SP4; Labvision/Neomarkers, Fremont, CA, USA); CD8, granzyme B (Thermo Fisher, Waltham, MA, USA); ALK (Cell
5
Signaling, Danvers, MA, USA); and PAX5 (Transduction Labs, San Diego, CA, USA). The Bond Refine Polymer detection system was used for visualization. Flow cytometry immunophenotypic analysis was performed on cell suspensions of tissue biopsy specimens or bone marrow aspirates using either a FACScanto II or FACSCalibur cytometer (Becton-Dickinson Biosciences, San Jose, CA, USA) as has been described. [15] Lymphocytes were gated for analysis using side scatter versus forward scatter and CD45 expression versus side scatter. The panel of monoclonal antibodies included reagents specific for CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD25, CD30, CD45, TCR alpha/beta, TCR gamma/delta (Becton-Dickinson Biosciences, San Jose, CA, USA).
Conventional Cytogenetic Analysis and Fluorescence in situ Hybridization Conventional cytogenetic analysis was performed on metaphase cells prepared from bone marrow aspirates or cell suspensions from tissue biopsy specimens as described previously. [16] Twenty Giemsa-banded metaphases were analyzed, and the results were reported using the 2016 International System for Human Cytogenetic Nomenclature. FISH analysis was performed using a LSI ALK dual color, breakapart rearrangement probe (Abbott Molecular, Des Plaines, IL, USA) on interphase nuclei obtained from bone marrow cells or tissue sections, according to the manufacturer’s instructions.
Statistical Analysis Statistical analyses were performed using the Graph-Pad Prism 7. Fisher’s exact test was utilized to compare the clinicopathologic features between CD8+ versus CD8-negative subgroups in patients with ALK+ or ALK-negative anaplastic large cell lymphoma, respectively.
6
Overall survival was calculated from the date of initial diagnosis to the date of death or last follow-up. Progression-free survival was calculated from the date of diagnosis to the date of progression/relapse or, if no progression/relapse, the date of death or last follow-up. Survival was analyzed using the Kaplan–Meier method and was compared using the log rank test. A p value of less than 0.05 was considered statistically significant.
7
Results Clinical Findings We identified 158 cases of ALCL that had CD8 results available including 67 ALK+ and 91 ALK-negative. Twenty-six cases were assessed by flow cytometry only, 26 cases were assessed by both flow cytometry and immunohistochemistry, and 106 cases were assessed only by immunohistochemistry. The CD8 results assessed by flow cytometry correlated with those assessed by immunohistochemistry (p = 0.0001, data not shown). There was no difference in CD8 positivity rate between ALK+ versus ALK-negative ALCL (19% vs 14%, p = 0.40). The clinical features of these patients are summarized in Table 1. The ALK+ ALCL cases included 13 (19%) that were CD8+ and 54 that were CD8negative. The ALK+ CD8+ subgroup included 7 men and 6 women with a median age of 22 years (range, 9-55 years) at the time of diagnosis. Nine of 11 (82%) patients had B symptoms. Lymphadenopathy was identified in 11 of 12 (92%) patients and 4 of 8 (50%) had extranodal involvement. Bone marrow was involved in 2 of 9 (22%) patients. Five of 8 (63%) fully staged patients had stage III or IV disease. Two of 5 (40%) patients had leukocytosis and 1 of 4 (25%) had absolute lymphocytosis. Two of 4 (50%) patients tested showed an elevated serum LDH level. None of the patients had an International Prognostic Index (IPI) score of > 3 (Table 1). The ALK+ CD8-negative subgroup included 54 patients, 38 men and 16 women with a median age of 28 years (range 4-75 years). The clinical features of the ALK+ CD8-negative subgroup were similar to the ALK+ CD8+ subgroup (all p > 0.05; Table 1). The ALK-negative ALCL cases included 13 (14%) that were CD8+ and 78 that were CD8-negative. As shown in Table 1, there were no significant differences in the clinical features between CD8+ versus CD8-negative subgroups of ALK-negative ALCL (all p > 0.05).
8
Pathologic Findings In ALK+ ALCL, the CD8+ cases included 4 (31%) with common (or classic) morphology and 9 (69%) with non-common morphologic patterns: 4 (31%) small cell, 2 (15%) lymphohistiocytic, and 3 (23%) composite (2 small cell/lymphohistiocytic, 1 small cell/common) (Table 2; Figures 1 and 3). The CD8+ subset of ALK+ ALCL cases more frequently showed non-common morphology (small cell and/or lymphohistiocytic variant) (69% vs. 13%, p = 0.0001). CD8+ cases also had variable expression of T cell antigens (Table 2): all cases were CD2+ (n = 10) and CD7+ (n = 9), 12 of 13 (92%) were CD3+, and 5 of 11 (45%) were CD5+. Eight of 12 67%) cases showed weak or partial CD3 positivity: 6 by immunohistochemistry and flow cytometry, 1 by immunohistochemistry, and 1 by flow cytometry. Using immunohistochemistry, 9 of 13 (69%) cases were positive for CD3, and by flow cytometry, 7 of 8 (88%) cases were positive for surface CD3. The CD8+ subgroup was significantly associated with more frequent CD3 expression, assessed by either immunohistochemistry (69% vs. 17%, p = 0.0005) or flow cytometry (88% vs. 20%, p = 0.0019). In addition, all CD8+ cases of ALK+ ALCL were positive for CD45 (n = 9) and granzyme B (n = 6), 5 of 6 (83%) were EMA+, and 3 of 6 (50%) were CD25+. In ALK+ ALCL, the CD8+ subgroup as compared with the CD8negative group was more often positive for CD2 (100% vs 45%, p = 0.001), CD3 (92% vs 24%, p = 0.0001), and CD7 (100% vs. 39%, p = 0.002), but less frequently positive for CD25 (50% vs. 100%, p = 0.02). There was no significant difference in the frequency of expression of CD5, CD45, granzyme B, and EMA between CD8+ versus CD8-negative ALK+ ALCL (all p > 0.05, Table 2).
9
In ALK-negative ALCL, all CD8+ neoplasms and 77 of 78 (99%) CD8-negative neoplasms had common morphologic features. Comparing the immunophenotypic features between the CD8+ versus CD8-negative subgroups, most features were similar except for CD3. All 13 CD8+ cases of ALK-negative ALCL were positive for CD3 (all cases by immunohistochemistry and 3/5 cases by flow cytometry). CD8+ cases of ALK-negative ALCL were more often positive for CD3 than CD8-negative cases (100% vs 48%, p = 0.0004; Table 2). CD8+ subgroup was also significantly associated with more frequent CD3 expression, assessed by immunohistochemistry (100% vs. 42%, p = 0.0001).
Treatment and Response Fifty-six patients with ALK+ ALCL and 71 patients with ALK-negative ALCL had treatment and follow-up information available. All patients were treated with various chemotherapy regimens over the time interval of this study, with or without stem cell transplant (SCT). In patients with ALK+ ALCL that were CD8+, 10 of 11 (91%) were treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or modified CHOP (Table 1). After initial induction chemotherapy, 6 of 11 (55%) patients achieved complete remission, but 9 of 11 (82%) patients relapsed. Eight of 11 (73%) patients received SCT: 4 autologous and 4 allogeneic. There was no significant difference in initial treatment or complete remission rate between the CD8+ versus CD8-negative ALK+ ALCL subgroups (all p > 0.05; Table 1); however, patients with CD8+ tumors more often received SCT than patients with CD8-negative tumors (73% vs 36%, p = 0.04). This result might be attributable to the higher relapse rate in CD8+ subgroup (82% vs 48%, p = 0.05). In patients with known time of relapse and SCT, 5 of 8 (63%) CD8+ patients versus 12 of 19 (63%) CD8-negative patients received SCT after their
10
disease relapsed; there was no statistical difference in SCT frequency between these groups (p = 1.0). In patients with ALK-negative ALCL, there was no significant difference in treatment, complete response rate, or relapse rate between the CD8+ versus CD8-negative subgroups (all p > 0.05, Table 1).
Outcome After a median clinical follow-up of 19 months (range, 0-186 months), 49 of 158 (31%) patients died. Ten of 26 (39%) patients with CD8+ ALCL died: 3 ALK+ and 7 ALK-negative. Survival analysis was performed in patients based on ALK status. CD8 expression did not affect overall survival (OS) in patients with either ALK+ or ALK-negative ALCL (p = 0.82 and p = 0.45, respectively; Figs. 4A and B). Furthermore, CD8 had no impact on progression-free survival (PFS) in patients with either ALK+ or ALK-negative ALCL (p = 0.12 and p = 0.69, respectively; Figs. 4C and D). In patients with ALK+ ALCL, the PFS curves of the CD8+ versus CD8-negative groups appear quite different, although there was no statistically significant difference with a p value of 0.12 (Fig. 4C). Since CD8 expression was associated with noncommon morphology in ALK+ ALCL, the possible role of non-common morphology in patient survival was investigated. Non-common morphology was found to have no impact on OS (p = 0.66) or PFS (p = 0.8) in patients with ALK+ ALCL (Figs.5A and B).
11
Discussion Anaplastic large cell lymphoma is thought to be derived from activated cytotoxic T cell precursors,[1] and is characterized by uniform CD30 reactivity; expression of T-cell markers such as CD2, CD4, and CD43; expression of cytotoxic antigens such as TIA1, granzyme B, and perforin; and clonal T-cell receptor (TCR) gene rearrangements.[3, 6, 7, 9, 10, 17, 18] Despite these common features, there are some immunophenotypic differences between ALK+ ALCL and ALK-negative ALCL. ALK+ ALCL cases are commonly EMA+ and often negative for CD3, T-cell receptors and BCL-2, whereas ALK-negative ALCL cases are more frequently positive for CD3, T-cell receptors and BCL-2 and less often EMA+. [3, 6, 7, 9, 10, 14, 18-21]. In both ALK+ ALCL and ALK-negative ALCL, CD8 is expressed uncommonly, and the clinicopathologic features of CD8+ cases are not well described in the literature. The reported rates of CD8 expression in ALCL vary in the literature, from 0 to 24%.[3, 6, 9, 10, 14, 18-20] In the current study of 158 patients, 16% of neoplasms were CD8+. To date, only one study by Abramov and colleagues correlated CD8 expression with pathologic features in ALCL.[14] Their study was focused on pediatric patients with ALK+ ALCL and CD8 was detected by immunofluorescence. CD8+ cases of ALK+ ALCL were found to more frequently express CD3 than CD8-negative cases, but there were no statistically significant difference in CD5 expression between these two groups. In this study, we show that cases of CD8+ ALK+ ALCL more frequently express CD2, CD3, CD7 and less often express CD25 expression compared with their CD8-negative counterparts. ALCL is known to express high levels of CD25.[20, 22] CD25 has been related to epigenetic down-regulation of the TCR signaling pathway; therefore, the loss of expression of TCR-associated molecules (such as CD3) is usually accompanied by strong CD25 expression.[23] The decreased frequency of CD25 observed in the
12
CD8+ subset of ALK+ ALCL cases in this study is likely related to CD3 being expressed frequently. In patients with ALK-negative ALCL, all of the CD8+ cases tested were positive for CD3. These data suggest that CD8 expression in ALCL is associated with a higher frequency of CD3 expression, independent of ALK status. It is of interest that CD8 expression was associated with CD2 and CD7 expression in ALK+ but not ALK-negative ALCL. This difference is likely associated with non-common morphology in the ALK+ ALCL, since non-common morphology was associated with expression of CD2 and CD7; data not shown). In a study of pediatric ALK+ ALCL by Abramov and colleagues, CD8 expression was also associated with non-common type morphology and, more importantly, a poorer outcome in multivariate analysis, suggesting that CD8 is an independent prognostic predictor of worse outcome.[14] In the ALK+ ALCL patient cohort we present, CD8 expression did not correlate with OS or PFS. However, CD8 expression in ALK+ ALCL tended to be associated with a higher relapse rate and the risk of relapse is likely related to the higher rate of SCT employed in the ALK+ ALCL patient subset with CD8+ lymphomas in this study. It is reasonable to hypothesize that SCT may overcome the adverse prognostic impact of CD8 expression in ALK+ ALCL patients. The higher relapse rate in patients with CD8+ ALK+ ALCL also may be attributable to the higher frequency of the small cell and/or lymphohistiocytic variant patterns. The small cell pattern shows a predominant population of small to medium-sized lymphoma cells with minimal cytoplasm. A few hallmark cells are usually present, often concentrating around blood vessels. The small cell pattern can be misdiagnosed as peripheral T cell lymphoma, not otherwise specified, without CD30 and ALK results. The lymphohistiocytic pattern is characterized by a minor population of hallmark cells usually around blood vessels and numerous admixed
13
histiocytes which may mask the neoplastic cells. The small cell and lymphohistiocytic patterns can appear alone or intermixed in the same tumor or in subsequent biopsy specimens, suggesting that these two patterns are closely related. Small cell and lymphohistiocytic patterns have been associated with a more aggressive course and adverse prognosis by others.[18, 24, 25] In a large series of childhood ALK+ ALCL cases, the presence of a small cell/lymphohistiocytic component was significantly associated with treatment failure in a multivariate analysis controlled for clinical characteristics.[18] However, in this study non-common morphology did not correlate with prognosis in patients with ALK+ ALCL. In conclusion, we assessed CD8 expression in 158 patients with systemic ALCL and compared the clinicopathologic features and outcome between patients with CD8+ versus CD8negative tumors. In cases of ALK+ ALCL, CD8 expression was associated with small cell/lymphohistiocytic morphology, a high frequency of expression of CD2, CD3 and CD7, and a lower frequency of CD25 positivity. In cases of ALK-negative ALCL, CD8 expression was also associated with a high frequency of CD3 expression, but not with other clinical or pathologic features. Although CD8 expression did not impact survival in this study, patients with CD8+ ALK+ ALCL had a higher relapse rate and more often were treated with stem cell transplant. Overall, the data presented suggest that CD8+ ALCL is biologically different from CD8-negative ALCL.
Disclosure/Conflicts of Interest The authors have disclosed that they have no relationships with, or financial interest in, any commercial companies pertaining to this article.
14
References 1. Swerdlow SH, Campo E, Pileri SA et al. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood 2016; 127: 2375-2390. 2. Vose J, Armitage J, Weisenburger D, International TCLP. International peripheral T-cell and natural killer/T-cell lymphoma study: pathology findings and clinical outcomes. J Clin Oncol 2008; 26: 4124-4130. 3. Savage KJ, Harris NL, Vose JM et al. ALK- anaplastic large-cell lymphoma is clinically and immunophenotypically different from both ALK+ ALCL and peripheral T-cell lymphoma, not otherwise specified: report from the International Peripheral T-Cell Lymphoma Project. Blood 2008; 111: 54965504. 4. Falini B, Pileri S, Zinzani PL et al. ALK+ lymphoma: clinico-pathological findings and outcome. Blood 1999; 93: 2697-2706. 5. Medeiros LJ, Elenitoba-Johnson KS. Anaplastic Large Cell Lymphoma. Am J Clin Pathol 2007; 127: 707-722. 6. Suzuki R, Kagami Y, Takeuchi K et al. Prognostic significance of CD56 expression for ALK-positive and ALK-negative anaplastic large-cell lymphoma of T/null cell phenotype. Blood 2000; 96: 2993-3000. 7. Sibon D, Fournier M, Briere J et al. Long-term outcome of adults with systemic anaplastic largecell lymphoma treated within the Groupe d'Etude des Lymphomes de l'Adulte trials. J Clin Oncol 2012; 30: 3939-3946. 8. ten Berge RL, de Bruin PC, Oudejans JJ et al. ALK-negative anaplastic large-cell lymphoma demonstrates similar poor prognosis to peripheral T-cell lymphoma, unspecified. Histopathology 2003; 43: 462-469. 9. Hsi ED, Said J, Macon WR et al. Diagnostic accuracy of a defined immunophenotypic and molecular genetic approach for peripheral T/NK-cell lymphomas. A North American PTCL study group project. Am J Surg Pathol 2014; 38: 768-775. 10. Parrilla Castellar ER, Jaffe ES, Said JW et al. ALK-negative anaplastic large cell lymphoma is a genetically heterogeneous disease with widely disparate clinical outcomes. Blood 2014; 124: 1473-1480. 11. Ellin F, Landstrom J, Jerkeman M, Relander T. Real-world data on prognostic factors and treatment in peripheral T-cell lymphomas: a study from the Swedish Lymphoma Registry. Blood 2014; 124: 1570-1577. 12. Gaiser T, Geissinger E, Schattenberg T et al. Case report: A unique pediatric case of a primary CD8 expressing ALK-1 positive anaplastic large cell lymphoma of skeletal muscle. Diagn Pathol 2012; 7: 38. 13. Tian C, Yu Y, Yang H et al. ALK-positive anaplastic large cell lymphoma with prominent bone involvement in a 13-year-old boy. Onco Targets Ther 2016; 9: 265-268. 14. Abramov D, Oschlies I, Zimmermann M et al. Expression of CD8 is associated with non-common type morphology and outcome in pediatric anaplastic lymphoma kinase-positive anaplastic large cell lymphoma. Haematologica 2013; 98: 1547-1553. 15. Xu J, Medeiros LJ, Saksena A et al. CD10-positive mantle cell lymphoma: clinicopathologic and prognostic study of 30 cases. Oncotarget 2018; 9: 11441-11450. 16. Hu Z, Medeiros LJ, Chen Z et al. Mantle Cell Lymphoma With MYC Rearrangement: A Report of 17 Patients. Am J Surg Pathol 2017; 41: 216-224. 17. Foss HD, Anagnostopoulos I, Araujo I et al. Anaplastic large-cell lymphomas of T-cell and null-cell phenotype express cytotoxic molecules. Blood 1996; 88: 4005-4011. 18. Lamant L, McCarthy K, d'Amore E et al. Prognostic impact of morphologic and phenotypic features of childhood ALK-positive anaplastic large-cell lymphoma: results of the ALCL99 study. J Clin Oncol 2011; 29: 4669-4676. 15
19. Muzzafar T, Wei EX, Lin P et al. Flow cytometric immunophenotyping of anaplastic large cell lymphoma. Arch Pathol Lab Med 2009; 133: 49-56. 20. Juco J, Holden JT, Mann KP et al. Immunophenotypic analysis of anaplastic large cell lymphoma by flow cytometry. Am J Clin Pathol 2003; 119: 205-212. 21. Rassidakis GZ, Sarris AH, Herling M et al. Differential expression of BCL-2 family proteins in ALKpositive and ALK-negative anaplastic large cell lymphoma of T/null-cell lineage. Am J Pathol 2001; 159: 527-535. 22. Delsol G, Al Saati T, Gatter KC et al. Coexpression of epithelial membrane antigen (EMA), Ki-1, and interleukin-2 receptor by anaplastic large cell lymphomas. Diagnostic value in so-called malignant histiocytosis. Am J Pathol 1988; 130: 59-70. 23. Ambrogio C, Martinengo C, Voena C et al. NPM-ALK oncogenic tyrosine kinase controls T-cell identity by transcriptional regulation and epigenetic silencing in lymphoma cells. Cancer Res 2009; 69: 8611-8619. 24. Spiegel A, Paillard C, Ducassou S et al. Paediatric anaplastic large cell lymphoma with leukaemic presentation in children: a report of nine French cases. Br J Haematol 2014; 165: 545-551. 25. Bayle C, Charpentier A, Duchayne E et al. Leukaemic presentation of small cell variant anaplastic large cell lymphoma: report of four cases. Br J Haematol 1999; 104: 680-688.
16
Figure Legends Figure 1. A case of CD8+ ALK+ ALCL. A. Typical morphology of ALCL. B-C. The lymphoma cells were weakly positive (subset, cytoplasmic) for CD3 (B) and negative for CD5 (C). D-F. The lymphoma cells were uniformly and strongly positive for CD30 (D, membranous staining), ALK (E, nuclear and cytoplasmic staining), and showed high Ki67 (F). Scattered small lymphocytes with strong positivity for CD3 (B) and CD5 (C) were the background reactive T cells. A. Hematoxylin-eosin stain, x400. B-F. Immunohistochemistry, x400.
Figure 2. T cell antigen expression shown by flow cytometric immunophenotypic analysis in the CD8+ ALK+ ALCL case described in Figure 1. The lymphoma cells (pink dots) showed increased side scatter (SSC) (A), were positive for CD45 (A), CD30 (B), CD8 (B), CD7 (C), CD2 (D), and were negative for surface CD3 (C), CD5 (D), CD4 (E), TCR A/B (F), and TCR G/D (F). The blue dots represent background reactive T cells which were negative for CD30 (B), but positive for CD3 (C), CD2 (D), CD5 (D), CD7 (C), with a normal CD4:CD8 ratio (B and E).
Figure 3. A case of CD8+ ALK+ ALCL, small cell pattern. A. At low magnification (x 200), the bone marrow biopsy showed no obvious lymphoma. However, small lymphoma cells were identified at high magnification (insert, x 1000, arrows). B-D. The lymphoma cells were positive for ALK (B), CD3 (C), and CD8 (D). A. Hematoxylin-eosin stain. B-D. Immunohistochemistry, x500.
Figure 4. CD8 expression did not correlate with overall survival (OS) or progression free survival (PFS) in patients with ALCL. A and C. Patients with ALK+ ALCL. B and D. Patients with ALK-negative ALCL.
17
Figure 5. Non-common morphology had no prognostic impact on (A) overall survival (OS) or (B) progression-free survival (PFS) in patients with ALK+ ALCL.
18
Table 1: Clinical Features of Patients with CD8+ and CD8-negative Anaplastic Large Cell Lymphoma
Male:Female Median Age (yrs, range) B symptoms Nodal presentation Extranodal involvement Bone marrow involvement Stage III or IV Elevated WBC Absolute lymphocytosis Elevated serum LDH IPI >3 Initial treatment CHOP or modified CHOP Other* Initial CR Relapse With SCT
ALK+ ALCL (n=67) CD8+ CD8-negative (n=13) (n=54) 1.2:1 (7/6) 2.4:1 (38/16) 22 (9-55) 28 (4-75) 82% (9/11) 67% (28/42) 92% (11/12) 80% (43/54) 50% (4/8) 60% (29/48) 22% (2/9) 14% (6/44) 63% (5/8) 66% (29/44) 40% (2/5) 48% (13/27) 25% (1/4) 8% (2/26) 50% (2/4) 42% (10/24) 0% (0/6) 18% (6/34) 91% (10/11) 9% (1/11) 55% (6/11) 82% (9/11) 73% (8/11)
73% (33/45) 27% (12/45) 77% (34/44) 48% (21/44) 36% (15/42)
$
P value 0.33 0.33 0.47 0.44 0.7 0.61 1.0 1.0 0.36 1.0 0.57 0.43 0.15 0.05 0.04
ALK-negative ALCL (n=91) CD8+ CD8-negative P value# (n=13) (n=78) 3.3:1 (10:3) 2:1 (52:26) 0.54 58(42-79) 60(21-95) 0.5 64% (7/11) 57% (33/58) 0.75 69% (9/13) 77% (54/70) 0.5 75% (9/12) 66% (43/65) 0.74 25% (3/12) 16% (10/61) 0.44 75% (9/12) 73% (44/60) 1.0 30% (3/10) 35% (15/43) 1.0 10% (1/10) 0% (0/42) 0.19 63% (5/8) 50% (18/36) 0.7 67% (6/9) 47% (23/49) 0.47 62% (8/13) 38% (5/13) 67% (8/12) 46% (6/13) 17% (2/12)
67% (39/58) 33% (19/58) 61% (35/57) 56% (32/57) 29% (16/56)
0.75 1.0 0.55 0.49
ALCL, anaplastic large cell lymphoma; $, ALK+ CD8+ ALCL compared with ALK+ CD8-negative ALCL; #, ALK-negative CD8+ ALCL compared with ALK-negative CD8-negative ALCL; WBC, white blood cells; LDH, lactate dehydrogenase; IPI, International Prognostic Index; CHOP, cyclophosphamide, doxorubicin, vincristine, and prednisone; *, including Hyper-CVAD (cyclophosphamide, vincristine, doxorubicin, and dexamethasone), EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin), ICE (ifosfamide, carboplatin, etoposide), BV (Brentuximab vedotin), and etc. R, rituximab; CR, complete response; SCT, stem cell transplant.
Table 2: Pathological Features of Patients with CD8+ and CD8-negative Anaplastic Large Cell Lymphoma ALK+ ALCL (n=67)
Morphologic type Common pattern Non-common pattern Immunophenotype CD2+ CD3+ (IHC and/or FC) CD3+ (IHC) Surface CD3+ (FC) CD5+ CD7+ CD25+ CD43+ CD45+ TCR A/B TCR G/D Granzyme B+ TIA1+ EMA+
ALK-negative ALCL (n=91) $
CD8-negative P value# (n=78)
CD8+ (n=13)
CD8-negative P value (n=54)
CD8+ (n=13)
31% (4/13) 69% (9/13)
87% (47/54) 13% (7/54)
1.0
0.0001
100% (13/13) 99% (77/78) 0% (0/13) 1% (1/78)
100% (10/10) 92% (12/13) 69% (9/13) 88% (7/8) 45% (5/11) 100% (9/9) 50% (3/6) 100% (1/1) 100% (9/9) 50% (2/3) 0% (0/3) 100% (6/6) 100% (1/1) 83% (5/6)
45% (18/40) 24% (12/50) 17% (8/48) 20% (4/20) 40% (18/45) 39% (13/33) 100% (13/13) 80% (24/30) 81% (26/32) 56% (5/9) 0% (0/7) 80% (8/10) 90% (9/10) 94% (17/18)
0.001 0.0001 0.0005 0.0019 0.75 0.002 0.02 1.0 0.31 1.0 1.0 0.47 1.0 0.45
90% (9/10) 100% (13/13) 100% (13/13) 60% (3/5) 36% (4/11) 63% (5/8) 25% (1/4) 25% (1/4) 100% (6/6) 40% (2/5) 0% (0/5) 29% (2/7) 14% (1/7) 17% (1/6)
0.67 0.0004 0.0001 1.0 1.0 0.10 0.12 0.12 1.0 1.0 1.0 0.24 0.1 0.65
77% (44/57) 48% (36/75) 42% (31/74) 48% (10/21) 36% (25/70) 27% (15/56) 75% (9/12) 75% (9/12) 88% (36/41) 31% (4/13) 8% (1/12) 56% (18/32) 53% (20/38) 32% (14/44)
ALCL, anaplastic large cell lymphoma; $, ALK+ CD8+ ALCL compared with ALK+ CD8-negative ALCL; #, ALK-negative CD8+ ALCL compared with ALK-negative CD8-negative ALCL; IHC, immunohistochemistry; FC, flow cytometry.
Figure 1
A
B CD3
C CD5
D CD30
E ALK
F Ki67
Figure 2 47.1% 47.1%
29.7% 13.8%5
A
CD8 PerCP-Cy5-5-A
10
196608
SSC-A
30.0%
131072
65536
2.5% 5
B
10
CD3 PE-Cy7-A
262144
104
103
10 3
10 4
10 5
45.6%
CD45 V500-A
24.9% 105
D
104
103
10.9% 10 5
-10 2 10 2
10 3
12.5%
10 4
2.4% 2.4%
-10 2 10 2
10 3
104
103
60.9% 10 5
10 4
CD2 APC-A
34.5%
-10 2 10 2
10 3
0.1% 7.2%
CD30 PE-A
F
104
103
38.2%
101 -102
10 5 38.2%
70.9%
10 4
37.9% 10 5 37.9%
10 4
CD7 APC-A
7.2% 105
E
101 -102
101 -102
5.2%
10 3
24.9%
CD4 FITC-A
CD5 PerCP-Cy5-5-A
10
103
CD30 PE-A
33.5% 0.4% 5
-10 2 10 2
TCR G/D PE-A
-10 2 10 2
104
101 -102
101 -102 0
C
-10 2 10 2
10 3
21.8% 10 5
10 4
TCR A/B FITC-A
Figure 3
A
B ALK
C CD3
D CD8
Figure 4
A
B
C
D
Figure 5
A
B
S0046-8177(20)30018-6
DOI:
https://doi.org/10.1016/j.humpath.2020.01.005
Reference:
YHUPA 4965
To appear in:
Human Pathology
Received Date: 20 September 2019 Revised Date:
14 January 2020
Accepted Date: 28 January 2020
Please cite this article as: Shen J, Medeiros LJ, Li S, Wang SA, Lin P, Khanlari M, Iyer SP, Yin CC, Tang G, Jorgensen JL, Hu S, Miranda RN, Xu J, CD8 expression in anaplastic large cell lymphoma (ALCL) correlates with non-common morphologic variants and T-cell antigen expression suggesting biological differences with CD8-negative ALCL, Human Pathology, https://doi.org/10.1016/ j.humpath.2020.01.005. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
CD8 expression in anaplastic large cell lymphoma (ALCL) correlates with non-common morphologic variants and T-cell antigen expression suggesting biological differences with CD8-negative ALCL Jing Shen1,2, L. Jeffrey Medeiros1, Shaoying Li1, Sa A. Wang1, Pei Lin1, Mahsa Khanlari1, Swaminathan P. Iyer3, C. Cameron Yin1, Guilin Tang1, Jeffrey L. Jorgensen1, Shimin Hu1, Roberto N. Miranda1, Jie Xu1 1 Department of Hematopathology, 3Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX 2
Department of Hematology, Capital Medical University Beijing Friendship Hospital, Beijing, China
Words of Abstract: 250 Number of Tables: 2 Number of Figures: 5 Running Title: CD8, anaplastic large cell lymphoma Key Words: CD8, anaplastic large cell lymphoma, pathology, prognosis Conflicts of Interest and Source of Funding: The authors have disclosed that they have no relationships with, or financial interest in, any commercial companies pertaining to this article. Part of the data has been presented at the Annual Meeting of United States and Canadian Academy of Pathology in 2019. Correspondence: Jie Xu, MD PhD Department of Hematopathology MD Anderson Cancer Center 1515 Holcombe Blvd, Unit 72 Houston, TX 77030 Phone: 713-794-1220 Fax: 713-792-8438 Email: [email protected]
1
ABSTRACT Anaplastic large cell lymphoma (ALCL) is a T cell neoplasm characterized by uniformly strong CD30 expression and common absence of T cell markers. Most ALCL cases express CD4, but a small subset of ALCL cases has been reported to express CD8. Little is known about the clinicopathologic and prognostic features of CD8+ ALCL. In this study, CD8 was assessed in 158 patients with systemic ALCL: CD8 was positive in 13 of 67 (19%) ALK+ and 13 of 91 (14%) ALK-negative neoplasms. In ALK+ ALCL, the CD8+ subgroup more often showed a non-common morphologic pattern (69% vs 13%, p = 0.0001) and was more often positive for CD2 (100% vs 45%, p = 0.001), CD3 (92% vs 24%, p = 0.0001), and CD7 (100% vs. 39%, p = 0.002), but less frequently positive for CD25 (50% vs. 100%, p = 0.02). ALK+ ALCL patients with CD8+ neoplasms also had a higher relapse rate (82% vs 48%, p = 0.05) and more often underwent stem cell transplant (73% vs 36%, p = 0.04). CD8 expression did not correlate with patient overall survival or progression-free survival regardless of ALK status (all p > 0.05). We conclude that CD8+ ALCL cases appear to be biologically different from the more common CD8-negative ALCL cases. Our data suggest that CD8 positivity in ALK+ ALCL helps to identify a patient subset more prone to relapse or more in need of stem cell transplant during their clinical course, although there was no impact on survival in this cohort.
2
Introduction Systemic anaplastic large cell lymphoma (ALCL) is a T cell neoplasm characterized by lymphoma cells that are usually large and pleomorphic with horseshoe-shaped nuclei and abundant cytoplasm (so-called “hallmark” cells), uniform and strong CD30 expression, a CD4+ cytotoxic immunophenotype, and aberrant loss of one or more T cell antigens. Patients with ALCL frequently have disease involving lymph nodes and extranodal sites, and many patients present with advanced stage disease and B symptoms. ALCL can be further classified into ALK+ and ALK-negative types.[1] ALK+ ALCL is characterized by ALK expression resulting from translocations involving ALK at 2p23, most commonly t(2;5)(p23;q35)/NPM1-ALK. Patients with ALK+ ALCL tend to be younger with a male predominance. These neoplasms are morphologically heterogeneous and five morphologic patterns are recognized in the current World Health Organization (WHO) classification: common, small cell, lymphohistiocytic, Hodgkin-like, and composite patterns.[1] Patients with ALK-negative ALCL lack abnormalities involving ALK. Compared to ALK+ ALCL, patients with ALK-negative ALCL are usually older, less often have extranodal involvement, and more often have an aggressive clinical course and poorer outcome. The long-term overall survival rates are 70-90% in patients with ALK+ ALCL, compared with < 50% in patients with ALK-negative ALCL.[2-11] CD8+ cases of ALCL have been reported, mostly in the form of case reports and small case series.[12, 13] The clinicopathologic features and outcome of patients with CD8+ ALCL are not well studied. One study in the literature using immunofluorescence showed that CD8 expression is associated with a non-common morphologic pattern and poorer outcome in pediatric patients with ALK+ ALCL.[14] We are not aware of a similarly designed study for patients with ALK-negative ALCL.
3
In this study, we assessed CD8 expression in ALK+ and ALK-negative ALCL cases by using flow cytometry immunophenotypic and/or immunohistochemical methods and correlated the results with clinicopathologic features and patient outcome.
4
Materials and Methods Case Selection We searched the database of the Department of Hematopathology at The University of Texas MD Anderson Cancer Center from January 1, 2007 through December 31, 2018 for cases of systemic ALCL that had CD8 results available by flow cytometry and/or immunohistochemistry. The diagnosis and sub-classification of ALCL were based on criteria specified in the 2016 WHO classification.[1] The diagnosis of ALK+ ALCL was confirmed by ALK expression by immunohistochemistry and/or t(2;5)(p23;q35) by conventional cytogenetics or ALK rearrangement by fluorescence in situ hybridization (FISH) analysis. Clinical information was obtained by review of medical records. This study was approved by the institutional review board.
Immunophenotypic Analysis Immunohistochemical studies were performed using formalin-fixed, paraffin-embedded (FFPE) tissue sections, either at the time of diagnosis or retrospectively for this study as described previously.[15] Immunohistochemical analysis was performed on an automated immunostainer (Leica Bond-Max IHC Stainer, San Diego, CA). Tissue sections, 4-µm-thick, were deparaffinized and underwent heat-induced antigen retrieval using the Bond Max Epitope Retrieval 1 solution for 15 minutes. The antibodies used were specific for CD2, CD7, EMA (Leica Biosystems, Newcastle, United Kingdom); CD3, CD20, CD43, CD45 (Dako, Carpinteria, CA, USA); CD4 (Cell Marque, Rocklin, CA, USA); CD5 (SP4; Labvision/Neomarkers, Fremont, CA, USA); CD8, granzyme B (Thermo Fisher, Waltham, MA, USA); ALK (Cell
5
Signaling, Danvers, MA, USA); and PAX5 (Transduction Labs, San Diego, CA, USA). The Bond Refine Polymer detection system was used for visualization. Flow cytometry immunophenotypic analysis was performed on cell suspensions of tissue biopsy specimens or bone marrow aspirates using either a FACScanto II or FACSCalibur cytometer (Becton-Dickinson Biosciences, San Jose, CA, USA) as has been described. [15] Lymphocytes were gated for analysis using side scatter versus forward scatter and CD45 expression versus side scatter. The panel of monoclonal antibodies included reagents specific for CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD25, CD30, CD45, TCR alpha/beta, TCR gamma/delta (Becton-Dickinson Biosciences, San Jose, CA, USA).
Conventional Cytogenetic Analysis and Fluorescence in situ Hybridization Conventional cytogenetic analysis was performed on metaphase cells prepared from bone marrow aspirates or cell suspensions from tissue biopsy specimens as described previously. [16] Twenty Giemsa-banded metaphases were analyzed, and the results were reported using the 2016 International System for Human Cytogenetic Nomenclature. FISH analysis was performed using a LSI ALK dual color, breakapart rearrangement probe (Abbott Molecular, Des Plaines, IL, USA) on interphase nuclei obtained from bone marrow cells or tissue sections, according to the manufacturer’s instructions.
Statistical Analysis Statistical analyses were performed using the Graph-Pad Prism 7. Fisher’s exact test was utilized to compare the clinicopathologic features between CD8+ versus CD8-negative subgroups in patients with ALK+ or ALK-negative anaplastic large cell lymphoma, respectively.
6
Overall survival was calculated from the date of initial diagnosis to the date of death or last follow-up. Progression-free survival was calculated from the date of diagnosis to the date of progression/relapse or, if no progression/relapse, the date of death or last follow-up. Survival was analyzed using the Kaplan–Meier method and was compared using the log rank test. A p value of less than 0.05 was considered statistically significant.
7
Results Clinical Findings We identified 158 cases of ALCL that had CD8 results available including 67 ALK+ and 91 ALK-negative. Twenty-six cases were assessed by flow cytometry only, 26 cases were assessed by both flow cytometry and immunohistochemistry, and 106 cases were assessed only by immunohistochemistry. The CD8 results assessed by flow cytometry correlated with those assessed by immunohistochemistry (p = 0.0001, data not shown). There was no difference in CD8 positivity rate between ALK+ versus ALK-negative ALCL (19% vs 14%, p = 0.40). The clinical features of these patients are summarized in Table 1. The ALK+ ALCL cases included 13 (19%) that were CD8+ and 54 that were CD8negative. The ALK+ CD8+ subgroup included 7 men and 6 women with a median age of 22 years (range, 9-55 years) at the time of diagnosis. Nine of 11 (82%) patients had B symptoms. Lymphadenopathy was identified in 11 of 12 (92%) patients and 4 of 8 (50%) had extranodal involvement. Bone marrow was involved in 2 of 9 (22%) patients. Five of 8 (63%) fully staged patients had stage III or IV disease. Two of 5 (40%) patients had leukocytosis and 1 of 4 (25%) had absolute lymphocytosis. Two of 4 (50%) patients tested showed an elevated serum LDH level. None of the patients had an International Prognostic Index (IPI) score of > 3 (Table 1). The ALK+ CD8-negative subgroup included 54 patients, 38 men and 16 women with a median age of 28 years (range 4-75 years). The clinical features of the ALK+ CD8-negative subgroup were similar to the ALK+ CD8+ subgroup (all p > 0.05; Table 1). The ALK-negative ALCL cases included 13 (14%) that were CD8+ and 78 that were CD8-negative. As shown in Table 1, there were no significant differences in the clinical features between CD8+ versus CD8-negative subgroups of ALK-negative ALCL (all p > 0.05).
8
Pathologic Findings In ALK+ ALCL, the CD8+ cases included 4 (31%) with common (or classic) morphology and 9 (69%) with non-common morphologic patterns: 4 (31%) small cell, 2 (15%) lymphohistiocytic, and 3 (23%) composite (2 small cell/lymphohistiocytic, 1 small cell/common) (Table 2; Figures 1 and 3). The CD8+ subset of ALK+ ALCL cases more frequently showed non-common morphology (small cell and/or lymphohistiocytic variant) (69% vs. 13%, p = 0.0001). CD8+ cases also had variable expression of T cell antigens (Table 2): all cases were CD2+ (n = 10) and CD7+ (n = 9), 12 of 13 (92%) were CD3+, and 5 of 11 (45%) were CD5+. Eight of 12 67%) cases showed weak or partial CD3 positivity: 6 by immunohistochemistry and flow cytometry, 1 by immunohistochemistry, and 1 by flow cytometry. Using immunohistochemistry, 9 of 13 (69%) cases were positive for CD3, and by flow cytometry, 7 of 8 (88%) cases were positive for surface CD3. The CD8+ subgroup was significantly associated with more frequent CD3 expression, assessed by either immunohistochemistry (69% vs. 17%, p = 0.0005) or flow cytometry (88% vs. 20%, p = 0.0019). In addition, all CD8+ cases of ALK+ ALCL were positive for CD45 (n = 9) and granzyme B (n = 6), 5 of 6 (83%) were EMA+, and 3 of 6 (50%) were CD25+. In ALK+ ALCL, the CD8+ subgroup as compared with the CD8negative group was more often positive for CD2 (100% vs 45%, p = 0.001), CD3 (92% vs 24%, p = 0.0001), and CD7 (100% vs. 39%, p = 0.002), but less frequently positive for CD25 (50% vs. 100%, p = 0.02). There was no significant difference in the frequency of expression of CD5, CD45, granzyme B, and EMA between CD8+ versus CD8-negative ALK+ ALCL (all p > 0.05, Table 2).
9
In ALK-negative ALCL, all CD8+ neoplasms and 77 of 78 (99%) CD8-negative neoplasms had common morphologic features. Comparing the immunophenotypic features between the CD8+ versus CD8-negative subgroups, most features were similar except for CD3. All 13 CD8+ cases of ALK-negative ALCL were positive for CD3 (all cases by immunohistochemistry and 3/5 cases by flow cytometry). CD8+ cases of ALK-negative ALCL were more often positive for CD3 than CD8-negative cases (100% vs 48%, p = 0.0004; Table 2). CD8+ subgroup was also significantly associated with more frequent CD3 expression, assessed by immunohistochemistry (100% vs. 42%, p = 0.0001).
Treatment and Response Fifty-six patients with ALK+ ALCL and 71 patients with ALK-negative ALCL had treatment and follow-up information available. All patients were treated with various chemotherapy regimens over the time interval of this study, with or without stem cell transplant (SCT). In patients with ALK+ ALCL that were CD8+, 10 of 11 (91%) were treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or modified CHOP (Table 1). After initial induction chemotherapy, 6 of 11 (55%) patients achieved complete remission, but 9 of 11 (82%) patients relapsed. Eight of 11 (73%) patients received SCT: 4 autologous and 4 allogeneic. There was no significant difference in initial treatment or complete remission rate between the CD8+ versus CD8-negative ALK+ ALCL subgroups (all p > 0.05; Table 1); however, patients with CD8+ tumors more often received SCT than patients with CD8-negative tumors (73% vs 36%, p = 0.04). This result might be attributable to the higher relapse rate in CD8+ subgroup (82% vs 48%, p = 0.05). In patients with known time of relapse and SCT, 5 of 8 (63%) CD8+ patients versus 12 of 19 (63%) CD8-negative patients received SCT after their
10
disease relapsed; there was no statistical difference in SCT frequency between these groups (p = 1.0). In patients with ALK-negative ALCL, there was no significant difference in treatment, complete response rate, or relapse rate between the CD8+ versus CD8-negative subgroups (all p > 0.05, Table 1).
Outcome After a median clinical follow-up of 19 months (range, 0-186 months), 49 of 158 (31%) patients died. Ten of 26 (39%) patients with CD8+ ALCL died: 3 ALK+ and 7 ALK-negative. Survival analysis was performed in patients based on ALK status. CD8 expression did not affect overall survival (OS) in patients with either ALK+ or ALK-negative ALCL (p = 0.82 and p = 0.45, respectively; Figs. 4A and B). Furthermore, CD8 had no impact on progression-free survival (PFS) in patients with either ALK+ or ALK-negative ALCL (p = 0.12 and p = 0.69, respectively; Figs. 4C and D). In patients with ALK+ ALCL, the PFS curves of the CD8+ versus CD8-negative groups appear quite different, although there was no statistically significant difference with a p value of 0.12 (Fig. 4C). Since CD8 expression was associated with noncommon morphology in ALK+ ALCL, the possible role of non-common morphology in patient survival was investigated. Non-common morphology was found to have no impact on OS (p = 0.66) or PFS (p = 0.8) in patients with ALK+ ALCL (Figs.5A and B).
11
Discussion Anaplastic large cell lymphoma is thought to be derived from activated cytotoxic T cell precursors,[1] and is characterized by uniform CD30 reactivity; expression of T-cell markers such as CD2, CD4, and CD43; expression of cytotoxic antigens such as TIA1, granzyme B, and perforin; and clonal T-cell receptor (TCR) gene rearrangements.[3, 6, 7, 9, 10, 17, 18] Despite these common features, there are some immunophenotypic differences between ALK+ ALCL and ALK-negative ALCL. ALK+ ALCL cases are commonly EMA+ and often negative for CD3, T-cell receptors and BCL-2, whereas ALK-negative ALCL cases are more frequently positive for CD3, T-cell receptors and BCL-2 and less often EMA+. [3, 6, 7, 9, 10, 14, 18-21]. In both ALK+ ALCL and ALK-negative ALCL, CD8 is expressed uncommonly, and the clinicopathologic features of CD8+ cases are not well described in the literature. The reported rates of CD8 expression in ALCL vary in the literature, from 0 to 24%.[3, 6, 9, 10, 14, 18-20] In the current study of 158 patients, 16% of neoplasms were CD8+. To date, only one study by Abramov and colleagues correlated CD8 expression with pathologic features in ALCL.[14] Their study was focused on pediatric patients with ALK+ ALCL and CD8 was detected by immunofluorescence. CD8+ cases of ALK+ ALCL were found to more frequently express CD3 than CD8-negative cases, but there were no statistically significant difference in CD5 expression between these two groups. In this study, we show that cases of CD8+ ALK+ ALCL more frequently express CD2, CD3, CD7 and less often express CD25 expression compared with their CD8-negative counterparts. ALCL is known to express high levels of CD25.[20, 22] CD25 has been related to epigenetic down-regulation of the TCR signaling pathway; therefore, the loss of expression of TCR-associated molecules (such as CD3) is usually accompanied by strong CD25 expression.[23] The decreased frequency of CD25 observed in the
12
CD8+ subset of ALK+ ALCL cases in this study is likely related to CD3 being expressed frequently. In patients with ALK-negative ALCL, all of the CD8+ cases tested were positive for CD3. These data suggest that CD8 expression in ALCL is associated with a higher frequency of CD3 expression, independent of ALK status. It is of interest that CD8 expression was associated with CD2 and CD7 expression in ALK+ but not ALK-negative ALCL. This difference is likely associated with non-common morphology in the ALK+ ALCL, since non-common morphology was associated with expression of CD2 and CD7; data not shown). In a study of pediatric ALK+ ALCL by Abramov and colleagues, CD8 expression was also associated with non-common type morphology and, more importantly, a poorer outcome in multivariate analysis, suggesting that CD8 is an independent prognostic predictor of worse outcome.[14] In the ALK+ ALCL patient cohort we present, CD8 expression did not correlate with OS or PFS. However, CD8 expression in ALK+ ALCL tended to be associated with a higher relapse rate and the risk of relapse is likely related to the higher rate of SCT employed in the ALK+ ALCL patient subset with CD8+ lymphomas in this study. It is reasonable to hypothesize that SCT may overcome the adverse prognostic impact of CD8 expression in ALK+ ALCL patients. The higher relapse rate in patients with CD8+ ALK+ ALCL also may be attributable to the higher frequency of the small cell and/or lymphohistiocytic variant patterns. The small cell pattern shows a predominant population of small to medium-sized lymphoma cells with minimal cytoplasm. A few hallmark cells are usually present, often concentrating around blood vessels. The small cell pattern can be misdiagnosed as peripheral T cell lymphoma, not otherwise specified, without CD30 and ALK results. The lymphohistiocytic pattern is characterized by a minor population of hallmark cells usually around blood vessels and numerous admixed
13
histiocytes which may mask the neoplastic cells. The small cell and lymphohistiocytic patterns can appear alone or intermixed in the same tumor or in subsequent biopsy specimens, suggesting that these two patterns are closely related. Small cell and lymphohistiocytic patterns have been associated with a more aggressive course and adverse prognosis by others.[18, 24, 25] In a large series of childhood ALK+ ALCL cases, the presence of a small cell/lymphohistiocytic component was significantly associated with treatment failure in a multivariate analysis controlled for clinical characteristics.[18] However, in this study non-common morphology did not correlate with prognosis in patients with ALK+ ALCL. In conclusion, we assessed CD8 expression in 158 patients with systemic ALCL and compared the clinicopathologic features and outcome between patients with CD8+ versus CD8negative tumors. In cases of ALK+ ALCL, CD8 expression was associated with small cell/lymphohistiocytic morphology, a high frequency of expression of CD2, CD3 and CD7, and a lower frequency of CD25 positivity. In cases of ALK-negative ALCL, CD8 expression was also associated with a high frequency of CD3 expression, but not with other clinical or pathologic features. Although CD8 expression did not impact survival in this study, patients with CD8+ ALK+ ALCL had a higher relapse rate and more often were treated with stem cell transplant. Overall, the data presented suggest that CD8+ ALCL is biologically different from CD8-negative ALCL.
Disclosure/Conflicts of Interest The authors have disclosed that they have no relationships with, or financial interest in, any commercial companies pertaining to this article.
14
References 1. Swerdlow SH, Campo E, Pileri SA et al. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood 2016; 127: 2375-2390. 2. Vose J, Armitage J, Weisenburger D, International TCLP. International peripheral T-cell and natural killer/T-cell lymphoma study: pathology findings and clinical outcomes. J Clin Oncol 2008; 26: 4124-4130. 3. Savage KJ, Harris NL, Vose JM et al. ALK- anaplastic large-cell lymphoma is clinically and immunophenotypically different from both ALK+ ALCL and peripheral T-cell lymphoma, not otherwise specified: report from the International Peripheral T-Cell Lymphoma Project. Blood 2008; 111: 54965504. 4. Falini B, Pileri S, Zinzani PL et al. ALK+ lymphoma: clinico-pathological findings and outcome. Blood 1999; 93: 2697-2706. 5. Medeiros LJ, Elenitoba-Johnson KS. Anaplastic Large Cell Lymphoma. Am J Clin Pathol 2007; 127: 707-722. 6. Suzuki R, Kagami Y, Takeuchi K et al. Prognostic significance of CD56 expression for ALK-positive and ALK-negative anaplastic large-cell lymphoma of T/null cell phenotype. Blood 2000; 96: 2993-3000. 7. Sibon D, Fournier M, Briere J et al. Long-term outcome of adults with systemic anaplastic largecell lymphoma treated within the Groupe d'Etude des Lymphomes de l'Adulte trials. J Clin Oncol 2012; 30: 3939-3946. 8. ten Berge RL, de Bruin PC, Oudejans JJ et al. ALK-negative anaplastic large-cell lymphoma demonstrates similar poor prognosis to peripheral T-cell lymphoma, unspecified. Histopathology 2003; 43: 462-469. 9. Hsi ED, Said J, Macon WR et al. Diagnostic accuracy of a defined immunophenotypic and molecular genetic approach for peripheral T/NK-cell lymphomas. A North American PTCL study group project. Am J Surg Pathol 2014; 38: 768-775. 10. Parrilla Castellar ER, Jaffe ES, Said JW et al. ALK-negative anaplastic large cell lymphoma is a genetically heterogeneous disease with widely disparate clinical outcomes. Blood 2014; 124: 1473-1480. 11. Ellin F, Landstrom J, Jerkeman M, Relander T. Real-world data on prognostic factors and treatment in peripheral T-cell lymphomas: a study from the Swedish Lymphoma Registry. Blood 2014; 124: 1570-1577. 12. Gaiser T, Geissinger E, Schattenberg T et al. Case report: A unique pediatric case of a primary CD8 expressing ALK-1 positive anaplastic large cell lymphoma of skeletal muscle. Diagn Pathol 2012; 7: 38. 13. Tian C, Yu Y, Yang H et al. ALK-positive anaplastic large cell lymphoma with prominent bone involvement in a 13-year-old boy. Onco Targets Ther 2016; 9: 265-268. 14. Abramov D, Oschlies I, Zimmermann M et al. Expression of CD8 is associated with non-common type morphology and outcome in pediatric anaplastic lymphoma kinase-positive anaplastic large cell lymphoma. Haematologica 2013; 98: 1547-1553. 15. Xu J, Medeiros LJ, Saksena A et al. CD10-positive mantle cell lymphoma: clinicopathologic and prognostic study of 30 cases. Oncotarget 2018; 9: 11441-11450. 16. Hu Z, Medeiros LJ, Chen Z et al. Mantle Cell Lymphoma With MYC Rearrangement: A Report of 17 Patients. Am J Surg Pathol 2017; 41: 216-224. 17. Foss HD, Anagnostopoulos I, Araujo I et al. Anaplastic large-cell lymphomas of T-cell and null-cell phenotype express cytotoxic molecules. Blood 1996; 88: 4005-4011. 18. Lamant L, McCarthy K, d'Amore E et al. Prognostic impact of morphologic and phenotypic features of childhood ALK-positive anaplastic large-cell lymphoma: results of the ALCL99 study. J Clin Oncol 2011; 29: 4669-4676. 15
19. Muzzafar T, Wei EX, Lin P et al. Flow cytometric immunophenotyping of anaplastic large cell lymphoma. Arch Pathol Lab Med 2009; 133: 49-56. 20. Juco J, Holden JT, Mann KP et al. Immunophenotypic analysis of anaplastic large cell lymphoma by flow cytometry. Am J Clin Pathol 2003; 119: 205-212. 21. Rassidakis GZ, Sarris AH, Herling M et al. Differential expression of BCL-2 family proteins in ALKpositive and ALK-negative anaplastic large cell lymphoma of T/null-cell lineage. Am J Pathol 2001; 159: 527-535. 22. Delsol G, Al Saati T, Gatter KC et al. Coexpression of epithelial membrane antigen (EMA), Ki-1, and interleukin-2 receptor by anaplastic large cell lymphomas. Diagnostic value in so-called malignant histiocytosis. Am J Pathol 1988; 130: 59-70. 23. Ambrogio C, Martinengo C, Voena C et al. NPM-ALK oncogenic tyrosine kinase controls T-cell identity by transcriptional regulation and epigenetic silencing in lymphoma cells. Cancer Res 2009; 69: 8611-8619. 24. Spiegel A, Paillard C, Ducassou S et al. Paediatric anaplastic large cell lymphoma with leukaemic presentation in children: a report of nine French cases. Br J Haematol 2014; 165: 545-551. 25. Bayle C, Charpentier A, Duchayne E et al. Leukaemic presentation of small cell variant anaplastic large cell lymphoma: report of four cases. Br J Haematol 1999; 104: 680-688.
16
Figure Legends Figure 1. A case of CD8+ ALK+ ALCL. A. Typical morphology of ALCL. B-C. The lymphoma cells were weakly positive (subset, cytoplasmic) for CD3 (B) and negative for CD5 (C). D-F. The lymphoma cells were uniformly and strongly positive for CD30 (D, membranous staining), ALK (E, nuclear and cytoplasmic staining), and showed high Ki67 (F). Scattered small lymphocytes with strong positivity for CD3 (B) and CD5 (C) were the background reactive T cells. A. Hematoxylin-eosin stain, x400. B-F. Immunohistochemistry, x400.
Figure 2. T cell antigen expression shown by flow cytometric immunophenotypic analysis in the CD8+ ALK+ ALCL case described in Figure 1. The lymphoma cells (pink dots) showed increased side scatter (SSC) (A), were positive for CD45 (A), CD30 (B), CD8 (B), CD7 (C), CD2 (D), and were negative for surface CD3 (C), CD5 (D), CD4 (E), TCR A/B (F), and TCR G/D (F). The blue dots represent background reactive T cells which were negative for CD30 (B), but positive for CD3 (C), CD2 (D), CD5 (D), CD7 (C), with a normal CD4:CD8 ratio (B and E).
Figure 3. A case of CD8+ ALK+ ALCL, small cell pattern. A. At low magnification (x 200), the bone marrow biopsy showed no obvious lymphoma. However, small lymphoma cells were identified at high magnification (insert, x 1000, arrows). B-D. The lymphoma cells were positive for ALK (B), CD3 (C), and CD8 (D). A. Hematoxylin-eosin stain. B-D. Immunohistochemistry, x500.
Figure 4. CD8 expression did not correlate with overall survival (OS) or progression free survival (PFS) in patients with ALCL. A and C. Patients with ALK+ ALCL. B and D. Patients with ALK-negative ALCL.
17
Figure 5. Non-common morphology had no prognostic impact on (A) overall survival (OS) or (B) progression-free survival (PFS) in patients with ALK+ ALCL.
18
Table 1: Clinical Features of Patients with CD8+ and CD8-negative Anaplastic Large Cell Lymphoma
Male:Female Median Age (yrs, range) B symptoms Nodal presentation Extranodal involvement Bone marrow involvement Stage III or IV Elevated WBC Absolute lymphocytosis Elevated serum LDH IPI >3 Initial treatment CHOP or modified CHOP Other* Initial CR Relapse With SCT
ALK+ ALCL (n=67) CD8+ CD8-negative (n=13) (n=54) 1.2:1 (7/6) 2.4:1 (38/16) 22 (9-55) 28 (4-75) 82% (9/11) 67% (28/42) 92% (11/12) 80% (43/54) 50% (4/8) 60% (29/48) 22% (2/9) 14% (6/44) 63% (5/8) 66% (29/44) 40% (2/5) 48% (13/27) 25% (1/4) 8% (2/26) 50% (2/4) 42% (10/24) 0% (0/6) 18% (6/34) 91% (10/11) 9% (1/11) 55% (6/11) 82% (9/11) 73% (8/11)
73% (33/45) 27% (12/45) 77% (34/44) 48% (21/44) 36% (15/42)
$
P value 0.33 0.33 0.47 0.44 0.7 0.61 1.0 1.0 0.36 1.0 0.57 0.43 0.15 0.05 0.04
ALK-negative ALCL (n=91) CD8+ CD8-negative P value# (n=13) (n=78) 3.3:1 (10:3) 2:1 (52:26) 0.54 58(42-79) 60(21-95) 0.5 64% (7/11) 57% (33/58) 0.75 69% (9/13) 77% (54/70) 0.5 75% (9/12) 66% (43/65) 0.74 25% (3/12) 16% (10/61) 0.44 75% (9/12) 73% (44/60) 1.0 30% (3/10) 35% (15/43) 1.0 10% (1/10) 0% (0/42) 0.19 63% (5/8) 50% (18/36) 0.7 67% (6/9) 47% (23/49) 0.47 62% (8/13) 38% (5/13) 67% (8/12) 46% (6/13) 17% (2/12)
67% (39/58) 33% (19/58) 61% (35/57) 56% (32/57) 29% (16/56)
0.75 1.0 0.55 0.49
ALCL, anaplastic large cell lymphoma; $, ALK+ CD8+ ALCL compared with ALK+ CD8-negative ALCL; #, ALK-negative CD8+ ALCL compared with ALK-negative CD8-negative ALCL; WBC, white blood cells; LDH, lactate dehydrogenase; IPI, International Prognostic Index; CHOP, cyclophosphamide, doxorubicin, vincristine, and prednisone; *, including Hyper-CVAD (cyclophosphamide, vincristine, doxorubicin, and dexamethasone), EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin), ICE (ifosfamide, carboplatin, etoposide), BV (Brentuximab vedotin), and etc. R, rituximab; CR, complete response; SCT, stem cell transplant.
Table 2: Pathological Features of Patients with CD8+ and CD8-negative Anaplastic Large Cell Lymphoma ALK+ ALCL (n=67)
Morphologic type Common pattern Non-common pattern Immunophenotype CD2+ CD3+ (IHC and/or FC) CD3+ (IHC) Surface CD3+ (FC) CD5+ CD7+ CD25+ CD43+ CD45+ TCR A/B TCR G/D Granzyme B+ TIA1+ EMA+
ALK-negative ALCL (n=91) $
CD8-negative P value# (n=78)
CD8+ (n=13)
CD8-negative P value (n=54)
CD8+ (n=13)
31% (4/13) 69% (9/13)
87% (47/54) 13% (7/54)
1.0
0.0001
100% (13/13) 99% (77/78) 0% (0/13) 1% (1/78)
100% (10/10) 92% (12/13) 69% (9/13) 88% (7/8) 45% (5/11) 100% (9/9) 50% (3/6) 100% (1/1) 100% (9/9) 50% (2/3) 0% (0/3) 100% (6/6) 100% (1/1) 83% (5/6)
45% (18/40) 24% (12/50) 17% (8/48) 20% (4/20) 40% (18/45) 39% (13/33) 100% (13/13) 80% (24/30) 81% (26/32) 56% (5/9) 0% (0/7) 80% (8/10) 90% (9/10) 94% (17/18)
0.001 0.0001 0.0005 0.0019 0.75 0.002 0.02 1.0 0.31 1.0 1.0 0.47 1.0 0.45
90% (9/10) 100% (13/13) 100% (13/13) 60% (3/5) 36% (4/11) 63% (5/8) 25% (1/4) 25% (1/4) 100% (6/6) 40% (2/5) 0% (0/5) 29% (2/7) 14% (1/7) 17% (1/6)
0.67 0.0004 0.0001 1.0 1.0 0.10 0.12 0.12 1.0 1.0 1.0 0.24 0.1 0.65
77% (44/57) 48% (36/75) 42% (31/74) 48% (10/21) 36% (25/70) 27% (15/56) 75% (9/12) 75% (9/12) 88% (36/41) 31% (4/13) 8% (1/12) 56% (18/32) 53% (20/38) 32% (14/44)
ALCL, anaplastic large cell lymphoma; $, ALK+ CD8+ ALCL compared with ALK+ CD8-negative ALCL; #, ALK-negative CD8+ ALCL compared with ALK-negative CD8-negative ALCL; IHC, immunohistochemistry; FC, flow cytometry.
Figure 1
A
B CD3
C CD5
D CD30
E ALK
F Ki67
Figure 2 47.1% 47.1%
29.7% 13.8%5
A
CD8 PerCP-Cy5-5-A
10
196608
SSC-A
30.0%
131072
65536
2.5% 5
B
10
CD3 PE-Cy7-A
262144
104
103
10 3
10 4
10 5
45.6%
CD45 V500-A
24.9% 105
D
104
103
10.9% 10 5
-10 2 10 2
10 3
12.5%
10 4
2.4% 2.4%
-10 2 10 2
10 3
104
103
60.9% 10 5
10 4
CD2 APC-A
34.5%
-10 2 10 2
10 3
0.1% 7.2%
CD30 PE-A
F
104
103
38.2%
101 -102
10 5 38.2%
70.9%
10 4
37.9% 10 5 37.9%
10 4
CD7 APC-A
7.2% 105
E
101 -102
101 -102
5.2%
10 3
24.9%
CD4 FITC-A
CD5 PerCP-Cy5-5-A
10
103
CD30 PE-A
33.5% 0.4% 5
-10 2 10 2
TCR G/D PE-A
-10 2 10 2
104
101 -102
101 -102 0
C
-10 2 10 2
10 3
21.8% 10 5
10 4
TCR A/B FITC-A
Figure 3
A
B ALK
C CD3
D CD8
Figure 4
A
B
C
D
Figure 5
A
B