Primary Cutaneous Diffuse Large B-Cell Lymphoma A ...

Anatomic Pathology / PRIMARY CUTANEOUS DIFFUSE LARGE B-CELL LYMPHOMA

Primary Cutaneous Diffuse Large B-Cell Lymphoma A Clinicopathologic Study of 15 Cases Thomas A. Hembury, MD,1 Benita Lee, MD,2 Randy D. Gascoyne, MD,2 Nicol Macpherson, MD,3 Bin Yang, MD, PhD,1 Nancy House, MD,5 L. Jeffrey Medeiros, MD,4 and Eric D. Hsi, MD1 Key Words: Cutaneous lymphoma; Diffuse large B-cell lymphoma; bcl-2; bcl-6; p53; CD5; CD10

Abstract Primary cutaneous diffuse large B-cell lymphoma (DLBCL) is an uncommon lymphoma. Some authors have suggested that large B-cell lymphoma can be segregated based on anatomic site, with tumors of the lower extremity being unique. We report 15 cases of primary cutaneous DLBCL. Each case was analyzed immunohistochemically using antibodies specific for CD3, CD5, CD10, CD20, bcl-2, bcl-6, and p53. Polymerase chain reaction analysis for t(14;18)(q32;q21) also was performed. There were 13 men and 2 women (median age, 64 years). Thirteen tumors were composed predominantly of centroblasts, and 2 were immunoblastic. There was a median followup of 72 months. Of the 4 patients with primary cutaneous DLBCL of the lower extremity (thigh, knee, leg), 2 (50%) experienced a recurrence and 1 patient died of disease. In the non–lower extremity cases, 18% (2/11) recurred and no patients died of disease. We conclude that primary cutaneous DLBCL usually occurs in elderly patients with a male predominance. Recurrences are common, but death of disease is rare.

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Approximately 25% of primary cutaneous lymphomas are B-cell lymphomas; large B-cell lymphoma accounts for many of these cases.1 The clinical and pathologic characterization of this entity has been difficult owing to the use of different classification systems.2-4 In particular, primary cutaneous diffuse large B-cell lymphoma (DLBCL) is a somewhat controversial entity. While most pathologists and hematologists have adopted the Revised European and American Classification of Lymphoid Neoplasms (REAL classification)5 and, by extension, the World Health Organization (WHO) classification,6 the European Organization for Research and Treatment of Cancer (EORTC) recently proposed a separate system for cutaneous lymphomas according to their clinical behavior.2 This has caused some degree of confusion since definitions in this latter system are at odds with the REAL and WHO systems. In the area of primary cutaneous B-cell lymphomas, one troublesome area for pathologists is the EORTC entity of primary cutaneous follicle center cell lymphoma, which may be a histopathologically heterogeneous entity that encompasses follicle center lymphoma and some cases of DLBCL (as defined by the REAL classification).7,8 It also has been proposed by the EORTC to separate large B-cell lymphoma of the leg from other cutaneous DLBCL.2 This type of lymphoma has been suggested to have a worse prognosis and to more often express bcl-2 protein compared with DLBCL in other cutaneous sites. 2,9,10 However, it is not clear whether some of these cases indeed represent follicle center lymphomas (with a follicular architecture) as defined by the REAL classification. In recent years, most studies of cutaneous large B-cell lymphomas have been done using classification schemes © American Society for Clinical Pathology

Anatomic Pathology / ORIGINAL ARTICLE

❚Table 1❚ Antibodies Used Antibody Anti-CD3 Anti-CD5 Anti-CD10 Anti-CD20 Anti–bcl-2 Anti–bcl-6 Anti-p53 Anti–cyclin D1 Anti-CD21 Anti-CD79a *

Clone

Dilution

Antigen Retrieval*

Polyclonal NCL-4C7 NCL-270 L26 1D5 N-3, polyclonal DO-7 DCS-6 1F8 JCB-117

1:5 1:10 1:5 1:100 Prediluted 1:20 1:20 Prediluted 1:10 1:10

HIERc HIERc HIERc HIERc HIERc HIERc HIERe HIERe Protease HIERc

Source Zymed, South San Francisco, CA Novocastra, Newcastle upon Tyne, England Novocastra DAKO, Carpinteria, CA Ventana, Tucson, AZ Santa Cruz, Santa Cruz, CA DAKO Ventana DAKO DAKO

HIERc is heat-induced epitope retrieval with a 10-mmol/L concentration of citrate, pH 6.0; HIERe, heat-induced epitope retrieval with EDTA, pH 8.0.

similar to the EORTC system.9-14 Thus, the clinical and pathologic features of primary cutaneous DLBCL, using the REAL classification criteria, are not well described. Yang et al4 recently have shown that the REAL classification is applicable to primary cutaneous B-cell lymphomas. We decided to focus on cases of primary cutaneous DLBCL as defined by the REAL and WHO classifications to investigate proposed differences in their clinical and immunophenotypic characteristics based on location and to determine whether the t(14;18)(q32;q21) is present in primary cutaneous DLBCL.

Materials and Methods Cases Cases of primary cutaneous DLBCLs were obtained from the 4 participating institutions. Fifteen cases of primary cutaneous DLBCL were included for the final study. All cases were selected based on a diffuse infiltrate of large, transformed cells with disease limited to the skin at the time of diagnosis and for at least 6 months following diagnosis. Staging procedures used to evaluate the patients were variable and most often included physical examination, CBC count, computed tomography scans, bone marrow aspirates, and biopsies. Additional procedures used to stage disease included lymphangiograms, chest radiographs, and bone scans. Of the 15 patients, 11 underwent physical examination, imaging studies, and bone marrow biopsy. One patient (case 9) underwent all of the aforementioned procedures, but no bone marrow biopsy was performed. One patient (case 6) refused staging and treatment after excisional biopsy of the lesion. Details of the initial staging for 2 patients (cases 10 and 15) were unavailable. Cases 6, 10, and 15 were not excluded from the study because all were treated by local therapy alone, and none developed extracutaneous disease during the follow-up period. © American Society for Clinical Pathology

There was no history of lymphoma in any of these patients. Five cases were part of a previous publication.4 Eighteen potential cases were excluded from the study for one or more of the following reasons: histologically having foci of follicular architecture, lack of adequate material, and equivocal initial staging such as rare lymphoid aggregates of uncertain significance in bone marrow or mildly, but not pathologically, enlarged lymph nodes shown by imaging studies. Immunohistochemical Analysis Immunohistochemical analysis on paraffin-embedded sections was performed in all cases using antibodies to CD3, CD5, CD10, CD20, bcl-2, bcl-6, and p53 using an automated immunostainer (Ventana Medical Systems, Tucson, AZ). Additional immunostaining was performed on selected cases using antibodies to CD79a, CD21, and cyclin D1. Positive and negative controls were run with the study cases and stained appropriately. Stains were considered positive when more than 10% of the tumor cells were immunoreactive for p53, bcl-2, and bcl-6. The remaining markers were considered positive when staining was seen in a majority of tumor cells. Details of the primary antibodies used are provided in ❚Table 1❚. Molecular Studies Polymerase chain reaction analysis of t(14;18)(q32;q21) was performed in all cases. Genomic DNA was extracted from formalin-fixed, paraffin-embedded sections using Proteinase K digestion and standard methods. Polymerase chain reaction (PCR) amplification of the beta-globin gene was used to assess suitability of DNA extracts. PCR analysis for the t(14;18)(q32;q21) translocation was performed using primer sets to bcl-2 major breakpoint region (mbr) (forward) 5'-GAG TTG CTT TAC GTG GCC TG-3' and JH consensus primer (reverse) 5'-ACC TGA GGA GAC GGT GAC C-3'. A nodal follicle center cell lymphoma known to carry the t(14;18)(q32;q21) involving bcl-2 mbr was used as a positive Am J Clin Pathol 2002;117:574-580

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❚Table 2❚ Clinical Information and Immunophenotype Case No./ Sex/Age (y) 1/M/71 2/M/64 3/M/53 4/M/62 5/M/40 6/M/68 7/M/59 8/M/44 9/F/82 10/M/82 11/M/36 12/F/62* 13/M/80 14/M/75 15/M/84

Location

Recurrence

Forearm lesion Breast nodule Back lesions Left arm lesions Back lesion Forehead lesion Chest wall lesion Scalp lesion Scalp nodule Groin nodule Back papules Thigh lesion Calf lesion Knee lesion Leg lesion

Yes, 12 mo No No No No No No No No No Yes, 4 mo Yes, 16 mo No No Yes, 14 mo

Follow-up (mo) 47 197 242 81 96 75 37 72 84 48 78 22 26 7 22

Treatment Chemotherapy Chemotherapy Chemotherapy Chemotherapy and radiation Chemotherapy and radiation Surgical excision Chemotherapy and radiation Chemotherapy and radiation Radiation Radiation Chemotherapy Chemotherapy and radiation Chemotherapy and radiation Surgical excision Radiation

bcl-2

bcl-6

p53

CD10

CD5

CD20

+ + – + – – + + + + – + – + +

– – + + + – + + – – + – + + –

+ – – + – – – – + + – – – – –

– – – – – – – + – – – – – – –

– + – – – – – – – – – – – – –

– + + + + + + + + + + + + + +

+, positive; –, negative. * Died of disease at 22 months. All other patients were alive at last follow-up.

control. Negative controls with no template DNA and normal patient DNA were included in each run. Forty-three cycles of amplification were run on the thermocycler. PCR products underwent electrophoresis on agarose gel and were stained with ethidium bromide. The test was considered positive when the gel contained 1 or 2 discrete narrow bands within the appropriate size range of 80 to 300 base pairs. Statistical Analysis The Fisher exact test, Kaplan-Meier analysis, and log-rank tests were performed using Statistica (StatSoft, Tulsa, OK).

Results Clinical Data The 15 cases included 13 men and 2 women (median age, 64 years). Four cases arose in the lower extremity (thigh, knee, leg). Eleven cases involved non–lower extremity sites, including head/neck (n = 3), trunk (n = 6), arm (n = 1), and forearm (n =1). Follow-up data were available for all cases, with a median follow-up of 72 months (range, 7-242 months). Of the 4 cases in the lower extremity group, 2 (50%) recurred during the follow-up period. One patient (case 12) died as a result of systemic progression of disease. She first had a recurrence in the skin of the leg and then developed pelvic lymph node involvement 16 months after initial diagnosis. She died 6 months later. The other patient in the lower extremity group (case 15) who had a recurrence had disease limited to the skin of the leg. In the non–lower extremity group, 2 (18%) of 11 tumors recurred. No patients in this group died of disease, and recurrences 576

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were limited to the skin. Case 1 recurred at the same site 1 year later, and a second recurrence appeared in the skin of the opposite forearm 3.5 years after the initial diagnosis. Case 11 had recurrent disease involving the skin of his back. Treatment methods were variable; 4 patients received chemotherapy alone, 3 received radiation alone, 6 received both chemotherapy and radiation, and 2 were treated by surgical excision only. Of the 10 patients receiving chemotherapy, all received regimens that contained anthracycline (doxorubicin [Adriamycin]). The clinical features are summarized in ❚Table 2❚. Histopathologic and Immunophenotypic Findings At low magnification, all cases showed a dense and diffuse infiltrate involving the entire dermis. There was no epidermotropism and no evidence of a follicular architecture. In some cases, the neoplastic cells tended to cluster around adnexal structures. Cytologically, 13 cases were composed of predominantly large, centroblastic cells ❚Image 1❚, while 2 contained a majority of immunoblasts ❚Image 2❚ (cases 9 and 10). Fourteen cases expressed CD20, and the 1 case negative for CD20 was strongly positive for CD79a. One case had a pattern similar to a T cell–rich B-cell lymphoma (case 6), as it was composed of large centroblastic cells in a background of small T lymphocytes.15 Case 6 was stained for CD21 and showed no evidence of dendritic cells suggestive of follicular architecture. One case expressed CD10, and 8 cases were positive for bcl-6 (1 coexpressed these markers). Of the 8 cases positive for bcl-6, 6 had staining in more than 50% of the tumor cells, while in 2 cases, between 10% and 50% of the cells stained. Ten cases expressed bcl-2 protein. More than 50% of the cells stained in 7 of these cases. In the remaining 3 cases, between 10% and 50% of the neoplastic © American Society for Clinical Pathology


A

B

❚Image 1❚ (Case 13) A, The dermis contains a diffuse infiltrate of neoplastic cells. There is no epidermotropism (H&E, original magnification ×100). B, The neoplastic cells have a predominantly centroblastic appearance (H&E, original magnification ×1,000).

❚Image 2❚ (Case 10) Upper left, The neoplastic cells have a predominantly immunoblastic appearance (H&E, original magnification ×1,000). The neoplastic cells stained positively for CD20 (upper right), p53 (lower left), and bcl-2 (lower right) (original magnification ×400).

cells stained. Four cases overexpressed p53. Three of the p53-positive cases displayed strong staining in the majority of tumor cells, while in 1 case, approximately 20% of tumor cells stained. Both of the cases that had an immunoblastic appearance stained positively for p53. All tumors were negative for CD3. One DLBCL was positive for CD5 but negative for cyclin D1.

Statistical Analysis Kaplan-Meier analysis showed 4-year relapse-free survival of 73% ❚Figure 1❚. We could find no significant difference in relapse-free survival for cases expressing bcl-2, p53, or bcl-6 (P > .10 for each). When the set of patients was divided into 2 groups based on site (lower extremity vs non–lower extremity), there was no obvious difference in types of therapy. Furthermore, there seemed to be no correlation between site of involvement (lower extremity vs non–lower extremity) and expression of bcl-2, bcl-6, CD5, or CD10. None of the cases on the lower extremity overexpressed p53; however, 4 (36 %) of 11 cases in the non–lower extremity group overexpressed p53. This difference in expression of p53 was not statistically significant (P > .10, Fisher exact test). We recognize the number of cases is small and that definitive conclusions about site would require more cases.

Cumulative Proportion Relapse-Free

Molecular Genetic Findings DNA was amplified in 14 of 15 cases, as indicated by amplification of the beta-globin gene. All 14 were negative for the t(14:18)(q32;q21) involving the bcl-2 mbr.

1.0 0.9 0.8 0.7 0.6 0.5

Discussion

0.4 0.3 0.2 0.1 0.0

0

50

100

150

200

250

Survival Time (Months)

❚Figure 1❚ Overall relapse-free survival. Circle indicates complete; +, censored.

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300

Primary cutaneous DLBCL is an uncommon lymphoma and as such has not been studied extensively. Characterization of this lymphoma has been made difficult, in our opinion, owing to differences in definition as specified in different classifications. As defined by the REAL classification and the WHO system, primary cutaneous DLBCLs are composed of large, transformed B cells without follicular architecture. The Am J Clin Pathol 2002;117:574-580

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cytologic features may be variable.5,6 Because of specific clinical features, namely an indolent clinical course that was observed for cutaneous lymphomas, the EORTC proposed a separate classification of cutaneous lymphomas based on their clinical behavior.2 In this system, it seems that cases diagnosed as DLBCL within the REAL and WHO classification systems might be placed into 2 groups based on location of involved skin.2,3,16 These EORTC groups include large Bcell lymphoma of the leg and cutaneous follicle center cell lymphoma. Support for this practice might exist at the phenotypic level, with some investigators reporting bcl-2 expression in large B-cell lymphoma of the leg, while large cell lymphomas at other cutaneous sites lack expression of this anti-apoptotic protein.10 Conversely, the latter EORTC group (follicle center cell lymphoma) might include follicle center lymphoma and diffuse large B-cell lymphoma as defined in the REAL classification. To better define the characteristics of primary cutaneous DLBCL, we report the morphologic, clinical, immunophenotypic, and molecular genetic features in a series of DLBCL primary cases in skin using the criteria of the REAL and WHO classifications. As in most series of primary cutaneous large B-cell lymphoma, primary cutaneous DLBCL in our series occurred in older adults, median age 64,4,16,17 and occurred most commonly on the head/neck and trunk.2,4,8,18 In agreement with Willemze and colleagues,16 we found a male predominance. However, this is not a universal finding.9,17,19 We are uncertain about the reason for this difference. We know of no sex bias in our case selection. Variation in case inclusion criteria might account for some differences, since some studies, such as the study by Geelen et al,9 may have included follicle center lymphomas. Geographic differences should not be discounted either. In our series of cases, the great majority of cases had centroblastic morphologic features. Two cases seemed to have numerous immunoblasts. In nodal DLBCL, some suggest that immunoblastic lymphoma has a worse prognosis than DLBCL without immunoblasts.20 We cannot substantively comment on this based on our results owing to a limited number of immunoblastic cases. This may be an area for future investigation. In no case could we detect, nor was there a history of, a lower cytologic grade of primary cutaneous lymphoma such as marginal zone or follicle center lymphoma. Review of several other larger series of primary cutaneous B-cell lymphomas4,9,12 suggests that the great majority of cases of primary cutaneous DLBCL are de novo lesions. The infiltrates usually diffusely involved the dermis and showed no epidermotropism. In some cases, a tendency to involve adnexal structures was noted. One case lacked CD20 but was found to express CD79a, a marker of B-cell lineage. 21 Only 1 (7%) of 15 cases expressed CD10, a percentage much lower than that seen in 578

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nodal DLBCL.22 This finding supports the contention that primary cutaneous DLBCLs are distinct from true follicle center lymphomas in which CD10 usually is expressed.4,12 Roughly 15% to 20% of nodal DLBCLs harbor the t(14;18)(q32;q21).23-25 We found no cases that contained this translocation involving the bcl-2 mbr by PCR. This finding is consistent with previous studies that reported the rarity of t(14;18)(q32;q21) in primary cutaneous lymphomas.26,27 Along with the CD10 data, this suggests that most primary cutaneous DLBCLs do not represent transformed follicle center lymphomas. Expression of bcl-2 was seen in 10 (67%) of 15 cases, a rate similar to or slightly higher than other series of nodal DLBCL.28-31 Expression of bcl-2 in these cases highlights the fact that bcl-2 protein can be occur in the absence of the t(14;18)(q32;q21).32 Expression of bcl-6 was seen in 8 (53%) of 15 cases, a rate slightly lower than reported in nodal DLBCL (71%).30 Overexpression of p53 was seen in 4 cases and is consistent with the finding of p53 immunoreactivity in only a minority of DLBCLs.33 This rate of p53 staining is higher than that recently reported by Fernandez-Vazquez and colleagues12; however, their criterion for positivity was more stringent than most. Although the number of cases is small, the 2 cases with morphologic features of immunoblastic large cell lymphoma both overexpressed p53 protein. This may be an area for future study. Importantly, we found no differences in immunophenotype, including bcl-2 expression, in cases of primary cutaneous DLBCL occurring on the lower extremity compared with those occurring at other cutaneous sites. This immunophenotypic heterogeneity also can be found in nodal DLBCL, and, as in that disease, it suggests a biologic heterogeneity.34 Our bcl-2 data differ from those of Geelen and colleagues9 who found that 100% of cases occurring on the lower extremity expressed bcl-2, while none of the cases on the head and trunk expressed bcl-2. We are uncertain of the exact reasons for this discrepancy. As alluded to earlier, it is possible that case definition and geographic factors may be involved. However, in support of our data, FernandezVazquez et al12 also found no difference in bcl-2 protein expression by site in primary cutaneous large B-cell lymphoma defined in a manner similar to that of Geelen et al9 and the EORTC.12 Both of those studies included cases that would be classified as follicle center lymphoma using the REAL classification criteria. From this observation and our data, it seems that bcl-2 expression is not a biologic factor that distinguishes DLBCL of the lower extremity from that found in other anatomic sites. As has been shown for primary cutaneous B-cell lymphomas in general, primary cutaneous DLBCL has an excellent prognosis,2,4 with only 1 patient dying of disease in © American Society for Clinical Pathology


our series. The median follow-up of our patients was 6 years. Primary cutaneous DLBCL, like other cutaneous B-cell lymphomas, seems to have an indolent and relapsing course but with uncommon dissemination to extracutaneous sites. In our series, relapse was seen in 4 of 15 patients. Three of the recurrences were limited to the skin, while 1 patient had progression of disease to involve lymph nodes. Because our number of cases is small, we could not adequately evaluate whether DLBCL of the lower extremity differed from other cutaneous DLBCLs in clinical outcome. However, in contrast with the findings of previous studies,10 FernandezVazquez et al12 found no difference in survival of patients with primary cutaneous large B-cell lymphoma based on disease location. Since our series is retrospective and patients received nonuniform treatment, we cannot make recommendations about therapy. This is, in fact, a common problem with primary cutaneous lymphomas. It seems to be the consensus that one must take into account patient factors such as age, general health, and extent of disease and that there is no recommended uniform therapy.18 We present our findings of a series of DLBCLs primary in skin. Clinically, the patients had features similar to other patients with primary cutaneous B-cell lymphomas, with lesions concentrated on the head/neck and trunk and usually an indolent clinical course. Relapse was frequent but death of disease was uncommon. These lymphomas rarely expressed CD10 and did not harbor the t(14;18)(q32;q21). Expression of bcl-2 protein and p53 was heterogeneous, as is the case for nodal DLBCL. Although we are limited by a small number of cases, we found no clinical or immunophenotypic differences between primary cutaneous DLBCL occurring in the lower extremity compared with other sites. Our definition of primary cutaneous DLBCL is different from that in some other series of cutaneous large B-cell lymphoma; we believe it to be more precise since it can then be distinguished from other entities such as follicle center lymphoma (REAL classification, follicular lymphoma in the WHO). This is important since many primary cutaneous follicle center lymphomas do indeed express CD10 and bcl-2 and harbor the t(14;18)(q32;q21).4,35,36 In an effort to more carefully define distinct biologic entities, precise definitions are essential. While it is clear that primary cutaneous lymphomas have distinct clinicopathologic features that seem to distinguish them from some of their nodal counterparts, recognizing them in the context of a broader framework seems appropriate. Additional studies with strict definitional criteria will be required to better understand the biology and clinical behavior of this type of lymphoma. Only then can we begin to make progress in defining useful biologic and clinical stratifications of primary cutaneous DLBCL, analogous to those being developed for nodal DLBCL.25,37 © American Society for Clinical Pathology

NOTE: While our manuscript was in review, a large series of cutaneous large B-cell lymphomas was published (Grange F, Bekkenk MW, Wechsler J, et al. Prognostic factors in cutaneous large B-cell lymphomas: a European multicenter study. J Clin Oncol. 2001;19:3602-3610). The authors suggest that location on the leg, round cell morphologic features of the large neoplastic cells, and the presence of multiple lesions were poor prognostic indicators. From the 1Department of Clinical Pathology, Cleveland Clinic Foundation, Cleveland OH; 2Department of Pathology, British Columbia Cancer Agency, Vancouver; 3Division of Medical Oncology, British Columbia Cancer Agency, Victoria; 4Department of Hematopathology, M.D. Anderson Cancer Center, Houston, TX; and 5Rabkin Dermatopathology, Pittsburgh, PA. Address reprint requests to Dr Hsi: Dept of Clinical Pathology, L-11, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195.

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