Lymphocytic Infiltrates and Subclinical Epithelial Tumor Extension in Patients With Chronic Leukemia and Solid-Organ Transplantation K HOSROW M EHRANY, MD, D AVID R B YRD , MD, R ANDALL K R OENIGK , MD, R OGER H W EENIG , MD, P K IM P HILLIPS , MD, T RI H N GUYEN , MD, AND C LARK C O TLEY, MD Division of Dermatologic Surgery, Mayo Clinic, Rochester, Minnesota Dense infiltrates in association with squamous cell carcinoma (SCC) or basal cell carcinoma (BCC) in patients with underlying chronic lymphocytic leukemia (CLL) may complicate pathologic interpretation of histologic margins OBJECTIVE The study was conducted to determine the frequency of identifying dense inflammatory infiltrates in frozen histologic sections during Mohs operation for BCC or SCC in patients with CLL and organ-transplant recipients, to characterize the infiltrate (reactive versus leukemic) in CLL, and to estimate the subclinical tumor extension in patients with CLL, transplant recipients, and control subjects undergoing Mohs procedure METHODS Frozen sections of head and neck BCC and SCC obtained during Mohs procedures in patients with CLL, organ transplant recipients, and a control group were reviewed retrospectively Biopsy specimens of CLL with dense infiltrates BACKGROUND were assessed with immunohistochemical stains Subclinical tumor extension (postoperative defect size minus preoperative tumor size) was evaluated in each group RESULTS Dense infiltrates were found in tumors of 20 of 55 patients with CLL (36%), of transplant recipients (13%), and of 105 controls (1%) In patients with CLL, 75% of the dense infiltrates were B-cell leukemic Compared with controls, the mean subclinical tumor extension was larger in patients with CLL (P 0.029) and in transplant recipients (P 0.55) CONCLUSION Dense leukemic infiltrates associated with BCC or SCC in CLL may complicate pathologic interpretation of Mohs surgical histologic margins and may be associated with larger postoperative defects relative to preoperative clinical tumor appearance In patients with CLL, as in transplant recipients, SCC seems more likely to develop than BCC K MEHRANY, MD, D R BYRD, MD, R K ROENIGK, MD, R H WEENIG, MD, P K PHILLIPS, MD, T NGUYEN, MD, AND C C OTLEY, MD HAVE INDICATED NO SIGNIFICANT INTEREST WITH COMMERCIAL SUPPORTERS CHRONIC LYMPHOCYTIC leukemia (CLL) is the most frequent form of leukemia in adults of Western countries, accounting for 25% of all leukemias.1 The risk for development of CLL progressively increases with age and is two times higher in men than in women.2 The estimated annual incidence of CLL in the United States ranges from 7,300 to 12,500 new cases, and the overall incidence rate is 2.3 per 100,000.3 Because immune function is impaired in CLL, patients have an increased risk for development of other malignant neoplasms Skin cancer is the most frequently associated malignancy in CLL, and squamous cell carcinoma (SCC) predominates over basal cell carcinoma (BCC).4 Lymphoma- and leukemiaassociated SCCs are unusually aggressive and have Address correspondence to: Randall K Roenigk, MD, Division of Dermatologic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN 55905 substantially higher rates of recurrence and metastasis.5,6 Microscopically controlled excisions of BCC and SCC therefore may represent the optimal technique for management of patients with CLL.5,6 For surgeons who perform the Mohs operation, dense lymphocytic infiltrates associated with nonmelanoma skin cancers may pose an intraoperative challenge for interpretation of histologic sections because they obscure possible residual tumor If the infiltrate represents inflammation caused by tumor, then further resection may be indicated, whereas leukemic infiltrates that not imply tumor would not require further resection.7 Leukemic infiltrates also complicate the pathologic findings by camouflaging tumor cells or trapping collagen in a manner that disguises tumor islands Our objective was to clarify the issues surrounding infiltrates in histologic sections from patients with CLL and to compare the findings with those in controls and in another immunosuppressed group r 2003 by the American Society for Dermatologic Surgery, Inc  Published by Blackwell Publishing, Inc ISSN: 1076-0512/02/$15.00/0  Dermatol Surg 2003;29:129–134 130 MEHRANY ET AL.: LYMPHOCYTIC INFILTRATES with severe skin cancers, namely, recipients of solidorgan transplants We sought (1) to determine the frequency of a dense inflammatory infiltrate during Mohs operation for BCC or SCC in patients with CLL and in organ transplant recipients, (2) to characterize the infiltrate (reactive vs leukemic) in patients with CLL, and (3) to estimate subclinical tumor extension in patients with CLL, transplant recipients, and controls undergoing the Mohs procedure Methods The study was approved by the Mayo Foundation Institutional Review Board The Mayo Clinic diagnostic index was used to identify all patients with a history of CLL who had undergone the Mohs operation The 56 patients identified (48 males and females) had a total of 76 BCCs or SCCs of the head and neck Eight representative patients (seven males and one female) who had received solid-organ transplants and who underwent the Mohs procedure for 53 BCCs or SCCs of the head and neck were randomly selected for comparative purposes The control group included 105 patients (70 males and 35 females) with a total of 105 tumors The control group included sequential patients undergoing Mohs procedure for BCCs or SCCs of the head and neck Dermatol Surg 29:2:February 2003 without a diagnosis of CLL or a history of organ transplantation All frozen sections from intraoperative Mohs layers were reviewed and assessed for the presence or absence of a dense inflammatory infiltrate We defined dense infiltrates as dense collections of mononuclear cell aggregates of approximately 50 cells or more closely associated with epithelial tumor collections (Figure 1) The nature of the infiltrate (leukemic vs reactive) was ascertained with immunostains for CD20, CD3, and CD5 on the tissue blocks of all original skin cancer biopsy specimens (pre-Mohs procedure) with dense infiltrates from patients with CLL (N 20) A retrospective chart review of operative notes from each group of patients was performed to determine subclinical tumor extension Subclinical tumor extension was defined as the maximal postoperative diameter of the defect after the Mohs procedure minus the maximal preoperative diameter of the lesion For statistical analysis, the Fisher exact test was used to compare the proportion of patients with a dense infiltrate in each group Because the distribution of subclinical tumor extension was highly skewed, a square-root transformation was applied Generalized estimating equation models with normal link function were fit to evaluate differences in the average subclinical extension between tumor types (BCC vs SCC) and the groups of comparison (CLL vs control Figure Dense infiltrate associated with SCC in frozen section obtained during Mohs procedure in a patient with CLL (hematoxylin and eosin; Â 25) Dermatol Surg 29:2:February 2003 MEHRANY ET AL.: LYMPHOCYTIC INFILTRATES and transplant recipients vs control) An exchangeable correlation structure was specified to model the correlation between multiple tumors within a patient All calculated P values were two sided, and P values less than 0.05 were considered statistically significant Results Patients with CLL were significantly more likely to have a dense infiltrate than controls (Po0.001) (Table 1) Organ transplant recipients also were more likely to have a dense infiltrate than controls, but this difference was not statistically significant (P 0.14) (Table 1) Of the 20 patients with CLL in whom dense infiltrates were identified on Mohs sections, 16 had tissue blocks of pre-Mohs biopsy specimens available for immunostaining In of these 16 patients, a dense infiltrate also was observed on the pre-Mohs biopsy specimen, and in of these patients, a leukemic infiltrate was confirmed by immunohistochemistry (lymphocyte coexpression of CD5 and CD20; Figure 2) The other two patients had reactive infiltrates The mean subclinical tumor extension was approximately twofold greater (6.7 mm) in patients with CLL than in control subjects (3.3 mm) (P 0.029) Transplant recipients also had larger mean subclinical tumor extension (4.1 vs 3.3 mm), but this difference was not statistically significant (P 0.55) (Table 2) The tumors that developed in patients with CLL and in organ transplant recipients were predominantly SCCs: 59% in the CLL group and 92% in the transplant group In comparison, only 24% of the tumors in controls were SCCs For this reason, patients were stratified and compared by tumor type to control for this intragroup difference (Table 3) The mean subclinical tumor extension was larger for SCC than for BCC, but this difference was not statistically significant (P 0.82) Table Presence of Dense Peritumoral Infiltrate Infiltrates Present Group CLL Transplant Control Number of Patients Number of Patients Percentage 55n 105 20 1 36.4 12.5 1.0 In one patient, Mohs slides were unavailable for review n 131 Discussion Patients with CLL have numerous factors contributing to impaired host immune function.2 These include low complement levels, hypogammaglobulinemia, altered leukemic cell expression of major histocompatibility complex class II antigens, impaired granulocyte function, functional defects in bystander T cells, and altered expression of T-cell receptor variable region genes The B cells of CLL also may be responsible for impaired host immune-suppressive factors These B cells can rapidly down-modulate expression of CD40 ligand (CD154) on activated T cells, in turn hindering activated T-cell interaction with bystander normal B lymphocytes or other antigen-presenting cells Such immunodeficiency may explain the increased risk for development of skin cancer in patients with CLL In addition, the unusually aggressive behavior of tumors with higher rates of recurrence and metastasis may arise from such underlying defects.2 As the most frequent CLL-associated malignancy, skin cancers have an 8- to 13-fold increase in incidence.4 Similar to transplant recipients, patients with CLL have an altered ratio of BCC to SCC The usual ratio of BCC to SCC is 4:1 in nonimmunocompromised patients The ratio is 3:8 in patients with CLL and 1:4 in transplant recipients.5,8 Our study identified BCC to SCC ratios of approximately 2:3 in the CLL group and 1:9 in the transplant group, which are similar to previously reported estimates To our knowledge, the CLL group from our study is the largest cohort evaluating the relationship between skin cancer and dense inflammatory infiltrates The increased ratio of SCC to BCC implies that SCC development may be more controlled by intact immunosurveillance mechanisms than in many other cancers; a compromise of immunosurveillance may permit uncontrolled proliferation, as in CLL- and transplant-associated immunosuppression Dense infiltrates associated with nonmelanoma skin cancers may be found in pathologic sections of patients with CLL, transplant recipients, or nonimmunocompromised patients Controlled, objective studies of this phenomenon remained lacking because there were only case reports of dense infiltrates in CLL-associated skin cancers Until now, the question remained whether this finding was common or just recall bias We found with statistical significance that more than one third of CLL-associated tumors have dense infiltrates Because of this high frequency, surgeons who perform Mohs procedures will regularly be challenged because tumor cells can be masked by infiltrate In the majority of cases, these infiltrates are leukemic cells rather than a specific reactive infiltrate The use of frozen or permanent section immunostains 132 MEHRANY ET AL.: LYMPHOCYTIC INFILTRATES Dermatol Surg 29:2:February 2003 Figure Immunostains of pre-Mohs biopsy specimen of basal cell carcinoma leukemic infiltrate ( Â 100.) (A) CD20 (B) CD5 (C) CD3 Note the prominent lymphocyte coexpression of CD20 and CD5 in the setting of sparse CD3 positivity may assist in assessing the presence of residual tumor within areas of dense infiltrate; we have not routinely used these techniques Dense infiltrates were not significantly increased in our transplant group compared with controls Thus, although both patients with CLL and transplant recipients are immunosuppressed, the tendency for development of nonmelanoma skin cancers in each group may arise from a different underlying mechanism In CLL, the immune response may be exaggerated yet dysfunctional, whereas in the transplant setting, there is an iatrogenic immune defect In evaluating the dense infiltrates associated with nonmelanoma skin cancers in patients with CLL, our Dermatol Surg 29:2:February 2003 MEHRANY ET AL.: LYMPHOCYTIC INFILTRATES 133 Table Subclinical Tumor Extension Subclinical Tumor Extension (mm) Group Number of Tumors Median Mean7SD Interquartile Range (25th and 75th Percentiles) 76 53 105 2 6.778.8 4.175.8 3.373.1 1.8 1.4 1.5 CLL Transplant Control Table Subclinical Tumor Extension, by Diagnosis and Tumor Type Subclinical Tumor Extension (mm) Group CLL Transplant Control Total Type of Tumor Number of Tumors BCC SCC BCC SCC BCC SCC BCC SCC 31 45 49 80 25 115 119 goal was to determine whether the cells represent an inflammatory response or a leukemic infiltrate Because 95% of CLL cases in the United States are B-cell phenotype, we used CD5 and CD20 immunohistochemical stains to define the nature of the infiltrates.3,9 A CD3 stain also was used to identify T-cell infiltrates, which would most likely represent an inflammatory response Because six of the eight original biopsy specimens with dense infiltrates stained strongly for CD5 and CD20, the majority of these cases represented leukemic B-cell infiltrates Subclinical tumor extension may represent the biologic outcome of unfettered tumor growth in the context of immunodeficiency Subclinical extension was significantly larger in the CLL group than controls The most likely explanation for this finding is that a dysfunctional immune system in the patients with CLL allowed for greater subclinical tumor growth Subclinical tumor extension was not significantly greater in the transplant group than in controls Although unexpected, this finding may be explainable In our experience, transplant recipients often present with multiple well-circumscribed SCCs that often have histologic and clinical features suggestive of viral induction These tumors rarely have considerable subclinical extension The greater numbers of these probable human papilloma virus–induced SCCs may have obfuscated the considerable subclinical extension Median 2.5 2 Mean7SD Interquartile Range (25th 75th Percentiles) 6.577.7 6.879.5 2.571.7 4.376.0 3.273.1 3.673.1 4.174.9 5.177.2 1.8 1.8 1.4 1.4 1.4 1.5 1.5 1.6 noted in ultraviolet light–induced SCCs in transplant recipients In addition to dysfunctional immunity, subclinical tumor extension in CLL may be enhanced by elaboration of cytokines and inflammatory mediators by the lymphocytic infiltrates Another possible explanation could be that patients with CLL not really have greater subclinical tumor extension Their larger postoperative defect relative to preoperative clinical appearance may be a consequence of extra tissue removal during Mohs operation to ensure clearance of tumor, which results from the obscuring of frozen section histologic analysis by dense infiltrates In conclusion, more than one third of patients with CLL had dense lymphocytic infiltrates associated with nonmelanoma skin cancers on Mohs sectioning These predominantly leukemic infiltrates may complicate interpretation of histologic sections and may partially explain the phenomenon of extensive subclinical tumor extension noted in these patients Transplant recipients had a trend toward increased subclinical tumor extension that did not seem to be 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patient with chronic lymphocytic leukemia: an intraoperative diagnostic challenge for the Mohs surgeon Dermatol Surg 1998;24:269–72 Berg D, Otley CC Skin cancer in organ transplant recipients epidemiology, pathogenesis, and management J Am Acad Dermatol 2002;47:1–17 Jaffe ES Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues Lyon: IARC Press, 2001  ... infiltrates associated with nonmelanoma skin cancers in patients with CLL, our Dermatol Surg 29:2:February 2003 MEHRANY ET AL.: LYMPHOCYTIC INFILTRATES 133 Table Subclinical Tumor Extension Subclinical. .. Mohs procedure in a patient with CLL (hematoxylin and eosin; Â 25) Dermatol Surg 29:2:February 2003 MEHRANY ET AL.: LYMPHOCYTIC INFILTRATES and transplant recipients vs control) An exchangeable... identified (48 males and females) had a total of 76 BCCs or SCCs of the head and neck Eight representative patients (seven males and one female) who had received solid-organ transplants and who underwent