Lymphocytic infiltrates and subclinical

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%), 1 of 8 transplant recipients (13%),
and 1 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 5 0.029) and in transplant recipients (P 5 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 do 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


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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 8
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 5 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 1. Dense infiltrate associated with SCC in frozen section obtained during Mohs procedure in a patient with CLL (hematoxylin and
eosin; Â 25).


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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 5 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 8 of these 16 patients, a dense
infiltrate also was observed on the pre-Mohs biopsy
specimen, and in 6 of these 8 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 5 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 5 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 5 0.82).

Table 1. Presence of Dense Peritumoral Infiltrate
Infiltrates Present
Group
CLL
Transplant
Control

Number of Patients

Number of Patients

Percentage

55n
8
105

20
1

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


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Dermatol Surg 29:2:February 2003

Figure 2. 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


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133

Table 2. Subclinical Tumor Extension
Subclinical Tumor Extension (mm)

Group

Number of Tumors

Median

Mean7SD

Interquartile Range
(25th and 75th Percentiles)

76
53
105

4
2
2

6.778.8

4.175.8
3.373.1

1.8
1.4
1.5

CLL
Transplant
Control

Table 3. 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
4
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
3
4
2.5
2
2
4
2
3

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 do 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 mediated by

similar mechanisms.

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