Langerhans Cells Related to Prognosis Patients With Laryngeal Carcinoma Oreste Gallo, MD; Giacinto Asprella Libonati, Carmelo Urso, MD; Roberto Bondi, MD \s=b\ Intratumoral and peritumoral infiltration of T-zone histiocytes, mainly of mature Langerhans cells, was investigated in 88 patients with squamous cell carcinoma of the larynx by immunohistochemical methods using polyclonal antibodies against S100 protein and lysozyme. Granulocytic and lymphoid inflammatory infiltration and its relationship to the presence of Langerhans cells were also evaluated. Langerhans cells were present within the cancer tissues and showed a relationship with lymphoid infiltrate. No significant correlation was present among the density of Langerhans cells and the site of neoplastic growth (supraglottic or subglottic), granulocytic inflammatory infiltration, histological tumor grade, or clini-
tumors in other sites, a laryn¬ tumor is often characterized
Likegeal
inflammatory reaction that fol¬ lows the growth of neoplastic ele¬ ments. This reaction has prognostic value,1"3 but its functional significance by
an
has still to be established.
Recently,
a
significant relationship
has been reported between prognosis and degree of infiltration of mononu¬ clear inflammatory cells within tumor tissues.4"6 It would seem that, if tumors Accepted for publication February 5,1991. From the Otorhinolaryngological Clinic II (Drs Gallo, Asprella Libonati, Gallina, and Fini-Storchi), and Institute of Pathological Anatomy (Drs Giannini, Urso, and Bondi), University of Florence
(Italy). Reprint requests to II Clinica Otorinolaringoiatrica, Universit\l=a`\di Firenze, Viale Morgagni 85, 50100 Firenze, Italia (Dr Gallo).
in
MD; Ezio Gallina, MD; Omero Fini-Storchi, MD; Augusto Giannini, MD;
cal stage. Patients with high or intermediate density of Langerhans cells survived longer than those with low density (mean survival, 61%, 62%, and 0%, respectively). The number of Langerhans cells was relevant in patients with evident infiltration of lymphocytes and plasma cells, according to their ability to present antigens to sensitized T cells. Our results indicate that the presence of high or intermediate density of Langerhans cells and of marked lymphoid inflammation may be considered favorable prognostic factors for patients with squamous cell carcinoma of the larynx. (Arch Otolaryngol Head Neck Surg.
1991;117:1007-1010)
are cells with particular immunohistochemical findings (lysozyme negative, S100 protein positive, OKT6\ OKT1") that are found in a direct relationship with lympho¬
tiocytes
cytes.1"8
Langerhans cells present little phagocytic ability and express several immunological markers such as FcIgG, C3 receptor, Ia/DR antigen, S100 protein, and T6 antigen.1315*20 In addi¬ tion, they show similar morphological
aspects such as lobate and convoluted nucleus, clear cytoplasm with dendritic processes, and distinct
cytoplasmic organelles (Birbeck's granules or LC granules) without tonofilaments and
are
desmosomes.1315
the site of the tumor growth.7 These cells with cytotoxic activity are specific lymphocytes and natural killer cells.8 T-Cell activation requires a particular antigen presentation. In this immuno¬ logical process, groups of cells, defined as antigen-presenting cells or accesso¬ ry cells, are involved.9 Accessory cells can present antigen associated with major histocompatibility complex class II polypeptides and can produce interleukin l.10"12 Langerhans cells (LCs) are a group of cells with accessory function.18"15 These cells, also defined as "T-zone
It has been demonstrated that an increased number of LCs is related to a more favorable prognosis in cancers of the rhinopharynx,21 oral cavity,22 stomach,23 lung,1617 and thyroid,24 and in T-cell lymphomas.25 It is well known, however, that Ia/DR accessory cells sensitize lymphocytes, both helper and suppressor, and may regulate their functional balance.26 Recently, many subpopulations of epidermal histiocytes have been dem¬ onstrated by immunohistochemical studies, using different antibodies to the immunological markers of LCs, and functional differences among them have been suggested.27"21' Therefore, it is possible that a quantitative increase of these cells may not always be linked
immunogenic in the host, the lym¬ phocytes with the most potent antitumor activity should be present within
histiocytes,"1617 are thought to present antigens to lymphocytes. T-Zone his-
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Fig 2.—S100 protein immunostain by peroxidase-antiperoxidase method, showing a cell with dendritic cytoplasm and Irregular nucleus, both of which were positive tor S100 protein. The specimen was obtained from the cancer tissue (peroxidase-antiperoxidase-S100, 1350).
Fig 1.—Langerhans cells in the cancer nest, with evidence of dendritic cytoplasmic processes interspersed among the neoplastic cells (peroxidase-antiperoxidase-S100, 500).
to
Density of Langerhans Cells According and
Lymphoid
to Tumor
Stage, Node Invasion,
Intermediate
High
Total 10
I
18 20
52
IV Node invasion 10
+
46
Lymphoid
infiltration
Slight (1+)
Moderate (2+)
Marked (3+) Total
22 14
0
10
42
36
26 42 20 88
3.—Global actuarial corrected survival according to Langerhans cell density. Light squares indicate low (1+J reaction for S100 protein; diamonds, intermediate (2 + ) reaction; and dark squares, high (3 + ) reaction.
Fig
100
co
We have previously shown the pres¬ of LCs and their correlation with lymphoid infiltrate in cancer of the larynx.31 In this study, we investigated the prognostic significance of the den¬ sity of LCs infiltrating tumor tissues in 88 patients with laryngeal cancer. Im¬ munological reaction in the host, in¬ volving these reactive lymphoreticular cells against tumor, has also been considered. PATIENTS AND METHODS The study group included 88 patients (86 men, two women), aged from 37 to 79 years (mean age, 62 years), affected by carcinoma ence
Tumor stage
24
functional activation of immune
responsiveness.3"
Infiltration
Density of Langerhans Cells Low
a
of the larynx and surgically treated at the Ear-Nose-Throat clinic of the University of Florence (Italy). Specimens from these pa¬ tients were examined at the Institute of Pathological Anatomy of the University of Florence. After surgical removal, the speci¬ mens were fixed in 10% phosphate-buffered formaldehyde solution (pH 7.4), embedded in paraffin, and processed conventionally. Consecutive sections 4 µ thick were stained with hematoxylin-eosin. Moreover, a peroxidase-antiperoxidase method was used employing antibodies against the S100 cyto¬ plasmic protein and lysozyme, as described by Nomori et al.21 (Antilysozyme antibody and the peroxidase-antiperoxidase kit used for immunostaining were purchased from
Dakopatts Ltd, Copenhagen, Denmark.)
Years
The paraffin-embedded (4-µ thick) sections were treated with 0.5% hydrogen peroxide in methanol for 30 minutes to eliminate endoge¬ nous peroxidase, and then washed in phos¬ phate-buffered saline (pH 7.2 to 7.4). Normal swine serum (1/20) was applied for 30 min¬ utes, followed by three washes in phosphatebuffered saline. The primary antibody to
Downloaded From: http://archotol.jamanetwork.com/ by a Georgetown University Medical Center User on 05/24/2015
SlOO protein (1/800)
or lysozyme (1/250) was applied at room temperature for 2 hours, then overnight at 4°C. After three washes in phosphate-buffered saline for 10 minutes each, antirabbit swine antibody (1/20) and peroxidase-antiperoxidase (1/100) were ap¬ plied. After the sections were washed, 3,3diaminobenzidine tetrahydrochloride solu¬ tion (0.04%) with 0.03% hydrogen peroxide
used for visualization. Of 88 patients, 81 had tumors of the su¬ praglottic region and seven had tumors of the subglottic region. All patients underwent to¬ tal (67) or partial (21) laryngectomy associat¬ ed with unilateral or bilateral radical neck dissection. Histological study of the tumors in all cases showed squamous cell carcinoma at different degrees of differentiation. Tumor staging was performed according to the 1987 Union International Contra Cancer criteria. Re¬ sults in our cases, grouped according to LC density, are given in the Table. The inflammatory reaction within and around the tumor was histologically classi¬ fied using a semiquantitative method as slight (1 + ), moderate (2 + ), and marked (3 + ); as lymphoid when lymphocytes, or less
was
100
oo
Years actuarial corrected survival according to granulocytic inflammatory infiltrate. Light squares indicate slight (1 + ) reaction; diamonds, moderate (2 + ) reaction; and dark squares, marked (3 + ) reaction.
Fig 4.—Global
100-
frequently plasma cells, were dominant; or as granulocytic when granulocytes and/or mac¬ rophages were prevalent. Langerhans cell density was rated as low (1 + ) when fewer than five cells per microscopic field ( 250 magnification) were counted on five random¬ ly selected fields; intermediate (2 + ) when six to 10 cells were counted; and high (3 + ) if than 10 cells were counted. A clinical follow-up was available for each patient. The period of observation ranged from 48 to 120 months. Survival curves, ob¬ more
tained by the actuarial method, were statisti¬ cally analyzed by the test.3
RESULTS cells were present in almost all of the specimens analyzed. These cells were identified based on their positive reaction with anti-SlOO protein antibodies, which outlined the typical spindly dendritic structure of the cytoplasm (Fig 1). In our 88 cases, 10 (11.4%) present¬ ed low, 42 (47.7%) intermediate, and 36 (40.9%) high density of LCs, respec¬ tively. Langerhans cells were inter¬ spersed within the tumor nests, and their cytoplasmic processes extended between tumor cells and lymphocytes (Fig 2). Rarely were LCs found around the tumor tissue. No significant relationship among the density of LCs and the site of
Langerhans
neoplastic growth (supraglottic
or
Years
Fig 5.—Global actuarial corrected survival according to lymphoid infiltrate. Light squares Indicate marked (3 + ) reaction; diamonds, moderate (2 + ) reaction; and dark squares, slight (1 + ) reaction.
subglottic), the histological grade (data not given), or the stage (Table)
was
cant statistical
found in tumors with moder¬ marked lymphoid infiltrate. In fact, 32 (88.9%) of 36 cases with high density of LCs also presented moder¬ ate (18 cases) or marked (14 cases) lymphoid infiltration, and eight (80%) of 10 cases with low density of LCs showed slight lymphoid infiltration
tumor
LCs
clinical
ate
present. A signifi¬
relationship between density of LCs and node invasion was not documented, although eight (80%) of 10 patients presenting low LC densi¬ ty had lymph node involvement, and 26 (72%) of 36 patients presenting high LC density had no neck métastases (Table).
The relationship between number of LCs and degree of inflammatory infil¬ tration was significant. High density of
was
or
(Table).
According to the density of LCs, three groups of patients were identi¬ fied. Survival curves were plotted for these groups and are given in Fig 3. The actuarial survival rate, at 5 years,
Downloaded From: http://archotol.jamanetwork.com/ by a Georgetown University Medical Center User on 05/24/2015
in the groups of patients with interme¬ diate or high density of LCs were 62% and 61%, respectively, whereas that of the group with low density of LCs was 0%. This difference between the plot¬ ted groups was shown to be statistical¬
ly significant (P
tumors in other sites, a laryn¬ tumor is often characterized
Likegeal
inflammatory reaction that fol¬ lows the growth of neoplastic ele¬ ments. This reaction has prognostic value,1"3 but its functional significance by
an
has still to be established.
Recently,
a
significant relationship
has been reported between prognosis and degree of infiltration of mononu¬ clear inflammatory cells within tumor tissues.4"6 It would seem that, if tumors Accepted for publication February 5,1991. From the Otorhinolaryngological Clinic II (Drs Gallo, Asprella Libonati, Gallina, and Fini-Storchi), and Institute of Pathological Anatomy (Drs Giannini, Urso, and Bondi), University of Florence
(Italy). Reprint requests to II Clinica Otorinolaringoiatrica, Universit\l=a`\di Firenze, Viale Morgagni 85, 50100 Firenze, Italia (Dr Gallo).
in
MD; Ezio Gallina, MD; Omero Fini-Storchi, MD; Augusto Giannini, MD;
cal stage. Patients with high or intermediate density of Langerhans cells survived longer than those with low density (mean survival, 61%, 62%, and 0%, respectively). The number of Langerhans cells was relevant in patients with evident infiltration of lymphocytes and plasma cells, according to their ability to present antigens to sensitized T cells. Our results indicate that the presence of high or intermediate density of Langerhans cells and of marked lymphoid inflammation may be considered favorable prognostic factors for patients with squamous cell carcinoma of the larynx. (Arch Otolaryngol Head Neck Surg.
1991;117:1007-1010)
are cells with particular immunohistochemical findings (lysozyme negative, S100 protein positive, OKT6\ OKT1") that are found in a direct relationship with lympho¬
tiocytes
cytes.1"8
Langerhans cells present little phagocytic ability and express several immunological markers such as FcIgG, C3 receptor, Ia/DR antigen, S100 protein, and T6 antigen.1315*20 In addi¬ tion, they show similar morphological
aspects such as lobate and convoluted nucleus, clear cytoplasm with dendritic processes, and distinct
cytoplasmic organelles (Birbeck's granules or LC granules) without tonofilaments and
are
desmosomes.1315
the site of the tumor growth.7 These cells with cytotoxic activity are specific lymphocytes and natural killer cells.8 T-Cell activation requires a particular antigen presentation. In this immuno¬ logical process, groups of cells, defined as antigen-presenting cells or accesso¬ ry cells, are involved.9 Accessory cells can present antigen associated with major histocompatibility complex class II polypeptides and can produce interleukin l.10"12 Langerhans cells (LCs) are a group of cells with accessory function.18"15 These cells, also defined as "T-zone
It has been demonstrated that an increased number of LCs is related to a more favorable prognosis in cancers of the rhinopharynx,21 oral cavity,22 stomach,23 lung,1617 and thyroid,24 and in T-cell lymphomas.25 It is well known, however, that Ia/DR accessory cells sensitize lymphocytes, both helper and suppressor, and may regulate their functional balance.26 Recently, many subpopulations of epidermal histiocytes have been dem¬ onstrated by immunohistochemical studies, using different antibodies to the immunological markers of LCs, and functional differences among them have been suggested.27"21' Therefore, it is possible that a quantitative increase of these cells may not always be linked
immunogenic in the host, the lym¬ phocytes with the most potent antitumor activity should be present within
histiocytes,"1617 are thought to present antigens to lymphocytes. T-Zone his-
Downloaded From: http://archotol.jamanetwork.com/ by a Georgetown University Medical Center User on 05/24/2015
Fig 2.—S100 protein immunostain by peroxidase-antiperoxidase method, showing a cell with dendritic cytoplasm and Irregular nucleus, both of which were positive tor S100 protein. The specimen was obtained from the cancer tissue (peroxidase-antiperoxidase-S100, 1350).
Fig 1.—Langerhans cells in the cancer nest, with evidence of dendritic cytoplasmic processes interspersed among the neoplastic cells (peroxidase-antiperoxidase-S100, 500).
to
Density of Langerhans Cells According and
Lymphoid
to Tumor
Stage, Node Invasion,
Intermediate
High
Total 10
I
18 20
52
IV Node invasion 10
+
46
Lymphoid
infiltration
Slight (1+)
Moderate (2+)
Marked (3+) Total
22 14
0
10
42
36
26 42 20 88
3.—Global actuarial corrected survival according to Langerhans cell density. Light squares indicate low (1+J reaction for S100 protein; diamonds, intermediate (2 + ) reaction; and dark squares, high (3 + ) reaction.
Fig
100
co
We have previously shown the pres¬ of LCs and their correlation with lymphoid infiltrate in cancer of the larynx.31 In this study, we investigated the prognostic significance of the den¬ sity of LCs infiltrating tumor tissues in 88 patients with laryngeal cancer. Im¬ munological reaction in the host, in¬ volving these reactive lymphoreticular cells against tumor, has also been considered. PATIENTS AND METHODS The study group included 88 patients (86 men, two women), aged from 37 to 79 years (mean age, 62 years), affected by carcinoma ence
Tumor stage
24
functional activation of immune
responsiveness.3"
Infiltration
Density of Langerhans Cells Low
a
of the larynx and surgically treated at the Ear-Nose-Throat clinic of the University of Florence (Italy). Specimens from these pa¬ tients were examined at the Institute of Pathological Anatomy of the University of Florence. After surgical removal, the speci¬ mens were fixed in 10% phosphate-buffered formaldehyde solution (pH 7.4), embedded in paraffin, and processed conventionally. Consecutive sections 4 µ thick were stained with hematoxylin-eosin. Moreover, a peroxidase-antiperoxidase method was used employing antibodies against the S100 cyto¬ plasmic protein and lysozyme, as described by Nomori et al.21 (Antilysozyme antibody and the peroxidase-antiperoxidase kit used for immunostaining were purchased from
Dakopatts Ltd, Copenhagen, Denmark.)
Years
The paraffin-embedded (4-µ thick) sections were treated with 0.5% hydrogen peroxide in methanol for 30 minutes to eliminate endoge¬ nous peroxidase, and then washed in phos¬ phate-buffered saline (pH 7.2 to 7.4). Normal swine serum (1/20) was applied for 30 min¬ utes, followed by three washes in phosphatebuffered saline. The primary antibody to
Downloaded From: http://archotol.jamanetwork.com/ by a Georgetown University Medical Center User on 05/24/2015
SlOO protein (1/800)
or lysozyme (1/250) was applied at room temperature for 2 hours, then overnight at 4°C. After three washes in phosphate-buffered saline for 10 minutes each, antirabbit swine antibody (1/20) and peroxidase-antiperoxidase (1/100) were ap¬ plied. After the sections were washed, 3,3diaminobenzidine tetrahydrochloride solu¬ tion (0.04%) with 0.03% hydrogen peroxide
used for visualization. Of 88 patients, 81 had tumors of the su¬ praglottic region and seven had tumors of the subglottic region. All patients underwent to¬ tal (67) or partial (21) laryngectomy associat¬ ed with unilateral or bilateral radical neck dissection. Histological study of the tumors in all cases showed squamous cell carcinoma at different degrees of differentiation. Tumor staging was performed according to the 1987 Union International Contra Cancer criteria. Re¬ sults in our cases, grouped according to LC density, are given in the Table. The inflammatory reaction within and around the tumor was histologically classi¬ fied using a semiquantitative method as slight (1 + ), moderate (2 + ), and marked (3 + ); as lymphoid when lymphocytes, or less
was
100
oo
Years actuarial corrected survival according to granulocytic inflammatory infiltrate. Light squares indicate slight (1 + ) reaction; diamonds, moderate (2 + ) reaction; and dark squares, marked (3 + ) reaction.
Fig 4.—Global
100-
frequently plasma cells, were dominant; or as granulocytic when granulocytes and/or mac¬ rophages were prevalent. Langerhans cell density was rated as low (1 + ) when fewer than five cells per microscopic field ( 250 magnification) were counted on five random¬ ly selected fields; intermediate (2 + ) when six to 10 cells were counted; and high (3 + ) if than 10 cells were counted. A clinical follow-up was available for each patient. The period of observation ranged from 48 to 120 months. Survival curves, ob¬ more
tained by the actuarial method, were statisti¬ cally analyzed by the test.3
RESULTS cells were present in almost all of the specimens analyzed. These cells were identified based on their positive reaction with anti-SlOO protein antibodies, which outlined the typical spindly dendritic structure of the cytoplasm (Fig 1). In our 88 cases, 10 (11.4%) present¬ ed low, 42 (47.7%) intermediate, and 36 (40.9%) high density of LCs, respec¬ tively. Langerhans cells were inter¬ spersed within the tumor nests, and their cytoplasmic processes extended between tumor cells and lymphocytes (Fig 2). Rarely were LCs found around the tumor tissue. No significant relationship among the density of LCs and the site of
Langerhans
neoplastic growth (supraglottic
or
Years
Fig 5.—Global actuarial corrected survival according to lymphoid infiltrate. Light squares Indicate marked (3 + ) reaction; diamonds, moderate (2 + ) reaction; and dark squares, slight (1 + ) reaction.
subglottic), the histological grade (data not given), or the stage (Table)
was
cant statistical
found in tumors with moder¬ marked lymphoid infiltrate. In fact, 32 (88.9%) of 36 cases with high density of LCs also presented moder¬ ate (18 cases) or marked (14 cases) lymphoid infiltration, and eight (80%) of 10 cases with low density of LCs showed slight lymphoid infiltration
tumor
LCs
clinical
ate
present. A signifi¬
relationship between density of LCs and node invasion was not documented, although eight (80%) of 10 patients presenting low LC densi¬ ty had lymph node involvement, and 26 (72%) of 36 patients presenting high LC density had no neck métastases (Table).
The relationship between number of LCs and degree of inflammatory infil¬ tration was significant. High density of
was
or
(Table).
According to the density of LCs, three groups of patients were identi¬ fied. Survival curves were plotted for these groups and are given in Fig 3. The actuarial survival rate, at 5 years,
Downloaded From: http://archotol.jamanetwork.com/ by a Georgetown University Medical Center User on 05/24/2015
in the groups of patients with interme¬ diate or high density of LCs were 62% and 61%, respectively, whereas that of the group with low density of LCs was 0%. This difference between the plot¬ ted groups was shown to be statistical¬
ly significant (P
