Neoadjuvant Immunotherapy of Oral Squamous Cell Carcinoma Modulates Intratumoral CD4/CD8 Ratio and Tumor Microenvironment: A Multicenter Pha
http://www.100md.com
《临床肿瘤学》
the National Institute of Oncology
Departments of Otolaryngology and Head and Neck Surgery and Dentistry and Oral Surgery, Semmelweis University
Department of Otolaryngology and Head and Neck Surgery, Uzsoki Hospital
District Hospital, Gyr, Budapest, Hungary
CEL-SCI Corporation, Vienna, VA
ABSTRACT
PURPOSE: To investigate the clinicopathologic effects of local neoadjuvant Leukocyte Interleukin Injection (LI) regimen in oral squamous cell carcinoma (OSCC) patients. Treatment regimen included LI 800 IU/d as interleukin-2 (IL-2), administered half peritumorally and half perilymphatically five times per week for 3 weeks; low-dose cyclophosphamide; indomethacin; zinc; and multivitamins.
PATIENTS AND METHODS: Thirty-nine patients diagnosed with T2-3N0-2M0 OSCC participated in the pathology portion of this phase II multicenter study (19 LI-treated patients and 20 historical controls). Clinical responses were determined by imaging. Paraffin-embedded tumor samples were obtained at surgery for all patients. Surgery for the LI-treated group was performed between days 14 and 54 after the end of treatment. Histologic evaluation, pathologic staging, necrosis, and American Joint Committee on Cancer grading were performed from hematoxylin and eosin sections. Immunohistochemistry and morphometry determined cellular infiltrate.
RESULTS: Two pathologically complete, two major (> 50%), and four minor responses (> 30% but < 50%) resulted from LI treatment (overall response rate, 42%). Histopathology showed that the intratumoral CD4+:CD8+ ratio was low (< 1) in patients not treated with LI (controls). An increase in tumor-infiltrating CD4+ and a decrease of CD8+ T cells was observed in LI-treated patients, leading to a significantly (P < .05) higher intratumoral CD4+:CD8+ ratio (> 2.5). This was paralleled by dendritic cell transition from tumor surface toward stromal interface (P < .05), with macrophage decrease and neutrophil accumulation, multifocal microscopic necrosis, and significant (P < .05) increase in tumor stroma of LI-treated patients compared with controls.
CONCLUSION: LI-treated OSCC patients were characterized by a markedly altered composition of tumor-infiltrating mononuclear cells, increased CD4+:CD8+ ratio, and increased tumor stroma to epithelial ratio, all of which were distinct from controls.
INTRODUCTION
Tumor-host interaction is a complex feature of tumor progression and is an increasingly important target for anticancer strategies.1 It involves cellular interactions between cancer cells, immune effector cells, and inflammatory cells, as well as cells of the tumor vasculature and the stroma. Because of this complex and multifaceted interaction, the therapeutic modulation of the function of individual members of this complex interactive network will most probably affect the function of the other members of the network, resulting in the modulation of tumor progression. Accordingly, it is prudent to pursue a more holistic approach to the analysis of the antitumor effects after chemotherapy, immunoadjuvant therapy, or other biologic-based therapies.
Immunotherapy is considered a relatively specific approach to cancer treatment in which activation of immunologic effector mechanisms are used to destroy cancer cells. The successful anticancer immunotherapeutic effect generally requires the expression of tumor and major histocompatibility complex (MHC) antigens on the target tumor cells, cancer cell sensitivity to effector mechanisms, and induced and/or augmented activity of the anticancer effectors.1,2 There are several feasible immune strategies to achieve these goals; one of these strategies is the use of cytokines to augment anticancer immune effector mechanisms. Oral squamous cell carcinoma (OSCC) was not considered, until recently, an immunotherapeutic target, but pioneer studies by Whiteside et al3 suggested that immunotherapeutic approaches could well be a new way to manage this otherwise highly aggressive cancer.
Local interleukin-2 (IL-2) treatment was introduced as a neoadjuvant immunobiotherapy applied before the surgical resection of the OSCC. Some therapeutic interventions used recombinant human IL-2 (rhIL-2), whereas others used a natural leukocyte-derived IL-2 preparation in different tumor types including head and neck cancer.4-7
Early data demonstrated highly variable response rates for local rhIL-2 administrations (6% to 65%).8 Phase III trials involving the local administration of rhIL-2 to OSCC patients resulted in a significant increase in disease-free survival as well as increased overall survival.9 Histologic examination of the tumor tissue obtained from rhIL-2–treated head and neck cancer patients revealed an enrichment in T-cell subsets paralleled by focal necrosis and occasional eosinophilic infiltration.3,6,10
Phase II clinical trials with natural IL-2 (in a mixture of naturally occurring cytokines) exhibited response rates of 30% to 50%.4,11-13 Histopathologic analysis indicated that this natural cytokine mixture (containing IL-2) induced predominantly T-lymphocytic infiltration of the tumor tissue and caused tumor fragmentation, which depended on the dose administered.4,12,13 A phase II trial with lower doses of Leukocyte Interleukin Injection (LI; also known as Multikine; CEL-SCI Corporation, Vienna, VA) showed similar response rates and induced CD3+CD25+ T-lymphocytic infiltration of tumor cell nests and entry of the cancer cells into cell cycle.14
These studies demonstrated that local treatment with a mixture of natural cytokines (including IL-2) was a feasible approach to the treatment of OSCC. However, the relationship between the induced alterations in the cellular infiltrate of the tumor, the histologically verified tumor response, and the clinically determined responses was not elucidated by these studies. Accordingly, in this phase II trial of LI as a neoimmunoadjuvant treatment before conventional therapy (surgery followed by radiotherapy) of OSCC, we investigated the relationship between LI administration and the cellular infiltrate in the tumor. These data were correlated to the clinicopathologic tumor responses induced by the locoregional administration of LI.
PATIENTS AND METHODS
Clinical Sites
The clinical investigation was conducted under the sponsorship of CEL-SCI Corporation and with the authorization of the Health Science Council–Ministry of Health, Budapest, Hungary. The clinical sites that participated in this clinical study were the National Institute of Oncology; the Departments of Otolaryngology and Head and Neck Surgery and Dentistry and Oral Surgery, Semmelweis University; the District Hospital, Gyr; and the Department of Otolaryngology and Head and Neck Surgery, Uzsoki Hospital, Budapest, Hungary. These clinical sites conducted the trial under a single clinical protocol approved by the Hungarian Ministry of Health and by the ethics committee of each institution.
Patients
A total of 41 patients were included in this study. Twenty-one patients (mean age, 59.4 years; range, 40 to 87 years) with previously untreated head and neck cancer were included in the treatment arm. The inclusion criteria were as follows: age older than 18 years; able and willing to provide written informed consent; histologically confirmed OSCC (locations: floor of the mouth, n = 7; tongue, n = 8; lip, n = 1; oropharynx, n = 1; bucca, n = 1; and gingiva, n = 1); no known distant (visceral) metastatic disease; and life expectancy of more than 6 months. Patients who were pregnant or who had radiation to the site of LI administration, duodenal or gastric ulcer, or asthma were excluded.
Of the 21 LI-treated patients, 19 were assessable. Two of the treated patients were enrolled and subsequently confirmed as having anaplastic carcinoma of the uvula and adenocarcinoma of the floor of the mouth, respectively. These two patients were administered the full course of therapy, including the administration of the investigational drug, but they were not part of the histopathology and immunohistochemistry analysis because they did not meet the criteria for having OSCC. All patients provided informed consent before enrollment onto this trial and before the initiation of any trial tests and procedures.
Historical controls were selected for the pathologic examination portion of this study exclusively from the pathology specimen repository of the National Institute of Oncology (Budapest, Hungary) and were matched to the specimens from the LI-treated group based on the size and location of the tumor, tumor stage, and sex and age of the patients. The control group consisted of 20 patients (mean age, 57.5 years; range, 40 to 77 years), all of whom had biopsy-confirmed OSCC of the oral cavity (locations: floor of mouth, n = 3; tongue, n = 12; and lip, n = 5).
Treatment Protocol
The local neoadjuvant treatment regimen in this trial was similar to that previously used by our group.14 The therapy (LI, cyclophosphamide, indomethacin, zinc sulfate, and multivitamins) and the schedules used were identical to those reported earlier.14 However, in this study, LI administration was performed in the following manner: one half of the daily dose (400 IU as IL-2) was injected peritumorally, and the other half (400 IU) was injected perilymphatically (sequentially and at the same visit) over a 3-week period, five times per week, reaching a cumulative dose of 12,000 IU as IL-2, which is a higher dose than the maximal doses tested in an earlier trial with LI.14 All LI injections were administered intradermally at the circumferential margin of the visible or palpable tumor mass, and the perilymphatic injection was administered at the posterior submandibular area at the jugular lymphatic chain, ipsilateral to the injected tumor.
The administration of LI was preceded by a single intravenous (bolus) infusion of cyclophosphamide 300 mg/m2 3 days before the first LI administration. Indomethacin (25 mg orally tid) was self-administered (with food), for a total daily dose of 75 mg, beginning 3 days after cyclophosphamide administration and until 24 hours before surgery. Zinc sulfate (50 mg, as elemental zinc) and a multivitamin supplement, both once daily, were self-administered beginning 3 days after cyclophosphamide administration and until 24 hours before surgery. Patients were counseled and encouraged to continue self-administration of the multivitamin and zinc regimen after surgical intervention.
Patient Examination
Before the inclusion in the trial, patients underwent a general assessment, and medical history was obtained prior to conducting a complete physical examination, hematologic and blood chemistry work-up, chest cardioradiography, and ECG. To visualize and measure oral cavity tumor, both physical measurement and magnetic resonance imaging (MRI) were performed. Oral cancer surface area (physical examination measurement) was measured in the longest dimension and at right angle to the longest dimension at the largest diameter of the palpable tumor. For MRI, coronal, axial, and sagittal scans of T1-and T2-weighted fast spin echo (FSE), inversion recovery FSE, and fat-saturated T1-weighted sequence with gadolinium were applied. Oral cancers were measured at the regions of highest diameter. MRI analysis of the OSCC was performed before and after LI treatment in each case and evaluated with a standard method. In the responder group, LI treatment decreased tumor size as well as T2 and inversion recovery FSE signal intensity and the level of contrast enhancement, and the margin of the lesions became less conspicuous. In the nonresponder group, MRI revealed no significant alteration in the tumor after LI treatment. In case of progression, tumor size was also shown to increase by pathology and morphometry. Patients were interviewed at each subsequent visit and were asked about quality of life (eg, level of pain, degree of tongue mobility, and so on) by the treating physician, who also assessed toxicity, at each visit.
Pathology
Pathologic evaluation was performed in a central pathology laboratory at the Department of Tumor Progression, National Institute of Oncology (Budapest, Hungary). A single pathology protocol governed this study and described the preparation and fixation of the surgically excised specimens and the gross, macroscopic and microscopic examination and histology and immunohistochemistry procedures, as described previously.14
Diagnosis of the oral lesions was based on an excision biopsy of the suspected lesion, and cancers classified as T2-3N0-2M0 were selected for immunotherapy with the LI treatment regimen, as described earlier.14,15 At the end of the LI treatment period and before surgery, clinical responses were evaluated as described earlier, and patients were scheduled for tumor resection. The excised tissue was placed in prelabeled containers with buffered formalin and fixed overnight before embedding the tissue in paraffin and preparing thin section slides for hematoxylin and eosin (HE) staining and immunohistochemistry.
Histology and American Joint Committee on Cancer grading were performed from HE-stained sections. The histopathologic analysis was performed on the following three different tumor regions: surface (R1), center (R2), and tumor-stroma interface (R3). Occurrence of necrotic tumor cells was also evaluated using HE slides. The percentage of the epithelial component versus the stroma in the OSCC tumors was determined by two methods; connective tissue was stained according to Mallory (trichrome staining), and the slides were measured for the area of cancer nests (tumor epithelia) by using ImagePro analysis software (Media Cybernetics, Silver Spring, MD). Separately, slides containing resected tissue were labeled for cytokeratine immunohistochemically using pan-cytokeratine antibody (A1A3+CK19; DAKO, Glostrup, Denmark), which labels cancer cells.
Characterization of the Mononuclear Cell Infiltrate
Mononuclear cells present in the close vicinity of tumor cell nests were determined by using immunohistochemistry performed on paraffin-embedded sections of the tumor samples after deparaffination and microwave antigen retrieval. Antibodies against the following markers were applied: myeloperoxidase, HLA class II (HLA-DP, DQ, DR), CD8, CD20, CD34, CD45R0, and CD68 (all from DAKO); CD4, CD25, and CD56 (Novocastra Laboratories Ltd, Newcastle on Tyne, United Kingdom); CD1a (Immunotech, Marseille, France); and CD134 (PharMingen, San Diego, CA). In all cases, negative control slides were prepared using isotype-matched nonimmune immunoglobulins.
Immunohistochemical labeling was performed with the DAKO LSAB-2 kit using a biotinylated antimouse/antirabbit immunoglobulin G linker and streptavidin-horseradish peroxidase to reveal specifically bound antibodies. The chromogen used was amino-ethyl-carbazol (red) label. Sections were counterstained for the nuclei with hematoxylin.
Evaluation of Tumor Samples
Tumor samples from the LI-treated group and control (non–LI-treated) group were evaluated for histologic changes and for the following infiltrating cells: T and B lymphocytes, natural killer cells, dendritic cells, macrophages and neutrophils, hematopoietic stem cells, and T-cell activation and memory cell markers. A magnification of x40 was used to select the area, and a magnification of x400 was used for the identification and quantitation of infiltrating cells. The density of mononuclear cells was determined based on the hot spot technique, which means that, in each studied tumor area, density was measured at the region of the highest tumor-infiltrating mononuclear cell density. This method minimizes the problem of the extreme heterogeneity of the cellular infiltrates in tissues.14
The histologic evaluation of each patient's tumor section was performed by three independent pathologists (J.T., C.F.-H., and B.D.), and, in case of disagreement, consensual determinations were reached. Morphometric measurements were performed by the same three pathologists and without the knowledge of the clinical background and treatment outcome of each of the patients.
Statistical Analysis
Pathology data were analyzed by analysis of variance (ANOVA) single-factor analysis and the 2 test, and P < .05 was considered to be statistically significant.
RESULTS
Clinicopathologic Response
No systemic or local toxicity or severe adverse events related to LI were reported in this trial. Furthermore, no complications related to LI at surgery, after surgery, or during wound healing were reported. Primary tumor samples of 19 LI-treated and 20 control (non–LI-treated) OSCC patients were evaluated. In the LI-treated group, surgical resection of the primary tumor was performed between days 14 and 54 after the end of the LI treatment (median time to surgery, 22 days). The histologic diagnosis, keratinization (Brodes scores), and tumor-node-metastasis stage of all the LI-treated and control patients are listed in Table 1. The two groups did not differ histologically or with respect to keratinization or tumor-node-metastasis stages.
In two of 19 LI-treated patients (patients 5 and 14; Table 1), it was not possible to detect any cancer tissue in the surgically resected tumor mass, and thus, these patients were considered to be complete responders (exhibiting 100% tumor reduction by histopathology as a result of LI treatment regimen). In two other LI-treated patients (patients 7 and 18; Table 1), the imaging technique verified more than 50% tumor volume reduction, which was considered a partial (major) response (two of 19 patients); and in four LI-treated patients (patients 4, 6, 9, and 12; Table 1), the volume reduction was proven to be more than 30%, which was considered a minor response (four of 19 patients). Progressive disease ( 40% tumor volume increase) occurred in only one patient (patient 17), whereas stable disease was detected in the rest of the patients (10 of 19 patients). Therefore, the objective response rate in the LI-treated group of OSCC patients was 21%, with an overall response of 42% (eight of 19 patients). The full pathologic analysis of the effect of LI treatment on OSCC was performed on the LI-treated nonresponder subgroup (stable disease and progressive disease, n = 11) as well as separately on the LI-treated responder subgroup (n = 6), excluding the two complete responders in whom no tumor tissue could be detected.
Tumor-Infiltrating Lymphocytes
We have observed that, similarly to our previously published data,14 CD20+ B cells were only present in the stroma of OSCC. In the tumor samples obtained after LI pretreatment, a nonsignificant decrease in B cells in OSCC, irrespective of the tumor area investigated, was observed. Clinical responses did not influence this pattern of B cells in the OSCC (data not shown).
In the control group, CD8+ cells infiltrating the tumor and stroma outnumbered the CD4+ cells (Fig 1A through 1D), and as a result, the CD4+:CD8+ ratio was well below 1 (at approximately 0.5; Fig 1E). In the LI-treated group, an increase in the density of CD4+ T cells and decrease in the density of CD8+ T cells was observed in the tumor stroma (Fig 1C). A similar trend was seen in the case of intraepithelial CD4+ and CD8+ cells (Fig 1D). It is of note that these changes were significant only in the case of responders to LI treatment (P < .05). As a consequence, the CD4+:CD8+ ratio was two- to eight-fold higher in the cellular infiltrate of the LI-treated tumors compared with the controls (Fig 1E). Although the highest CD4+ and the lowest CD8+ cellular infiltrate densities were detected in the responder to LI treatment subgroup, the difference from nonresponders to LI treatment was only statistically significant (P < .05) in the tumor stroma segment of the tumor, as can be seen by the CD4+:CD8+ ratio.
Evaluation of the number of activated T cells based on the expression of CD25 and OX40 (CD134) molecules revealed a relatively low mean density of cells expressing these markers in the infiltrates of OSCC (especially in the case of OX40) and no significant differences in their frequency between the control and LI-treated groups (Fig 2A and 2B). However, using available frozen samples and immunofluorescence, we observed that CD25+CD4+ T cells were more frequent in the LI-treated group (10 of 10 patients; range, 14% to 61% of CD4+ cells) but were only detected occasionally in the control group (one of nine patients). We also determined the density of CD45R0+ memory cells and found that their density was highly similar in the control and LI-treated patients both in the tumor stroma and intraepithelially (Fig 2C). The density of CD45R0+ lymphoid cells was, at an average, approximately 60% of T cells (CD4+ + CD8+), suggesting that a high proportion of tumor-infiltrating T lymphocytes were memory cells. Similar to our previously reported findings,14 CD34+ hematopoietic stem cells and CD56+ natural killer cells were not found in the stroma or tumor epithelial nests of the OSCC, irrespective of the tumor area or the patient population investigated (control or LI-treated patients).
We demonstrated that CD1a+ dendritic cells were almost exclusively located in the tumor epithelial nests (both in the current study and in our previous study14). In the LI responder subgroup, we observed a differential effect of LI treatment on the distribution of dendritic cells in the three areas of OSCC investigated; no change was observed in the center of the tumor (R2), a decrease (P < .05) in CD1a+ dendritic cells was detected at the surface (R1), and an increase (P < .05) in CD1a+ dendritic cells was present at the tumor-stroma interface (R3), compared with control (non–LI-treated) patients (Fig 3A).
Inflammatory Cells
Neutrophils were present both in the tumor stroma and in the tumor epithelial nests, which was similar to macrophage distribution in the tumor, whereas eosinophils were found in the tumor stroma exclusively. The presence of neutrophil and macrophage infiltrate in the resected tumor specimens was determined by staining for myeloperoxidase and CD68 markers, respectively.
Analysis of the macrophage density indicated a downmodulation of the stromal presence of CD68+ macrophages in the LI responder group exclusively, and this trend was even more pronounced intraepithelially in the LI-treated group (P < .002 for the LI responder subgroup, and P < .01 for LI nonresponders; Fig 3B). However, LI-treated patients exhibited increased neutrophil migration into the cancer cell nests (Fig 4A through 4C), and neutrophil density was also pronounced in the tumor stroma in the LI responder subgroup (Fig 4C). In the LI-treated group, increased intratumoral infiltration by neutrophils was observed exclusively in patients with multifocal microscopic necrosis (see Tumor Necrosis section).
The density of eosinophils was found to be highly heterogeneous in both OSCC groups (LI-treated and control patients). Morphometric analysis revealed no significant alterations in eosinophilic cell population in the tumor stroma in the LI-treated patients (data not shown). However, a more detailed analysis of the patients revealed that tumors with high eosinophil density (> 50 cells per high-power field) were twice as numerous in the LI-treated group (nine of 19 patients; 47%) compared with the controls (five of 20 patients; 25%).
Tumor Necrosis
Macroscopic cancer necrosis foci in the surgically obtained samples occurred in the control group exclusively. However, multifocal microscopic necrosis was markedly more frequent in the LI-treated group (10 of 17 patients; Tables 1 and 2 and Fig 5B) compared with the control group (four of 20 patients, P < .05; Tables 1 and 2 and Fig 5A), and there was no significant difference in this respect between the responder and nonresponder (to LI treatment) subgroups.
Tumor Stroma to Cancer Cell Nest Ratio
The percentage of the area of cancer cell nests relative to tumor stroma was determined in OSCC patients and revealed a significant effect of LI treatment. The resected tissue samples were stained with the Mallory trichrome technique, where cancer cell nests were labeled distinctly pink, and the proportion of the stroma in the tumor tissue was determined by morphometry (Figs 6A and B). Data indicate that, in the LI-treated group, the proportion of the collagenous stroma in tumor tissue was significantly increased (Fig 6C) compared with the proportion in tumor tissue of control patients (P < .05), indicating a reduction of cancer cell nest area.
Analysis of the patterns of fibrosis in the OSCC samples showed that collagen fibers accumulated around the cancer cell nests and appeared as periepithelial staining or were found in between the cancer cell nests in the tumor stroma (Figs 6A and B). Periepithelial collagenosis was similar in frequency in the tumors of both the control and LI-treated patients, whereas interstitial fibrosis was highly significantly (P < .001) more frequent in the LI-treated group (12 of 17 patients; 70%) compared with the controls (two of 20 patients; 10%), and the nonresponder and responder (to LI treatment) subgroups did not differ significantly in this respect (Table 3).
HLA Class II Expression
HLA class II expression of OSCC was also determined by immunohistochemistry. Tumor-infiltrating cells exhibited a strong HLA II surface staining in both the LI-treated and control patients (data not shown), but the cancer cell expression was heterogeneous. In the control group, approximately half of the patients (11 of 20 patients) had HLA II–negative tumor samples, whereas the other half (nine of 20 patients) contained tumors with at least focal or strong membrane HLA II expression (Table 4). A similar pattern was observed in the LI-treated cohort (eight of 17 patients’ tumors were HLA II positive). However, in the LI-treated group, HLA II–positive tumors belonged exclusively to the nonresponder group, whereas the cancer cells of all LI responder patients were negative for HLA II antigen expression (P < .05; Table 4).
DISCUSSION
Recently, we reported that LI administered to advanced primary OSCC patients as a first-line treatment before conventional therapy (surgery followed by radiation therapy) induced alterations in infiltrating immune cells rather than in inflammatory cells.14 We observed changes in the T-cell population that migrated into the cancer cell nests, and the infiltrating T cells were found to be activated at the lowest LI dose administered, as was demonstrated by increased CD25 expression.14 However, because the treatment regimen (low-dose LI, 400 IU as IL-2, 3 days a week for 2 weeks) did not result in massive necrosis of the tumor tissue, there was no marked change in the ratio of connective tissue to the tumor epithelial component.14
In this study, a higher dose (800 IU/d as IL-2, 5 days a week, extended for 3 weeks) of LI was administered to OSCC patients as first-line treatment before the initiation of conventional therapy (surgery followed by radiotherapy). In the LI-treated patients, a significant (P < .05) increase in tumor-infiltrating CD4+ T cells, with a concomitant decrease in CD8+ T cells (in situ), was observed, resulting in a marked difference in the CD4+:CD8+ ratio in the OSCC. Although the controls were characterized by a low CD4+:CD8+ ratio (< 1, with CD8+ T-cell predominance), a significantly higher CD4+:CD8+ ratio was observed in both the LI-treated nonresponder and responder (to LI treatment) subgroups (two- to eight-fold increase, with a CD4+ T-cell predominance). Of note is the fact that the stromal CD4+:CD8+ ratio was significantly (P < .05) higher in the LI responders compared with the nonresponders, suggesting a possible clinical importance to this phenomenon. A proportion of these CD4+ T cells carried the activation marker CD25, whereas the expression of OX40, a marker of freshly activated CD4+ T cells that has been detected on a portion of tumor-infiltrating lymphocytes in several cancers including head and neck carcinomas,16,17 was rare in the T-cell infiltrate in this study. This would suggest a later phase of the T-cell activation process and may be a result of the relatively long period (range, 14 to 54 days; median, 22 days) between the end of LI therapy and the surgical removal of the tumors in the LI-treated group. However, double-labeling studies indicated that a large proportion of the CD4+ and CD8+ T cells were CD45R0+ (data not shown), suggesting that they were memory T cells. Parallel to these changes, intraepithelial dendritic cells shifted from the surface of the OSCC to the tumor-stroma interface. Similarly, in a previous report on the local effect of IL-2 infusion in head and neck cancer, a selective effect on CD4+ T lymphocytes and dendritic cells was observed, with marked increase in CD25+ cells only at lower doses of IL-2 administration.18
LI-treated OSCC patients were characterized by a markedly altered pattern of inflammatory cells, suggesting that an acute inflammatory reaction was mediated predominantly by neutrophils and, to a smaller extent, eosinophils, rather than by macrophages. Other studies (in melanoma patients) showed that the accumulation of neutrophil leukocytes at vaccination and tumor sites was prevalent after vaccination with granulocyte-macrophage colony-stimulating factor (GM-CSF)–producing autologous tumor cells.19,20 Because LI preparations contain several cytokines, including GM-CSF,14,15 this could be an explanation for the observed increased neutrophil infiltration in the LI-treated patients compared with controls.
In parallel to the complex alterations in the tumor-infiltrating cellular components of the host antitumor response, multifocal necrosis of cancer cell nests and an increase in the proportion of connective tissue were detected in the LI-treated OSCC patients compared with the nontreated controls. These changes may reflect the aftermath of an antitumor immune response raised against the OSCC, which was induced by LI neoadjuvant administration.
As a result of the local neoadjuvant administration of LI to the OSCC patients, the overall response rate in the LI-treated group was 42% (21% of these patients were considered to have an objective response, with two pathologic complete responses). Our results indicate that, although some degree of antitumor reactivity could be elicited in most patients, this does not necessarily translate into an objective clinical response in all patients.
One of the most compelling findings of this study is illustrated by the fact that a successful immune response to OSCC may require the reversal of the inherently low intratumoral CD4+:CD8+ ratio (as seen in non–LI-treated controls). The predominance of CD8+ cells over CD4+ cells in solid tumors is not specific for OSCC. Similar low CD4+:CD8+ ratios have been reported in basal cell carcinoma21 and cervical22 and breast cancers.23 High percentages of CD4+ T cells or high CD4+:CD8+ ratios correlated with better prognosis or response to immunotherapy in melanoma,24 B-cell non-Hodgkin's lymphoma,25 renal cell carcinoma,26 and cervical carcinoma.27 This suggests that an acquired immunosuppressed state of cancer patients, induced by the tumor, is a common phenomenon, reflecting the general poor immune response in cancer patients. Furthermore, CD4+ T lymphocytes play a central role in initiating and maintaining antitumor immunity by providing help for CD8+ cytotoxic T lymphocytes, stimulating antigen-presenting cells, and sustaining immunologic memory or, in some cases, by performing effector functions via cytokine secretion or direct cytolysis.28-30 In the case of oral carcinomas treated with LI, it is not yet clear which are the specific mediators (in LI) that elicit the antitumor immune response. However, the increased density of intraepithelial leukocytes in parallel to microscopic necrosis after LI treatment may suggest that the activation of nonspecific antitumor mechanisms (in OSCC patients) is equally important to produce clinicopathologically detectable destruction of the established tumor.
Histopathologic comparison of tumors in the LI-treated nonresponder subgroup to the tumors of the responder subgroup (in which clinical and histopathologic regressions were observed in eight of 19 patients) revealed that most of the studied immunologic parameters, from immune cell composition to inflammatory cell patterns, were similar. These data suggest that the LI-treated group responded homogenously to the treatment but that the anticancer effects of LI treatment were heterogeneous. A plausible explanation for this discrepancy could be the individual differences in immunosensitivity of OSCC tumors (differences in tumor antigens and/or HLA composition) and/or the level of impairment of functional immune response in the different patients.
Data from other studies indicate that the lack of expression of MHC class I antigens31 or costimulatory molecules32 on the target (tumor) cells, FasL expression,33 and signaling defects34 can all contribute to impaired immune response to the tumor. In this study, analysis of HLA class II expression by OSCC cancer cells indicated that the level of expression was highly similar between the LI-treated and control groups ( 45% of patients were positive for HLA class II antigen expression). This suggested that LI treatment did not induce or enhance the expression of HLA II molecules on the tumor compared with controls. However, within the LI-treated cohort, all HLA II–positive patients belonged exclusively to the nonresponder subgroup, whereas all the LI responder patients were negative. This finding, although derived from a small number of patients, may indicate that HLA class II expression on OSCC cells may be associated with an unfavorable clinical outcome of disease or with a diminished ability to respond to immunotherapy. Similar findings were reported in malignant melanoma where the expression of MHC class II constitutes a poor prognostic factor,35 which may be a result of the induction of T-cell anergy by MHC class II–expressing tumor cells in the absence of costimulatory signals.36 As was described in melanomas, head and neck cancers lack the expression of the costimulatory molecules B7.1 and B7.2,32 and therefore, the expression of MHC class II molecules on head and neck (squamous cell carcinoma) tumor cells may induce immunologic tolerance. Therefore, we suggest that exclusive targeting of the immune system without a concurrent attempt to modulate the sensitivity of the cancer cell target may not yield an improvement of the overall response rate to an immunologically based treatment.
We hypothesize that LI mode of action involves the combined activity of the different cytokines present in LI14 inducing a cascade of events as follows. First, tumor necrosis factors present in LI (such as tumor necrosis factor-alpha) attack the tumor to release tumor antigens. Second, antigen-presenting cells (eg, dendritic cells) transport the newly released tumor antigens to lymph nodes. Third, lymphoproliferative cytokines (present in LI administered peritumorally and perilymphatically; eg, IL-1 and IL-2) induce a marked polyclonal expansion of tumor-specific T cells, primarily in lymph nodes. Fourth, LI recruits CD4+ T cells from local lymph nodes via chemotactic factors and reverts the balance of intratumoral CD4+ and CD8+ cells in favor of CD4+ T cells, which further upregulates the antitumor immune response, resulting in tumor cell necrosis. Fifth, LI recruits neutrophils from the circulation (via GM-CSF, also present in LI), which propagates the destruction of the tumor cell nests. And sixth, either LI-derived cytokines or the de novo cytokine production by the tumor-infiltrating cells induce massive local fibrosis.
Our data supports the notion that these immune-mediated processes continue long after the cessation of LI administration, as evidenced by the immunohistopathologic changes observed (in the tumor) 14 to 54 days after the end of LI treatment. The efficacy of this neoadjuvant treatment protocol for patients with advanced primary OSCC resulted in a marked reduction in tumor mass (in 42% of the patients) compared with baseline tumor measurements. These findings, taken together with our previous report,14 indicate that the LI neoadjuvant immunotherapy regimen for OSCC is a clinically viable approach to the management of OSCC. The results of larger efficacy trials (planned for the near future) may promote the introduction of LI into the current treatment protocols of OSCC.
Authors' Disclosures of Potential Conflicts of Interest
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Consultant/Advisory Role: Eyal Talor, TEVA Pharm. Stock Ownership: Eyal Talor, CEL-SCI Corp, TEVA Pharm. Research Funding: József Tímár, CEL-SCI Corp. For a detailed description of these categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and Disclosures of Potential Conflicts of Interest found in Information for Contributors in the front of each issue.
NOTES
Supported in part by the Ministry of Education (NKFP 1/48/2001, J.T. and M.K.).
Previously published in abstract form (with modifications) in the ASCO Proceedings (Proc Am Soc Clin Oncol 22:189s, 2004 [abstr 2605]).
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Verastegui E, Barrera JL, Zinser J, et al: A natural cytokine mixture (IRX-2) and interference with immune suppression induce immune mobilization and regression of head and neck cancer. Int J Immunopharmacol 19:619-627, 1997
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Departments of Otolaryngology and Head and Neck Surgery and Dentistry and Oral Surgery, Semmelweis University
Department of Otolaryngology and Head and Neck Surgery, Uzsoki Hospital
District Hospital, Gyr, Budapest, Hungary
CEL-SCI Corporation, Vienna, VA
ABSTRACT
PURPOSE: To investigate the clinicopathologic effects of local neoadjuvant Leukocyte Interleukin Injection (LI) regimen in oral squamous cell carcinoma (OSCC) patients. Treatment regimen included LI 800 IU/d as interleukin-2 (IL-2), administered half peritumorally and half perilymphatically five times per week for 3 weeks; low-dose cyclophosphamide; indomethacin; zinc; and multivitamins.
PATIENTS AND METHODS: Thirty-nine patients diagnosed with T2-3N0-2M0 OSCC participated in the pathology portion of this phase II multicenter study (19 LI-treated patients and 20 historical controls). Clinical responses were determined by imaging. Paraffin-embedded tumor samples were obtained at surgery for all patients. Surgery for the LI-treated group was performed between days 14 and 54 after the end of treatment. Histologic evaluation, pathologic staging, necrosis, and American Joint Committee on Cancer grading were performed from hematoxylin and eosin sections. Immunohistochemistry and morphometry determined cellular infiltrate.
RESULTS: Two pathologically complete, two major (> 50%), and four minor responses (> 30% but < 50%) resulted from LI treatment (overall response rate, 42%). Histopathology showed that the intratumoral CD4+:CD8+ ratio was low (< 1) in patients not treated with LI (controls). An increase in tumor-infiltrating CD4+ and a decrease of CD8+ T cells was observed in LI-treated patients, leading to a significantly (P < .05) higher intratumoral CD4+:CD8+ ratio (> 2.5). This was paralleled by dendritic cell transition from tumor surface toward stromal interface (P < .05), with macrophage decrease and neutrophil accumulation, multifocal microscopic necrosis, and significant (P < .05) increase in tumor stroma of LI-treated patients compared with controls.
CONCLUSION: LI-treated OSCC patients were characterized by a markedly altered composition of tumor-infiltrating mononuclear cells, increased CD4+:CD8+ ratio, and increased tumor stroma to epithelial ratio, all of which were distinct from controls.
INTRODUCTION
Tumor-host interaction is a complex feature of tumor progression and is an increasingly important target for anticancer strategies.1 It involves cellular interactions between cancer cells, immune effector cells, and inflammatory cells, as well as cells of the tumor vasculature and the stroma. Because of this complex and multifaceted interaction, the therapeutic modulation of the function of individual members of this complex interactive network will most probably affect the function of the other members of the network, resulting in the modulation of tumor progression. Accordingly, it is prudent to pursue a more holistic approach to the analysis of the antitumor effects after chemotherapy, immunoadjuvant therapy, or other biologic-based therapies.
Immunotherapy is considered a relatively specific approach to cancer treatment in which activation of immunologic effector mechanisms are used to destroy cancer cells. The successful anticancer immunotherapeutic effect generally requires the expression of tumor and major histocompatibility complex (MHC) antigens on the target tumor cells, cancer cell sensitivity to effector mechanisms, and induced and/or augmented activity of the anticancer effectors.1,2 There are several feasible immune strategies to achieve these goals; one of these strategies is the use of cytokines to augment anticancer immune effector mechanisms. Oral squamous cell carcinoma (OSCC) was not considered, until recently, an immunotherapeutic target, but pioneer studies by Whiteside et al3 suggested that immunotherapeutic approaches could well be a new way to manage this otherwise highly aggressive cancer.
Local interleukin-2 (IL-2) treatment was introduced as a neoadjuvant immunobiotherapy applied before the surgical resection of the OSCC. Some therapeutic interventions used recombinant human IL-2 (rhIL-2), whereas others used a natural leukocyte-derived IL-2 preparation in different tumor types including head and neck cancer.4-7
Early data demonstrated highly variable response rates for local rhIL-2 administrations (6% to 65%).8 Phase III trials involving the local administration of rhIL-2 to OSCC patients resulted in a significant increase in disease-free survival as well as increased overall survival.9 Histologic examination of the tumor tissue obtained from rhIL-2–treated head and neck cancer patients revealed an enrichment in T-cell subsets paralleled by focal necrosis and occasional eosinophilic infiltration.3,6,10
Phase II clinical trials with natural IL-2 (in a mixture of naturally occurring cytokines) exhibited response rates of 30% to 50%.4,11-13 Histopathologic analysis indicated that this natural cytokine mixture (containing IL-2) induced predominantly T-lymphocytic infiltration of the tumor tissue and caused tumor fragmentation, which depended on the dose administered.4,12,13 A phase II trial with lower doses of Leukocyte Interleukin Injection (LI; also known as Multikine; CEL-SCI Corporation, Vienna, VA) showed similar response rates and induced CD3+CD25+ T-lymphocytic infiltration of tumor cell nests and entry of the cancer cells into cell cycle.14
These studies demonstrated that local treatment with a mixture of natural cytokines (including IL-2) was a feasible approach to the treatment of OSCC. However, the relationship between the induced alterations in the cellular infiltrate of the tumor, the histologically verified tumor response, and the clinically determined responses was not elucidated by these studies. Accordingly, in this phase II trial of LI as a neoimmunoadjuvant treatment before conventional therapy (surgery followed by radiotherapy) of OSCC, we investigated the relationship between LI administration and the cellular infiltrate in the tumor. These data were correlated to the clinicopathologic tumor responses induced by the locoregional administration of LI.
PATIENTS AND METHODS
Clinical Sites
The clinical investigation was conducted under the sponsorship of CEL-SCI Corporation and with the authorization of the Health Science Council–Ministry of Health, Budapest, Hungary. The clinical sites that participated in this clinical study were the National Institute of Oncology; the Departments of Otolaryngology and Head and Neck Surgery and Dentistry and Oral Surgery, Semmelweis University; the District Hospital, Gyr; and the Department of Otolaryngology and Head and Neck Surgery, Uzsoki Hospital, Budapest, Hungary. These clinical sites conducted the trial under a single clinical protocol approved by the Hungarian Ministry of Health and by the ethics committee of each institution.
Patients
A total of 41 patients were included in this study. Twenty-one patients (mean age, 59.4 years; range, 40 to 87 years) with previously untreated head and neck cancer were included in the treatment arm. The inclusion criteria were as follows: age older than 18 years; able and willing to provide written informed consent; histologically confirmed OSCC (locations: floor of the mouth, n = 7; tongue, n = 8; lip, n = 1; oropharynx, n = 1; bucca, n = 1; and gingiva, n = 1); no known distant (visceral) metastatic disease; and life expectancy of more than 6 months. Patients who were pregnant or who had radiation to the site of LI administration, duodenal or gastric ulcer, or asthma were excluded.
Of the 21 LI-treated patients, 19 were assessable. Two of the treated patients were enrolled and subsequently confirmed as having anaplastic carcinoma of the uvula and adenocarcinoma of the floor of the mouth, respectively. These two patients were administered the full course of therapy, including the administration of the investigational drug, but they were not part of the histopathology and immunohistochemistry analysis because they did not meet the criteria for having OSCC. All patients provided informed consent before enrollment onto this trial and before the initiation of any trial tests and procedures.
Historical controls were selected for the pathologic examination portion of this study exclusively from the pathology specimen repository of the National Institute of Oncology (Budapest, Hungary) and were matched to the specimens from the LI-treated group based on the size and location of the tumor, tumor stage, and sex and age of the patients. The control group consisted of 20 patients (mean age, 57.5 years; range, 40 to 77 years), all of whom had biopsy-confirmed OSCC of the oral cavity (locations: floor of mouth, n = 3; tongue, n = 12; and lip, n = 5).
Treatment Protocol
The local neoadjuvant treatment regimen in this trial was similar to that previously used by our group.14 The therapy (LI, cyclophosphamide, indomethacin, zinc sulfate, and multivitamins) and the schedules used were identical to those reported earlier.14 However, in this study, LI administration was performed in the following manner: one half of the daily dose (400 IU as IL-2) was injected peritumorally, and the other half (400 IU) was injected perilymphatically (sequentially and at the same visit) over a 3-week period, five times per week, reaching a cumulative dose of 12,000 IU as IL-2, which is a higher dose than the maximal doses tested in an earlier trial with LI.14 All LI injections were administered intradermally at the circumferential margin of the visible or palpable tumor mass, and the perilymphatic injection was administered at the posterior submandibular area at the jugular lymphatic chain, ipsilateral to the injected tumor.
The administration of LI was preceded by a single intravenous (bolus) infusion of cyclophosphamide 300 mg/m2 3 days before the first LI administration. Indomethacin (25 mg orally tid) was self-administered (with food), for a total daily dose of 75 mg, beginning 3 days after cyclophosphamide administration and until 24 hours before surgery. Zinc sulfate (50 mg, as elemental zinc) and a multivitamin supplement, both once daily, were self-administered beginning 3 days after cyclophosphamide administration and until 24 hours before surgery. Patients were counseled and encouraged to continue self-administration of the multivitamin and zinc regimen after surgical intervention.
Patient Examination
Before the inclusion in the trial, patients underwent a general assessment, and medical history was obtained prior to conducting a complete physical examination, hematologic and blood chemistry work-up, chest cardioradiography, and ECG. To visualize and measure oral cavity tumor, both physical measurement and magnetic resonance imaging (MRI) were performed. Oral cancer surface area (physical examination measurement) was measured in the longest dimension and at right angle to the longest dimension at the largest diameter of the palpable tumor. For MRI, coronal, axial, and sagittal scans of T1-and T2-weighted fast spin echo (FSE), inversion recovery FSE, and fat-saturated T1-weighted sequence with gadolinium were applied. Oral cancers were measured at the regions of highest diameter. MRI analysis of the OSCC was performed before and after LI treatment in each case and evaluated with a standard method. In the responder group, LI treatment decreased tumor size as well as T2 and inversion recovery FSE signal intensity and the level of contrast enhancement, and the margin of the lesions became less conspicuous. In the nonresponder group, MRI revealed no significant alteration in the tumor after LI treatment. In case of progression, tumor size was also shown to increase by pathology and morphometry. Patients were interviewed at each subsequent visit and were asked about quality of life (eg, level of pain, degree of tongue mobility, and so on) by the treating physician, who also assessed toxicity, at each visit.
Pathology
Pathologic evaluation was performed in a central pathology laboratory at the Department of Tumor Progression, National Institute of Oncology (Budapest, Hungary). A single pathology protocol governed this study and described the preparation and fixation of the surgically excised specimens and the gross, macroscopic and microscopic examination and histology and immunohistochemistry procedures, as described previously.14
Diagnosis of the oral lesions was based on an excision biopsy of the suspected lesion, and cancers classified as T2-3N0-2M0 were selected for immunotherapy with the LI treatment regimen, as described earlier.14,15 At the end of the LI treatment period and before surgery, clinical responses were evaluated as described earlier, and patients were scheduled for tumor resection. The excised tissue was placed in prelabeled containers with buffered formalin and fixed overnight before embedding the tissue in paraffin and preparing thin section slides for hematoxylin and eosin (HE) staining and immunohistochemistry.
Histology and American Joint Committee on Cancer grading were performed from HE-stained sections. The histopathologic analysis was performed on the following three different tumor regions: surface (R1), center (R2), and tumor-stroma interface (R3). Occurrence of necrotic tumor cells was also evaluated using HE slides. The percentage of the epithelial component versus the stroma in the OSCC tumors was determined by two methods; connective tissue was stained according to Mallory (trichrome staining), and the slides were measured for the area of cancer nests (tumor epithelia) by using ImagePro analysis software (Media Cybernetics, Silver Spring, MD). Separately, slides containing resected tissue were labeled for cytokeratine immunohistochemically using pan-cytokeratine antibody (A1A3+CK19; DAKO, Glostrup, Denmark), which labels cancer cells.
Characterization of the Mononuclear Cell Infiltrate
Mononuclear cells present in the close vicinity of tumor cell nests were determined by using immunohistochemistry performed on paraffin-embedded sections of the tumor samples after deparaffination and microwave antigen retrieval. Antibodies against the following markers were applied: myeloperoxidase, HLA class II (HLA-DP, DQ, DR), CD8, CD20, CD34, CD45R0, and CD68 (all from DAKO); CD4, CD25, and CD56 (Novocastra Laboratories Ltd, Newcastle on Tyne, United Kingdom); CD1a (Immunotech, Marseille, France); and CD134 (PharMingen, San Diego, CA). In all cases, negative control slides were prepared using isotype-matched nonimmune immunoglobulins.
Immunohistochemical labeling was performed with the DAKO LSAB-2 kit using a biotinylated antimouse/antirabbit immunoglobulin G linker and streptavidin-horseradish peroxidase to reveal specifically bound antibodies. The chromogen used was amino-ethyl-carbazol (red) label. Sections were counterstained for the nuclei with hematoxylin.
Evaluation of Tumor Samples
Tumor samples from the LI-treated group and control (non–LI-treated) group were evaluated for histologic changes and for the following infiltrating cells: T and B lymphocytes, natural killer cells, dendritic cells, macrophages and neutrophils, hematopoietic stem cells, and T-cell activation and memory cell markers. A magnification of x40 was used to select the area, and a magnification of x400 was used for the identification and quantitation of infiltrating cells. The density of mononuclear cells was determined based on the hot spot technique, which means that, in each studied tumor area, density was measured at the region of the highest tumor-infiltrating mononuclear cell density. This method minimizes the problem of the extreme heterogeneity of the cellular infiltrates in tissues.14
The histologic evaluation of each patient's tumor section was performed by three independent pathologists (J.T., C.F.-H., and B.D.), and, in case of disagreement, consensual determinations were reached. Morphometric measurements were performed by the same three pathologists and without the knowledge of the clinical background and treatment outcome of each of the patients.
Statistical Analysis
Pathology data were analyzed by analysis of variance (ANOVA) single-factor analysis and the 2 test, and P < .05 was considered to be statistically significant.
RESULTS
Clinicopathologic Response
No systemic or local toxicity or severe adverse events related to LI were reported in this trial. Furthermore, no complications related to LI at surgery, after surgery, or during wound healing were reported. Primary tumor samples of 19 LI-treated and 20 control (non–LI-treated) OSCC patients were evaluated. In the LI-treated group, surgical resection of the primary tumor was performed between days 14 and 54 after the end of the LI treatment (median time to surgery, 22 days). The histologic diagnosis, keratinization (Brodes scores), and tumor-node-metastasis stage of all the LI-treated and control patients are listed in Table 1. The two groups did not differ histologically or with respect to keratinization or tumor-node-metastasis stages.
In two of 19 LI-treated patients (patients 5 and 14; Table 1), it was not possible to detect any cancer tissue in the surgically resected tumor mass, and thus, these patients were considered to be complete responders (exhibiting 100% tumor reduction by histopathology as a result of LI treatment regimen). In two other LI-treated patients (patients 7 and 18; Table 1), the imaging technique verified more than 50% tumor volume reduction, which was considered a partial (major) response (two of 19 patients); and in four LI-treated patients (patients 4, 6, 9, and 12; Table 1), the volume reduction was proven to be more than 30%, which was considered a minor response (four of 19 patients). Progressive disease ( 40% tumor volume increase) occurred in only one patient (patient 17), whereas stable disease was detected in the rest of the patients (10 of 19 patients). Therefore, the objective response rate in the LI-treated group of OSCC patients was 21%, with an overall response of 42% (eight of 19 patients). The full pathologic analysis of the effect of LI treatment on OSCC was performed on the LI-treated nonresponder subgroup (stable disease and progressive disease, n = 11) as well as separately on the LI-treated responder subgroup (n = 6), excluding the two complete responders in whom no tumor tissue could be detected.
Tumor-Infiltrating Lymphocytes
We have observed that, similarly to our previously published data,14 CD20+ B cells were only present in the stroma of OSCC. In the tumor samples obtained after LI pretreatment, a nonsignificant decrease in B cells in OSCC, irrespective of the tumor area investigated, was observed. Clinical responses did not influence this pattern of B cells in the OSCC (data not shown).
In the control group, CD8+ cells infiltrating the tumor and stroma outnumbered the CD4+ cells (Fig 1A through 1D), and as a result, the CD4+:CD8+ ratio was well below 1 (at approximately 0.5; Fig 1E). In the LI-treated group, an increase in the density of CD4+ T cells and decrease in the density of CD8+ T cells was observed in the tumor stroma (Fig 1C). A similar trend was seen in the case of intraepithelial CD4+ and CD8+ cells (Fig 1D). It is of note that these changes were significant only in the case of responders to LI treatment (P < .05). As a consequence, the CD4+:CD8+ ratio was two- to eight-fold higher in the cellular infiltrate of the LI-treated tumors compared with the controls (Fig 1E). Although the highest CD4+ and the lowest CD8+ cellular infiltrate densities were detected in the responder to LI treatment subgroup, the difference from nonresponders to LI treatment was only statistically significant (P < .05) in the tumor stroma segment of the tumor, as can be seen by the CD4+:CD8+ ratio.
Evaluation of the number of activated T cells based on the expression of CD25 and OX40 (CD134) molecules revealed a relatively low mean density of cells expressing these markers in the infiltrates of OSCC (especially in the case of OX40) and no significant differences in their frequency between the control and LI-treated groups (Fig 2A and 2B). However, using available frozen samples and immunofluorescence, we observed that CD25+CD4+ T cells were more frequent in the LI-treated group (10 of 10 patients; range, 14% to 61% of CD4+ cells) but were only detected occasionally in the control group (one of nine patients). We also determined the density of CD45R0+ memory cells and found that their density was highly similar in the control and LI-treated patients both in the tumor stroma and intraepithelially (Fig 2C). The density of CD45R0+ lymphoid cells was, at an average, approximately 60% of T cells (CD4+ + CD8+), suggesting that a high proportion of tumor-infiltrating T lymphocytes were memory cells. Similar to our previously reported findings,14 CD34+ hematopoietic stem cells and CD56+ natural killer cells were not found in the stroma or tumor epithelial nests of the OSCC, irrespective of the tumor area or the patient population investigated (control or LI-treated patients).
We demonstrated that CD1a+ dendritic cells were almost exclusively located in the tumor epithelial nests (both in the current study and in our previous study14). In the LI responder subgroup, we observed a differential effect of LI treatment on the distribution of dendritic cells in the three areas of OSCC investigated; no change was observed in the center of the tumor (R2), a decrease (P < .05) in CD1a+ dendritic cells was detected at the surface (R1), and an increase (P < .05) in CD1a+ dendritic cells was present at the tumor-stroma interface (R3), compared with control (non–LI-treated) patients (Fig 3A).
Inflammatory Cells
Neutrophils were present both in the tumor stroma and in the tumor epithelial nests, which was similar to macrophage distribution in the tumor, whereas eosinophils were found in the tumor stroma exclusively. The presence of neutrophil and macrophage infiltrate in the resected tumor specimens was determined by staining for myeloperoxidase and CD68 markers, respectively.
Analysis of the macrophage density indicated a downmodulation of the stromal presence of CD68+ macrophages in the LI responder group exclusively, and this trend was even more pronounced intraepithelially in the LI-treated group (P < .002 for the LI responder subgroup, and P < .01 for LI nonresponders; Fig 3B). However, LI-treated patients exhibited increased neutrophil migration into the cancer cell nests (Fig 4A through 4C), and neutrophil density was also pronounced in the tumor stroma in the LI responder subgroup (Fig 4C). In the LI-treated group, increased intratumoral infiltration by neutrophils was observed exclusively in patients with multifocal microscopic necrosis (see Tumor Necrosis section).
The density of eosinophils was found to be highly heterogeneous in both OSCC groups (LI-treated and control patients). Morphometric analysis revealed no significant alterations in eosinophilic cell population in the tumor stroma in the LI-treated patients (data not shown). However, a more detailed analysis of the patients revealed that tumors with high eosinophil density (> 50 cells per high-power field) were twice as numerous in the LI-treated group (nine of 19 patients; 47%) compared with the controls (five of 20 patients; 25%).
Tumor Necrosis
Macroscopic cancer necrosis foci in the surgically obtained samples occurred in the control group exclusively. However, multifocal microscopic necrosis was markedly more frequent in the LI-treated group (10 of 17 patients; Tables 1 and 2 and Fig 5B) compared with the control group (four of 20 patients, P < .05; Tables 1 and 2 and Fig 5A), and there was no significant difference in this respect between the responder and nonresponder (to LI treatment) subgroups.
Tumor Stroma to Cancer Cell Nest Ratio
The percentage of the area of cancer cell nests relative to tumor stroma was determined in OSCC patients and revealed a significant effect of LI treatment. The resected tissue samples were stained with the Mallory trichrome technique, where cancer cell nests were labeled distinctly pink, and the proportion of the stroma in the tumor tissue was determined by morphometry (Figs 6A and B). Data indicate that, in the LI-treated group, the proportion of the collagenous stroma in tumor tissue was significantly increased (Fig 6C) compared with the proportion in tumor tissue of control patients (P < .05), indicating a reduction of cancer cell nest area.
Analysis of the patterns of fibrosis in the OSCC samples showed that collagen fibers accumulated around the cancer cell nests and appeared as periepithelial staining or were found in between the cancer cell nests in the tumor stroma (Figs 6A and B). Periepithelial collagenosis was similar in frequency in the tumors of both the control and LI-treated patients, whereas interstitial fibrosis was highly significantly (P < .001) more frequent in the LI-treated group (12 of 17 patients; 70%) compared with the controls (two of 20 patients; 10%), and the nonresponder and responder (to LI treatment) subgroups did not differ significantly in this respect (Table 3).
HLA Class II Expression
HLA class II expression of OSCC was also determined by immunohistochemistry. Tumor-infiltrating cells exhibited a strong HLA II surface staining in both the LI-treated and control patients (data not shown), but the cancer cell expression was heterogeneous. In the control group, approximately half of the patients (11 of 20 patients) had HLA II–negative tumor samples, whereas the other half (nine of 20 patients) contained tumors with at least focal or strong membrane HLA II expression (Table 4). A similar pattern was observed in the LI-treated cohort (eight of 17 patients’ tumors were HLA II positive). However, in the LI-treated group, HLA II–positive tumors belonged exclusively to the nonresponder group, whereas the cancer cells of all LI responder patients were negative for HLA II antigen expression (P < .05; Table 4).
DISCUSSION
Recently, we reported that LI administered to advanced primary OSCC patients as a first-line treatment before conventional therapy (surgery followed by radiation therapy) induced alterations in infiltrating immune cells rather than in inflammatory cells.14 We observed changes in the T-cell population that migrated into the cancer cell nests, and the infiltrating T cells were found to be activated at the lowest LI dose administered, as was demonstrated by increased CD25 expression.14 However, because the treatment regimen (low-dose LI, 400 IU as IL-2, 3 days a week for 2 weeks) did not result in massive necrosis of the tumor tissue, there was no marked change in the ratio of connective tissue to the tumor epithelial component.14
In this study, a higher dose (800 IU/d as IL-2, 5 days a week, extended for 3 weeks) of LI was administered to OSCC patients as first-line treatment before the initiation of conventional therapy (surgery followed by radiotherapy). In the LI-treated patients, a significant (P < .05) increase in tumor-infiltrating CD4+ T cells, with a concomitant decrease in CD8+ T cells (in situ), was observed, resulting in a marked difference in the CD4+:CD8+ ratio in the OSCC. Although the controls were characterized by a low CD4+:CD8+ ratio (< 1, with CD8+ T-cell predominance), a significantly higher CD4+:CD8+ ratio was observed in both the LI-treated nonresponder and responder (to LI treatment) subgroups (two- to eight-fold increase, with a CD4+ T-cell predominance). Of note is the fact that the stromal CD4+:CD8+ ratio was significantly (P < .05) higher in the LI responders compared with the nonresponders, suggesting a possible clinical importance to this phenomenon. A proportion of these CD4+ T cells carried the activation marker CD25, whereas the expression of OX40, a marker of freshly activated CD4+ T cells that has been detected on a portion of tumor-infiltrating lymphocytes in several cancers including head and neck carcinomas,16,17 was rare in the T-cell infiltrate in this study. This would suggest a later phase of the T-cell activation process and may be a result of the relatively long period (range, 14 to 54 days; median, 22 days) between the end of LI therapy and the surgical removal of the tumors in the LI-treated group. However, double-labeling studies indicated that a large proportion of the CD4+ and CD8+ T cells were CD45R0+ (data not shown), suggesting that they were memory T cells. Parallel to these changes, intraepithelial dendritic cells shifted from the surface of the OSCC to the tumor-stroma interface. Similarly, in a previous report on the local effect of IL-2 infusion in head and neck cancer, a selective effect on CD4+ T lymphocytes and dendritic cells was observed, with marked increase in CD25+ cells only at lower doses of IL-2 administration.18
LI-treated OSCC patients were characterized by a markedly altered pattern of inflammatory cells, suggesting that an acute inflammatory reaction was mediated predominantly by neutrophils and, to a smaller extent, eosinophils, rather than by macrophages. Other studies (in melanoma patients) showed that the accumulation of neutrophil leukocytes at vaccination and tumor sites was prevalent after vaccination with granulocyte-macrophage colony-stimulating factor (GM-CSF)–producing autologous tumor cells.19,20 Because LI preparations contain several cytokines, including GM-CSF,14,15 this could be an explanation for the observed increased neutrophil infiltration in the LI-treated patients compared with controls.
In parallel to the complex alterations in the tumor-infiltrating cellular components of the host antitumor response, multifocal necrosis of cancer cell nests and an increase in the proportion of connective tissue were detected in the LI-treated OSCC patients compared with the nontreated controls. These changes may reflect the aftermath of an antitumor immune response raised against the OSCC, which was induced by LI neoadjuvant administration.
As a result of the local neoadjuvant administration of LI to the OSCC patients, the overall response rate in the LI-treated group was 42% (21% of these patients were considered to have an objective response, with two pathologic complete responses). Our results indicate that, although some degree of antitumor reactivity could be elicited in most patients, this does not necessarily translate into an objective clinical response in all patients.
One of the most compelling findings of this study is illustrated by the fact that a successful immune response to OSCC may require the reversal of the inherently low intratumoral CD4+:CD8+ ratio (as seen in non–LI-treated controls). The predominance of CD8+ cells over CD4+ cells in solid tumors is not specific for OSCC. Similar low CD4+:CD8+ ratios have been reported in basal cell carcinoma21 and cervical22 and breast cancers.23 High percentages of CD4+ T cells or high CD4+:CD8+ ratios correlated with better prognosis or response to immunotherapy in melanoma,24 B-cell non-Hodgkin's lymphoma,25 renal cell carcinoma,26 and cervical carcinoma.27 This suggests that an acquired immunosuppressed state of cancer patients, induced by the tumor, is a common phenomenon, reflecting the general poor immune response in cancer patients. Furthermore, CD4+ T lymphocytes play a central role in initiating and maintaining antitumor immunity by providing help for CD8+ cytotoxic T lymphocytes, stimulating antigen-presenting cells, and sustaining immunologic memory or, in some cases, by performing effector functions via cytokine secretion or direct cytolysis.28-30 In the case of oral carcinomas treated with LI, it is not yet clear which are the specific mediators (in LI) that elicit the antitumor immune response. However, the increased density of intraepithelial leukocytes in parallel to microscopic necrosis after LI treatment may suggest that the activation of nonspecific antitumor mechanisms (in OSCC patients) is equally important to produce clinicopathologically detectable destruction of the established tumor.
Histopathologic comparison of tumors in the LI-treated nonresponder subgroup to the tumors of the responder subgroup (in which clinical and histopathologic regressions were observed in eight of 19 patients) revealed that most of the studied immunologic parameters, from immune cell composition to inflammatory cell patterns, were similar. These data suggest that the LI-treated group responded homogenously to the treatment but that the anticancer effects of LI treatment were heterogeneous. A plausible explanation for this discrepancy could be the individual differences in immunosensitivity of OSCC tumors (differences in tumor antigens and/or HLA composition) and/or the level of impairment of functional immune response in the different patients.
Data from other studies indicate that the lack of expression of MHC class I antigens31 or costimulatory molecules32 on the target (tumor) cells, FasL expression,33 and signaling defects34 can all contribute to impaired immune response to the tumor. In this study, analysis of HLA class II expression by OSCC cancer cells indicated that the level of expression was highly similar between the LI-treated and control groups ( 45% of patients were positive for HLA class II antigen expression). This suggested that LI treatment did not induce or enhance the expression of HLA II molecules on the tumor compared with controls. However, within the LI-treated cohort, all HLA II–positive patients belonged exclusively to the nonresponder subgroup, whereas all the LI responder patients were negative. This finding, although derived from a small number of patients, may indicate that HLA class II expression on OSCC cells may be associated with an unfavorable clinical outcome of disease or with a diminished ability to respond to immunotherapy. Similar findings were reported in malignant melanoma where the expression of MHC class II constitutes a poor prognostic factor,35 which may be a result of the induction of T-cell anergy by MHC class II–expressing tumor cells in the absence of costimulatory signals.36 As was described in melanomas, head and neck cancers lack the expression of the costimulatory molecules B7.1 and B7.2,32 and therefore, the expression of MHC class II molecules on head and neck (squamous cell carcinoma) tumor cells may induce immunologic tolerance. Therefore, we suggest that exclusive targeting of the immune system without a concurrent attempt to modulate the sensitivity of the cancer cell target may not yield an improvement of the overall response rate to an immunologically based treatment.
We hypothesize that LI mode of action involves the combined activity of the different cytokines present in LI14 inducing a cascade of events as follows. First, tumor necrosis factors present in LI (such as tumor necrosis factor-alpha) attack the tumor to release tumor antigens. Second, antigen-presenting cells (eg, dendritic cells) transport the newly released tumor antigens to lymph nodes. Third, lymphoproliferative cytokines (present in LI administered peritumorally and perilymphatically; eg, IL-1 and IL-2) induce a marked polyclonal expansion of tumor-specific T cells, primarily in lymph nodes. Fourth, LI recruits CD4+ T cells from local lymph nodes via chemotactic factors and reverts the balance of intratumoral CD4+ and CD8+ cells in favor of CD4+ T cells, which further upregulates the antitumor immune response, resulting in tumor cell necrosis. Fifth, LI recruits neutrophils from the circulation (via GM-CSF, also present in LI), which propagates the destruction of the tumor cell nests. And sixth, either LI-derived cytokines or the de novo cytokine production by the tumor-infiltrating cells induce massive local fibrosis.
Our data supports the notion that these immune-mediated processes continue long after the cessation of LI administration, as evidenced by the immunohistopathologic changes observed (in the tumor) 14 to 54 days after the end of LI treatment. The efficacy of this neoadjuvant treatment protocol for patients with advanced primary OSCC resulted in a marked reduction in tumor mass (in 42% of the patients) compared with baseline tumor measurements. These findings, taken together with our previous report,14 indicate that the LI neoadjuvant immunotherapy regimen for OSCC is a clinically viable approach to the management of OSCC. The results of larger efficacy trials (planned for the near future) may promote the introduction of LI into the current treatment protocols of OSCC.
Authors' Disclosures of Potential Conflicts of Interest
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Consultant/Advisory Role: Eyal Talor, TEVA Pharm. Stock Ownership: Eyal Talor, CEL-SCI Corp, TEVA Pharm. Research Funding: József Tímár, CEL-SCI Corp. For a detailed description of these categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and Disclosures of Potential Conflicts of Interest found in Information for Contributors in the front of each issue.
NOTES
Supported in part by the Ministry of Education (NKFP 1/48/2001, J.T. and M.K.).
Previously published in abstract form (with modifications) in the ASCO Proceedings (Proc Am Soc Clin Oncol 22:189s, 2004 [abstr 2605]).
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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