Kazuaki Chikamatsu

Clinical significance of p53 functional loss in squamous cell carcinoma of the oropharynx

We examined the frequency of p53 mutations in 38 oropharyngeal squamous cell carcinomas (SCC), using both a yeast functional assay and a conventional immunohistochemical staining method (IHC) to detect p53 mutations.We also explored the clinical importance of p53 mutations in oropharyngeal SCC. An accumulation of p53 protein was detected in 17 of the 38 (45%) tumors by IHC, whereas the yeast‐based assay detected 6 additional p53 mutations, for a total of 23 tumors (61%) with p53 mutations. The cDNA sequencing analysis revealed that the 6 mutations undetected by IHC consisted of 3 frameshift, 1 nonsense and 2 missense mutations. Thus, the yeast functional assay was more sensitive than conventional IHC for detecting p53 mutations. Subsequently, the relationship between p53 mutations and the clinico‐pathological parameters in oropharyngeal SCC was evaluated using the results of the functional assay. Mutation of p53 was not associated with the patient age, sex, tumor stage or degree of tumor cell differentiation. Interestingly, heavy drinking had a significant positive correlation with the p53 mutation, but heavy smoking did not, suggesting that prolonged exposure to alcohol is more related to p53 mutation in oropharyngeal SCC than to tobacco consumption. Radiation sensitivity was examined by comparing tumor size on magnetic resonance images before and after completion of therapy with 45 Gy radiation, in the 18 cases of T2 oropharyngeal SCC that were initially treated by radiotherapy. The results showed that tumors with wild‐type p53 decreased in size significantly compared to those with mutant p53. In 33 patients treated with curative intent, the overall survival after the completion of therapy was better in patients with a wild‐type p53 tumor than in patients with a mutant p53 tumor. We conclude that p53 mutation is associated with radiation resistance and a decreased probability of survival in oropharyngeal SCC. Int. J. Cancer (Pred. Oncol.) 89:187–193, 2000.

Expression of the MAGE-1, -2, -3, -4, and -6 Genes in Non-Squamous Cell Carcinoma Lesions of the Head and Neck

The messenger RNA level of several MAGE genes, some of which have been proven to encode tumor rejection antigens recognized by cytotoxic T lymphocytes, were examined in 41 benign and malignant lesions of the head and neck region. By a reverse transcription-polymerase chain reaction assay and Southern blot hybridization, MAGE-1, -2, -3, -4, and -6 genes were expressed in 25%, 41.7%, 33.3%, 8.3% and 33.3% of 12 non-squamous cell carcinomas, respectively. These tumors consisted of 6 papillary adenocarcinomas, 3 adenoid cystic carcinomas, 2 adenocarcinomas, and 1 mucoepidermoid tumor. Of 7 non-Hodgkins lymphomas, one case from the oropharynx and 2 from the nasopharynx expressed for the MAGE-1 and MAGE-2 genes, respectively. In contrast, none of 12 benign tumors expressed any of these MAGE genes. Interestingly, of 10 other lesions including hyperplasia, keratosis, and ulcer, one histologically diagnosed as dysplasia expressed the MAGE-2, -3, -4, and -6 genes. These results suggest that the MAGE genes may be expressed in malignant tumors and precancerous lesions but not in benign tumors. In addition, non-squamous cell carcinomas may be suitable targets for specific immunotherapy against MAGE gene products.

Analysis of T cell receptor variability in fresh tumor-infiltrating lymphocytes from human head and neck cancer

In this study, we analyzed T cell receptor (TCR) gene rearrangements in tumor‐infiltrating lymphocytes (TIL) freshly obtained from 15 patients with head and neck cancer using the reversely transcribed polymerase chain reaction (RT‐PCR) method. These TILs showed preferential expression of Vα10, Vα8 and Vα1, detected in 13 (87%), 11 (73%), and 9 cases (60%), respectively. The TCRVβ gene revealed diversity without preferential usage. The head and neck region is exposed to bacteria and viruses, so it is possible that the tumor site can become infected and accumulate T cells involved in infection and inflammation. Therefore, we also investigated TCR gene usage in T cells infiltrating in chronic sinusitis mucosa to address the question of whether the Vα1, Vα8, and Vα10 subfamilies are characteristic in TIL from squamous cell carcinoma of head and neck. TCR Vα10 gene usage was also the most common in Vα segment in T cells infiltrating the sinus mucosa, but Vα and Vα8 were not detected in the T cells in sinusitis. These results indicate that the Vα10 subfamily, the preferred T cell population in both TIL and T cells in inflammatory disease, might he involved mainly in inflammation or infection. On the other hand, Vα1 and Vα8 appear to be relatively specific populations for antitumor immunity in head and neck cancer.

The role of major histocompatibility complex expression on head and neck cancer cells in the induction of autologous cytotoxic T lymphocytes

Using head and neck tumors, we studied the role of HLA class I and DR antigens on tumor cells in cytotoxic T lymphocyte (CTL) induction. Expression of major histocompatibility complex (MHC) antigens was investigated by two-color flow cytometry analysis and for this study we used the tumor cells, over 50% of which expressed both HLA class I and DR antigens on their surface. In seven cases, tumor cells were divided into three groups according to the specificity of monoclonal antibodies (mAb) to MHC to study the role of MHC antigens on tumor cells in CTL induction: one was not blocked (MHC double-positive tumor), a second was blocked by anti-class I mAb (class-Ingative DR-positive tumor) and third was blocked by anti-DR mAb (class-I-positive DR-negative tumor). Subsequently, these tumors were used to stimulate an autologous mixed lymphocyte/tumor cell culture for 5 days (MLTC) followed by further cultivation with interleukin-2 for 12 days. The induced autologous tumor killer cells were most cytotoxic when non-treated tumors, which consist mainly of cells that are both HLA-class I and DR-positive, were used as stimulator cells. When the tumor cells blocked by anti-DR mAb were used as stimulators, autologous tumor killer activity was lower than that induced by tumor cells blocked by anti-class-I mAb. Moreover, cytolysis by autologous tumor killer cells induced by stimulation of non-treated tumor cells was blocked during the effector phase, 26.6%–42.3% and 32.7%–53.8% by anti-class-I and anti-DR mAb respectively, suggesting that majority of the autologous tumor killer cells are MHC-restricted CD8+ or CD4+ CTL. These results suggest that both MHC class I and class II antigens on head and neck tumor cells play a critical role in inducing CTL.

Functional and T Cell Receptor Gene Usage Analysis of Cytotoxic T Lymphocytes in Fresh Tumor‐infiltrating Lymphocytes from Human Head and Neck Cancer

Twenty‐one cytotoxic T lymphocyte (CTL) clones or lines that killed autologous tumor cells, but not allogeneic tumor, K562, or Daudi cells, were established from fresh tumor‐infiltrating lymphocytes of two individuals (HP‐1 and HP‐2) with head and neck cancer by limiting dilution in the presence of recombinant interleukin‐2. Sixteen (76%) of these 21 clones or lines comprised CD4+ CTLs and the other five comprised CD8+ CTLs. These observations suggest that autologous tumor cell‐specific CD4+ CD8− and CD4− CD8+ CTLs are present in vivo at the tumor site in head and neck cancer. Analysis of T cell receptor (TCR) gene arrangements in 20 of the 21 CTL isolates with reverse transcriptase and the polymerase chain reaction revealed that five of 12 and five of eight isolates from HP‐1 and HP‐2, respectively, were clones, the other isolates being lines comprised of two or more clones. Each CTL clone showed a different combination of Vα and Vβ gene expression, suggesting that more than five different tumor‐associated antigens may be expressed on head and neck cancer cells. In spite of the diversity of TCR αβ combinations, TCR Vα1, Vα3, Vα8, Vα10, Vβ8, and Vβ9, and Vβ17 were also frequently expressed in both patients. These data suggest that specific CTLs proliferate oligoclonally and contribute to the specific immune response against head and neck cancer in vivo.

Angioimmunoblastic lymphadenopathy-like T-cell lymphoma A case report and immunologic study

BACKGROUND Angioimmunoblastic lymphadenopathy (AILD) is rare in the head and neck and its definition remains controversial. METHOD A case of AILD with an ulcer of the lateral pharyngeal wall was studied for viral infection, immunohistologic findings and T-cell receptor (TCR) V beta rearrangement. RESULTS We observed elevation of antibodies against herpes simplex virus and herpes zoster virus as well as Epstein-Barr virus considered closely associated with AILD. The affected neck lymph node showed a preponderance of T-cells, predominantly CD4+ over CD8+ T-cells and all V beta gene families were expressed in the T-cells without enhancement of any particular TCR gene usage. CONCLUSION Viral infection may occur easily in patients with AILD, possibly owing to immunodeficiency. Assessment of TCR V beta gene usage indicated T-cells to non-specifically become lymphomatous in AILD-like T-cell lymphoma.

Ambient mass spectrometry-based detection system for tumor cells in human blood

Circulating tumor cells (CTCs) are a rare cell population in peripheral blood that lead to distant metastasis in many types of cancer. Most current CTC-detection methods, including antibody-based detection, have shortcomings such as false-positive and false-negative findings. These limitations must be addressed to achieve CTC detection with high accuracy and reproducibility; and therefore, clinical utility. Herein, we propose a novel method of tumor cell detection in human blood samples using ambient mass spectrometry (MS). Probe electrospray ionization (PESI)-MS was used to obtain mass spectra containing 1,136 peaks from peripheral blood mononuclear cells (PBMCs) spiked with different numbers of SAS tumor cells by. Similarities between the spectral patterns of PBMCs spiked with different numbers of tumor cells, and those from PBMCs alone, were statistically reduced with increasing numbers of SAS tumor cells. Intriguingly, the influences of a few hundred tumor cells could be observed in the changes to the spectral peaks of PBMCs. Moreover, 16 individual peaks, with positive and negative correlations between peak intensities and the number of SAS tumor cells, were identified as tumor-associated metabolites. In conclusion, we successfully used PESI-MS to detect tumor cells present among very large numbers of PBMCs without the need for enrichment using antibodies for tumor cell markers. Our data strongly suggest that this method may be clinically suitable for CTC detection.

Abstract 4627: Prognostic significance and population shift of peripheral monocytes in patients with oropharyngeal squamous cell carcinoma

Evidence has accumulated that chronic inflammation plays a critical role in carcinogenesis, tumor growth, and metastasis. To date, several inflammatory biomarkers are considered as potential prognostic factors in various cancers. The aim of the present study was to investigate the prognostic significance of the pretreatment levels of inflammatory biomarkers in patients with oropharyngeal squamous cell carcinoma (OPSCC). A total of 67 patients newly diagnosed as OPSCC was finally included. The neutrophil, lymphocyte, monocyte and platelet counts recorded before treatment was initiated, then lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) were calculated. These inflammatory biomarkers and clinical parameters including age, differentiation, T-stage, N-stage, TNM-stage, smoking, and drinking were compared with progression-free survival (PFS) and overall survival (OS) using cox regression. Elevated monocyte count and advanced T-stage were significantly associated with poor PFS; in addition, these were independent prognostic factors for PFS. Elevated monocyte count and low LMR were significantly associated with poor OS; moreover, low LMR was an independent prognostic factor for OS. Of note, monocyte count and LMR were associated with prognosis more intensively in patients with p16-negative OPSCC. Following these results, we evaluated the peripheral blood mononuclear cells of 14 patients with OPSCC collected before treatments. CD14+ CD86+ HLR-DR+ cells were gated as monocytes, then divided into three subsets according to the expression of CD14 and CD16. The proportion of intermediate monocytes was lower and that of classical monocytes was higher in OPSCC patients than in healthy donors, especially in patients with elevated monocyte count. Furthermore, the expression level of PD-L1 was higher in patients than in healthy donors on all subsets. In conclusion, our data suggests that elevated monocyte count and low LMR seem to be useful biomarkers for predicting prognosis of OPSCC; furthermore, the population shift of monocytes may relate to poor prognosis of OPSCC. Citation Format: Hideyuki Takahashi, Koichi Sakakura, Kazuaki Chikamatsu. Prognostic significance and population shift of peripheral monocytes in patients with oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4627. doi:10.1158/1538-7445.AM2017-4627

Abstract 5177: Primary tumor induces lymphangiogenesis and immune suppression in sentinel lymph nodes

The main factor that affects the prognosis of patients with squamous cell carcinoma of the head and neck (SCCHN) is regional lymph node metastases, which usually spreads first to the sentinel lymph nodes (SLNs). Recent studies using animal models have demonstrated that tumor cells can induce lymphangiogenesis in SLNs before metastasizing. In this study, we investigated the association of SLN lymphangiogenesis with the status of primary tumors. Moreover, the immunological status of SLNs was also examined. The expression of lymphatic-specific markers, including VEGFR-3, Prox-1, and LYVE-1 in 23 metastasis-negative SLNs obtained from 10 patients with SCCHN was investigated using quantitative real-time RT-PCR. Moreover, primary tumors obtained from patients were evaluated for the expression of VEGF-A, -C, and -D by immunohistochemistry. The level of LYVE-1 expression in SLNs was significantly higher than in control lymph nodes from patients with non-cancerous diseases. Moreover, SLNs from patients with VEGF-C-positive tumor showed a significantly higher expression of VEGFR-3 than those from patients with VEGF-C-negative tumor. VEGFR-3 is expressed mainly in lymphatic endothelium but also in a subset of antigen-presenting cells including immature dendritic cells (DCs), therefore; we furthermore examined the infiltration of DCs into SLN by immunohistochemistry. Interestingly, S-100+ and CD1a+ immature DCs infiltrated significantly into SLNs compared to non-SLNs in patients with SCCHN, suggesting that predominant migration of immature DCs, which suppress T-cell activation and support regulatory T cell development, would contribute to inhibit rather than develop anti-tumor immunity. Our findings suggest that primary tumor actively induces lymphangiogenesis in SLNs prior to the onset of metastases, and where tumor-derived VEGF-C plays an important role. Moreover, active lymphangiogenesis may concomitantly lead to down-regulation of SLN immunity. Thus, both lymphangiogenesis and immune suppression in SLNs are likely the basis of its susceptibility to tumor metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5177. doi:10.1158/1538-7445.AM2011-5177

Abstract 5314: Immunoregulatory properties of CD44+ cancer stem-like cells in squamous cell carcinoma of the head and neck

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC There is increasing evidence in certain malignancies that tumor cells are organized into a hierarchy originating from a small population of cancer stem cells (CSCs) that possesses extensive proliferative and self-renewal potential, and that these CSCs are responsible for maintaining the tumors. In squamous cell carcinoma of the head and neck (SCCHN), CD44+ cells with significant tumorigenic potential have been identified as CSCs; however, the immunological properties of such CSCs have not yet been elucidated. In this study, we investigated the immunological functions of CD44+ cells from a SCCHN cell line. Analysis of flow cytometry demonstrated that the expression of HLA-A2 molecules was deficient in the parental cell line, whereas the CD44+ cell population restored HLA-A2 expression, although at very low levels. Moreover, CD44+ cells exhibited weak HLA class II expression on the cell surface. Interestingly, down-regulation of TAP2 was found in CD44+ cells. The CD44+ cell population produced significantly higher levels of IL-8, G-CSF, and TGF-β than the CD44- cell population. Moreover, CD44+ cells have been shown to not only more strongly inhibit the proliferation of T cells activated with anti-CD3/CD28 mAb, but also to more efficiently induce CD4+CD25+FOXP3+ Treg cells and myeloid-derived suppressor cells as compared with CD44- cells. Additionally, CD44+ cells also suppressed Th1 responses (IFN-γ production by PBMCs) and enhanced regulatory T cell responses (IL-10 production by PBMCs). Thus, CSCs may have higher immunosuppressive ability promoting evasion from immunosurveillance; therefore, the development of novel immunotherapeutic strategies to efficiently overcome CSCs-driven immune suppression is urgently needed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5314.