Phorbol dibutyrate plus ionomycin improves the generation of cytotoxic T cells from draining lymph nodes of patients with advanced head and neck cancer.

Fifty-one cervical nodes from 19 patients with advanced head and neck cancer were stimulated with phorbol dibutyrate and ionomycin (PDBu + Io) to determine the effect of such stimulation on the generation of cytotoxic T cells and whether this stimulation could bypass the need for autologous tumor stimulation. Lymphocytes stimulated with PDBu + Io demonstrated a sixfold greater in vitro expansion and significantly increased DNA synthesis. Whereas fresh lymphocytes displayed no cytotoxicity, stimulation with PDBu + Io and culture in interleukin-2 (IL-2) led to significant cytotoxicity equivalent to that of lymphocytes stimulated with autologous tumor and IL-2. T cells with the greatest cytotoxicity were generated from patients with nodal metastases. In patients with stage IV tumors, effector cells demonstrating greater lysis of natural killer-resistant targets (Daudi cells) were associated with higher rates of recurrence (50% versus 12%, respectively, p < 0.001). Stimulation with PDBu + Io augments growth and proliferation of lymphocytes from draining lymph nodes and preserves cytotoxicity without the need for autologous tumor. Excluding the need for antigenic stimulation by autologous tumor may prove useful in adoptive immunotherapy procedures.     

The nodal neck level of sentinel lymph nodes in mucosal head and neck cancer.

Sentinel node biopsy is emerging as a successful means of identifying subclinical lymph node disease in mucosal head and neck cancer. Sentinel node studies in melanoma and breast cancer have identified sentinel nodes at unusual sites and the technique is redefining our understanding of dynamic lymphatic flow. In this study, the sentinel nodes in mucosal head and neck malignancies were mapped according to their site within the neck and this was correlated with tumour site within the oral cavity. Fifty-two necks were explored for sentinel nodes from tumours located in the tongue (23 cases), floor of mouth (12 cases), palate (six cases), retromolar trigone (five cases), alveolus (three cases), buccal mucosa (two cases), tonsil (two cases) and lip (one case). In total, 124 sentinel nodes were found in levels I-V. Two hot spots were found in the tonsils and were not excised, two nodes were located in level IIB, four nodes were found in level IV, three in the contralateral neck and one in level V. The sentinel nodes located at unusual sites would not have been excised in a supraomohyoid neck dissection and the study has improved our understanding of dynamic lymph flow from tumours.     

Vascular interventions in head and neck cancer patients as a marker of poor survival

Objective

Head and neck cancer can involve the surrounding vasculature and require technically challenging vascular interventions. These interventions can be complicated by tumor invasion, history of prior surgery, and history of radiation therapy. Our aim was to examine patients with vascular interventions in association with head and neck cancer to determine outcomes and best practice.

Interleukin-2 injected around tumor-draining lymph nodes in head and neck cancer

Twenty patients with recurrent, inoperable head and neck squamous cell carcinoma received perilymphatic injections of natural interleukin-2 (nIL-2) for 10 days. Ten patients received 200 units (U) of nIL-2; five 1,000 U; and five 5,000 U. Irrespective of the location of the recurrence, the injections were always performed 1.5 cm below the insertion of the sternocleidomastoid muscle on the mastoid. When the ipsilateral lymphatic chain was still present, they were performed on the same side as the tumor site, whereas when it had been stripped as a result of previous surgery, they were contralateral. Patients who had undergone bilateral neck dissection were injected on the tumor side. Whenever possible, the treatment was repeated after 45-day intervals. In 13 patients (65%) with bilateral or contralateral lymph nodes, complete or partial disappearance of the lesion was observed. Despite these marked responses, the tumor always relapsed, and subsequent IL-2 courses were poorly effective. There were no systemic disturbances during or after treatment, but only moderate local swelling and pain.     

乳腺癌淋巴结微小转移状态下前哨与非前哨淋巴结免疫细胞的改变

目的观察乳腺癌淋巴结微小转移状态下前哨淋巴结(SLN)与非前哨淋巴结(Non- SLN)免疫细胞密度的改变。方法53例符合研究标准的女性乳腺癌患者按SLN不同转移状态分为阴性、游离肿瘤细胞及微转移3组。从各组随机选择6对SLN和Non-SLN与DC-LAMP,CD68,CD123,Foxp3抗体行免疫组织化学染色,半定量评估染色细胞密度改变;所有SLN和Non-SLN蜡块切片均行与DC-LAMP抗体免疫反应的免疫组织学检查以确认成熟树突状细胞(DC)。蔡司图像分析系统定量分析每个淋巴结DC-LAMP阳性细胞的相对密度(DC-LAMP阳性细胞面积/淋巴结面积)。Wicoxon检验和Mann-Whitney检验分别用于DC-LAMP阳性细胞的相对密度的组内和组间比较。结果DC-LAMP阳性细胞密度改变显著。组内比较显示阴性组和微转移组SLN内DC-LAMP阳性细胞平均密度较Non-SLN高(P<0.05);组间比较显示微转移组SLN和Non-SLN内DC-LAMP阳性细胞密度较阴性组显著升高(P<0.05)。结论SLN和Non-SLN内DC-LAMP 细胞平均密度在淋巴结肿瘤转移形成早期发生改变,揭示SLN在肿瘤与引流淋巴结免疫相互作用中起重要作用。     

Clinical relevance of occult tumor cells in lymph nodes from gastric cancer patients

The current method for staging in gastric cancer is not sufficient as even after a complete primary tumor resection, patients with node-negative gastric cancer suffer from disease recurrence. In this study, the relation between disease recurrence and the presence of occult tumor cells (OTC) in lymph nodes from gastric cancer patients was evaluated. In a case-control design, lymph nodes from 40 cases (disease recurrence) and 41 controls (no disease recurrence and followed for at least five years) with gastric cancer were examined for the presence of OTC, that comprised micrometastases (MM; >0.2 mm and < or =2.0 mm) and isolated tumor cells (ITC; < or =0.2 mm). The original hematoxylin and eosin-stained sections of all lymph nodes from cases and controls were previously considered as tumor-negative by the local pathologist. Fresh hematoxylin and eosin-stained sections were screened by conventional microscopy. Histologic sections stained by immunohistochemistry with anticytokeratin antibodies CAM5.2 were screened by conventional and automated microscopy. Tumor cells were detected in lymph nodes from 40 of 81 (49%) patients. There was no significant difference in the presence of OTC, MM, or ITC between the case and control groups (P = 0.658, P = 0.691, P = 0.887, respectively). However, significantly more cases presented with 20% or more OTC-positive lymph nodes (P = 0.015). A multivariate logistic regression analysis showed that examination of less than five lymph nodes (odds ratio, 13.8; 95% confidence interval, 1.6-120.6, P = 0.018) was the only significant independent risk factor for disease recurrence, especially for locoregional disease recurrence (odds ratio, 20.4; 95% confidence interval, 2.2-190.8, P = 0.008). A similar analysis for distant disease recurrence showed a percentage of 20% or more OTC-positive lymph nodes to be the only significant independent risk factor (odds ratio, 15.6, 95% confidence interval, 1.6-151.4, P = 0.018). The sensitivity of immunohistochemistry evaluated by microscopy to identify cases with 20% or more OTC-positive lymph nodes increased from 8% for conventional microscopy to 22% for automated microscopy (McNemar's test, P = 0.063). The mere presence of OTC-positive lymph nodes in gastric cancer patients did not predict disease recurrence. However, the number of examined lymph nodes and the percentage of OTC-positive lymph nodes were independent risk factors for locoregional disease recurrence and distant disease recurrence, respectively. Automated microscopy was essential in identifying patients with 20% or more OTC-positive lymph nodes. Therefore, a maximum number of lymph nodes should be removed and meticulously examined for OTC to identify high-risk patients. These patients should be considered for additional treatment.     

CD 133 and CXCR4 colon cancer cells as a marker for lymph node metastasis

Introduction

Colorectal cancer (CRC) stem cells or tumor-initiating cells (Co-TIC) are implicated in both cancer recurrence and extranodal metastasis. CD133 and CXCR4 are specific cell surface markers that are indicators of Co-TIC. The presence of lymph node (LN) metastases is one of the strongest negative prognostic factors for CRC patients. We examined the relationship between the Co-TIC markers CD133 and CXCR4 and LN involvement in CRC.

Methods

CRC cells were isolated via enzymatic digestion. CD133⁺, CXCR4⁺, and double-positive CRC cells were detected by fluorescence-activated cell sorting analysis. The percentages of CD133⁺, CXCR4⁺, and double-positive cells were identified and correlated to the number and percentage of positive LN on staging.

Results

Twenty-seven samples underwent fluorescence-activated cell sorting analysis. The mean percentage of CD133⁺ cells was 3.94% (range 0.15%–19.06%). The mean percentage of CXCR4⁺ cells was 6.15% (range 0%–27.11%). The mean percentage of CD133⁺CXCR4⁺ cells was 0.45% (range 0%–2.08%). Thirteen patients had LN metastasis: 8 N1 disease and 5 N2 disease. The correlation coefficients between the percentage of Co-TIC marker–positive cells and percentage of positive LN were r = 0.58 (P = 0.0016) for CD133⁺ cells, r = 0.36 (P = 0.5868) for CXCR4⁺ cells, and r = 0.56 (P = 0.0022) for double-positive cells.

Discussion

Our results show CD133⁺ and CD133⁺CXCR4⁺ cancer cells correlate with the presence of LN metastasis in CRC. Further studies will examine whether these markers can give consistent prognostic information and may help to develop novel diagnostic and therapeutic options.     

Hierarchical immunosuppression of regional lymph nodes in patients with head and neck squamous cell carcinoma.

Patients with head and neck squamous cell carcinomas (HNSCCs) manifest defects in cell-mediated immune function. Previous studies in this laboratory have demonstrated regional alterations in the immunocompetence of draining lymph nodes (LNs) in HNSCC patients. In this investigation, we studied functional activity of lymphocytes from lymph nodes in different locations in the radical neck dissections (RNDs) from patients undergoing operations for HNSCC. Lymphocytes from nodes close to the primary tumor ("near" lymph nodes or NLN) exhibited a significant decrease in interleukin-2 (IL-2)-activated cytotoxicity when compared to lymphocyts from distant nodes ("far" lymph nodes or FLN). In addition, co-culture experiments suggested the existence of a soluble regulatory factor, produced by lymph nodes, that inhibited the development of lymphokine-activated killer (LAK) cells in vitro. Further experiments with conditioned supernatants from the lymph node cells confirmed the presence of this soluble inhibitory factor. The inhibitory effect is significantly greater in NLNs than in FLNS. This hierarchical phenomenon suggests a regional network of immunosuppression in HNSCC patients. It is likely that tumor- and lymph node-induced suppression plays a role in limiting the efficacy of current immunotherapy protocols in human beings. A greater understanding of mechanisms of local inhibition of immune function will aid in improving adoptive immunotherapy for treatment of cancers in human beings.     

Occult tumor cells in lymph nodes as a predictor for tumor relapse in pancreatic adenocarcinoma

Background and aims Occurrence of tumor relapse is frequent in patients with pancreatic cancer despite the absence of residual tumor detectable at primary surgery and in histopathological examination. Therefore, it has to be assumed that current tumor staging procedures fail to identify minimal amounts of disseminated tumor cells, which might be precursors of subsequent metastatic relapse. The aim of this study was to assess the prognostic impact of minimal tumor cell spread detected in lymph nodes classified as “tumor-free” in routine histopathologic evaluation. Materials and methods A total of 154 “tumor-free” lymph nodes from 59 patients with pancreatic cancer who underwent intentionally curative tumor resection were examined by immunohistochemistry for disseminated tumor cells. Results Fifty (32.5%) of the “tumor-free” lymph nodes obtained from 36 (61%) patients displayed disseminated tumor cells. Multivariate survival analysis revealed that the presence of disseminated tumor cells in “tumor-free” lymph nodes is an independent prognostic factor for both a significantly reduced relapse-free survival (p = 0.03) and overall survival (p = 0.02). Conclusions The frequent occurrence and prognostic impact of immunohistochemically identifiable disseminated tumor cells in lymph nodes of patients with operable pancreatic cancer supports the need for a refined staging system of excised lymph nodes, which should include immunohistochemical examination.     

Prognostic impact of isolated tumor cells and micrometastases in axillary lymph nodes of breast cancer patients

With the introduction of the sentinel node (SN) procedure, the detection frequency of nodal isolated tumor cells and micrometastases has increased. We reviewed the literature on prognostic significance of these small nodal metastases. All studies before the SN era and all studies using the SN procedure that reported outcome in relation to presence of isolated tumor cells and/or micrometastases were included. Studies before the SN era were divided in 'cohort' and 'occult metastases' studies. The SN studies were divided in single-centre studies and in one multicentre cohort study. In the pre-SN cohort studies, axillary lymph node metastases of 2 mm or less were associated with reduced overall survival with an adjusted pooled hazard ratio of 1.44 (95%CI 1.29-1.62). In the pre-SN occult metastases studies, occult nodal metastases were associated with a pooled relative risk of deaths after 5 years of 1.45 (95%CI 1.11-1.88). In single-centre SN studies, using multivariate analyses, the presence of micrometastases was associated with a hazard ratio for disease events of 1.43 to 1.89 as compared to node-negative disease. The largest SN study, including nearly 2000 patients with isolated tumor cells or micrometastases, reported an adjusted hazard ratio for disease-events of 1.50 (95%CI 1.15-1.94) and 1.56 (95%CI 1.15-2.13), respectively, in patients who had not received systemic therapy. We conclude that isolated tumor cells and micrometastases are associated with increased risk of disease-events of about 1.5 compared to node-negative disease. Therefore, we recommend to consider the use of adjuvant systemic therapy in these patients.     

Methylation-specific PCR for DNA-based detection of occult tumor cells in lymph nodes of prostate cancer patients

OBJECTIVES: To assess the feasibility of methylation-specific PCR (MSP) for the detection of promoter hypermethylation of the detoxifying glutathione-S-transferase P1 gene (GSTP1) to detect occult prostate cancer cells in lymph nodes (LNs). METHODS: Paraffin-embedded pelvic LNs from 20 patients with pT2pN0M0R0 prostate cancer who developed PSA relapse were assessed by MSP. In 18 of the patients, samples of the primary tumor were obtained for MSP. In 19 patients, bone marrow (BM) aspirates were analyzed preoperatively for disseminated tumor cells by immunocytochemistry (mAb A45-B/B3). In 16 patients, biopsies of the anastomotic region were performed following PSA relapse. As a negative control GSTP1 methylation status was also assessed in LNs from 9 patients for whom an autopsy was performed for non-cancer-related causes. RESULTS: All primary tumors displayed GSTP1 hypermethylation (HM). Preoperative BM assessment showed disseminated tumor cells in 8/20 cases (40%). In 4 patients, biopsies of the vesico-urethral anastomosis showed local tumor recurrence. The LNs in the cancer patients showed GSTP1 HM in 18/20 cases (90%) versus 1/9 patients (11.1%) in the non-cancer cohort (p<0.0001). CONCLUSION: GSTP1 HM appears to be well suited for molecular staging of prostate cancer and accurately detects disseminated tumor cells in LNs, which was seen in 90% of the patients with PSA relapse. However, the limited number of patients and the finding of benign and malignant prostatic tissue at the vesico-urethral anastomosis as a putative local source of PSA recurrence does not allow us to draw conclusions on the prognostic significance of our findings, yet.     

Detection of disseminated tumor cells in the lymph nodes of colorectal cancer patients using a real-time polymerase chain reaction assay

Introduction Postoperative treatment for colorectal cancer depends on tumor stage as defined by the International Union Against Cancer (UICC). Adjuvant chemotherapy is not recommended in patients without lymph node involvement (UICC stages I and II). As many as 20–30% of these patients, however, will develop recurrence. Aims and objectives We conducted this study to determine the presence of disseminated tumor cells in the lymph nodes by quantitative real-time polymerase chain reaction (QRT-PCR) for cytokeratin 20 (CK20) in an attempt to provide supplementary information compared to histopathological findings. Materials and methods Using a standard QRT-PCR assay, we examined primary tumors and 391 lymph nodes from 31 patients with completely resected colorectal cancer. Results Of the 31 primary tumors, 29 were positive for CK20 by QRT-PCR. Discussion An examination of the lymph nodes from the 29 patients with CK20-positive primary tumors revealed that 35 (92.1% sensitivity) of the 38 histopathologically positive lymph nodes and 54 (16.7%) of the 324 histopathologically negative lymph nodes were positive by molecular analysis. CK20 expression was detected in 10 (100%) of 10 patients with a histopathologically positive lymph node status (pN1). In 9 (47.4%) of 19 patients with negative histopathological results (pN0), we detected a CK20 mRNA signal in at least one lymph node. Whereas eight patients with histopathologically negative lymph nodes could be upstaged on the basis of the molecular findings, no patient would be downstaged. Conclusion Our results suggest that QRT-PCR for CK20 is a useful tool for the quantitative detection of micrometastases in the regional lymph nodes. We introduce a standardized procedure that integrates a molecular diagnostic technique in the clinical staging.     

Cyclin D1 expression is predictive of occult metastases in head and neck cancer patients with clinically negative cervical lymph nodes

BACKGROUND: The aim of this study was to investigate the value of p53 and cyclin D1 gene expression in predicting the risk of occult lymph node metastases in patients with head and neck squamous cell carcinoma (HNSCC). METHODS: The expression of cyclin D1 and p53 was evaluated by means of immunohistochemical analysis in 32 HNSCC patients with clinically and radiologically negative lymph nodes in whom metastatic involvement was subsequently demonstrated at histologic examination (pN+). A group of 64 head and neck cancer patients with histologically negative laterocervical lymph nodes (pN0) was used as a control. RESULTS: Cyclin D1 and p53 expression were observed respectively in 42 (43.7%) and 48 cases (50%). Cyclin D1 expression significantly correlated with tumor extension and advanced clinical stage (p =.002 and p =.001, respectively). At univariate regression analysis, cyclin D1 expression significantly correlated with the presence of occult lymph node metastases (p =. 0007), and it remained an independent predictor at multivariate regression analysis (p =.0059). CONCLUSIONS: Our study indicates that the expression of cyclin D1 correlates with the presence of occult cervical metastases in head and neck carcinoma patients, thus suggesting that its immunohistochemical evaluation in biopsy samples may be used as an additional tool for identifying patients to be treated with elective neck dissection.     

Proliferation index as a prognostic marker in hemangiopericytoma of the head and neck

BACKGROUND: Hemangiopericytoma (HPC) of the head and neck is a rare neoplasm whose biologic behavior is difficult to predict by means of conventional histologic parameters. METHODS: H & E-stained sections from 12 cases of HPC were reviewed. Proliferation index was assessed using an immunoperoxidase stain for MIB-1 (Ki-67). RESULTS: The study group consisted of 4 adult men, 5 adult women, and 1 infant male. Necrosis, hypercellularity, and pleomorphism were found in 1, 5, and 6 case(s), respectively. The mitotic index per 10 high power fields varied from 0-1 to 15. Proliferation indices using MIB-1 ranged from 2.6% to 52.5%. Clinical follow-up revealed 3 cases with recurrence all possessing proliferation indices of approximately 10%. CONCLUSIONS: Standard histomorphologic features may be inadequate predictors of clinical outcome. A proliferation index of 10% or greater may indicate a more aggressive subset of HPC of the head and neck.     

Detection of occult tumor cells in lymph nodes from bladder cancer patients by MUC7 nested RT-PCR

OBJECTIVE: Systemic progression is the prevalent form of bladder tumor recurrence after radical cystectomy. The detection of occult bladder tumor cells in histopathologically normal lymph nodes could be of prognostic value. We examined the possibility that mucin 7 (MUC7) RNA might reflect the presence of occult tumor cells in lymph nodes from bladder cancer patients. We used the polymerase chain reaction (RT-PCR), a highly sensitive assay, to monitor MUC7 RNA. METHODS: We collected 240 pelvic lymph nodes from 25 bladder cancer patients undergoing radical cystectomy. We also obtained 20 lymph nodes from patients with prostate cancer and interstitial cystitis to use as negative controls. Each lymph node was divided in two parts to provide tissue for both histopathological and PCR analysis. RESULTS: 166/240 lymph nodes from bladder cancer patients were usable for MUC7 RT-PCR. By conventional histopathology, six of these nodes contained metastases. MUC7 RT-PCR analysis was positive for five of the six histologically proven lymph node metastases. Histopathological reevaluation of the sixth node revealed tumor in an adjacent vein, not in the lymph node, itself. In contrast, 46/160 (29%) histologically classified normal lymph nodes (pN0) from 17 bladder cancer patients were positive for MUC7. All 20 lymph nodes from control patients were MUC7-negative. CONCLUSION: MUC7 RT-PCR is a specific and sensitive method for the detection of occult tumor cells in lymph nodes from bladder cancer patients. Long-term observation will be necessary to evaluate the clinical value of MUC7 as a prognostic indicator of lymph node metastasis and disease progression.     

Unexpected lymph node pathology in neck dissection for head and neck cancer

The pathological findings in 62 patients with head and neck cancers who underwent neck dissection during a 12-month period are presented. Histological confirmation of metastatic disease was obtained in 46 cases (74%). In the remaining 16 cases (26%), there was no evidence of metastasis from the primary tumor. In 7 cases (11%), there was unexpected pathology in the cervical lymph nodes which was not related to the primary tumor. Accurate clinical staging of head and neck tumors is made increasingly more difficult in such cases where unrelated or dual lymph node pathology exists.     

Metastases to lymph nodes of the head and neck from an unknown primary site

Sixty-four patients with a metastasis to the head and neck from an unknown primary site were reviewed. Survival as a function of cell type, stage of disease, and mode of treatment was analyzed. Survival correlated best with stage of disease prior to treatment.