Prognostic impact of mRNA levels of LGR5 transcript variants in OSCC patients.

BACKGROUND: The human leucine-rich, repeat-containing G protein-coupled receptor 5 (LGR5) is a stem cell marker in numerous adult tissues and is overexpressed in a large number of human carcinoma including colon cancer, breast cancer and oral squamous cell carcinomas (OSCC). The role of the full length transcript (LGR5FL) in progression and prognosis of several cancers was reported. However, the biological function of three splice variants of LGR5 (LGR5Δ5, LGR5Δ8 and LGR5Δ5-8) has yet to be thoroughly investigated. METHODS: Seventy-eight frozen tumor samples from adult OSCC patients were studied using quantitative real-time TaqMan™ PCR analysis. The mRNA levels of full length LGR5, the splice variant of LGR5 lacking exon 5 (LGR5Δ5), the splice variant of LGR5 lacking exon 8 (LGR5Δ8) and the mRNA level of all known transcript variants together (LGR5all) were quantified and correlated to overall and disease-specific survival of OSCC patients, clinical parameters and the mRNA level of different tumor-associated markers. RESULTS: An elevated level of tumoral LGR5Δ5 mRNA, but not LGR5FL, LGR5Δ8 or LGR5all mRNA was significantly associated with a poor prognosis for the overall and disease-specific survival of OSCC patients (hazard ratio (HR) = 2.0; p = 0.02; 95% CI: 1.1-3.7; HR = 3.2; p = 0.01; 95% CI: 1.3-8.0; multivariable Cox regression), respectively. Additionally, a higher tumoral level of LGR5Δ5 mRNA in primary tumors was associated with the occurrence of regional lymph node metastases in OSCC patients (odds ratio (OR) = 3.1; p = 0.022; 95% CI: 1.2-7.9; binary logistic regression). Furthermore, the mRNA levels of all investigated LGR5 transcript variants were significantly correlated with the mRNA expression of Wnt-target genes and markers of epithelial-to-mesenchymal transition (EMT). CONCLUSION: The mRNA level of the LGR5 splice variant LGR5Δ5 is an independent negative prognostic marker for overall and disease-specific survival and metastasis in OSCC patients. Additionally, we suggest, all LGR5 transcript variants are involved in the EMT process mainly through activating the Wnt-signalling pathway.

Investigation of the Prognostic Role of Carbonic Anhydrase 9 (CAIX) of the Cellular mRNA/Protein Level or Soluble CAIX Protein in Patients with Oral Squamous Cell Carcinoma.

s: Carbonic anhydrase 9 (CAIX) is an important protein that stabilizes the extracellular pH value and is transcriptionally regulated by hypoxia-inducible factor 1 (HIF1), but more stable than HIF1α. Here we show a comparative study that examines the prognostic value of CA9 mRNA, CAIX protein of tumor cells and secreted CAIX protein for oral squamous cell carcinoma (OSCC) patients. Tumor samples from 72 OSCC patients and 24 samples of normal tissue were analyzed for CA9 mRNA levels. A total of 158 OSCC samples were stained for CAIX by immunohistochemistry and 89 blood serum samples were analyzed by ELISA for soluble CAIX protein content. Survival analyses were performed by Kaplan⁻Meier and Cox's regression analysis to estimate the prognostic effect of CA9/CAIX in OSCC patients. The CA9 mRNA and CAIX protein levels of tumor cells correlated with each other, but not with those of the secreted CAIX protein level of the blood of patients. ROC curves showed a significant (p < 0.001) higher mRNA-level of CA9 in OSCC samples than in adjacent normal tissue. Cox's regression analysis revealed an increased risk (i) of death for patients with a high CA9 mRNA level (RR = 2.2; p = 0.02), (ii) of locoregional recurrence (RR = 3.2; p = 0.036) at higher CA9 mRNA levels and (iii) of death at high CAIX protein level in their tumors (RR = 1.7; p = 0.066) and especially for patients with advanced T4-tumors (RR = 2.0; p = 0.04). However, the secreted CAIX protein level was only as a trend associated with prognosis in OSCC (RR = 2.2; p = 0.066). CA9/CAIX is an independent prognostic factor for OSCC patients and therefore a potential therapeutic target.

Coexpression of hypoxia-inducible factor-1α and glucose transporter-1 is associated with poor prognosis in oral squamous cell carcinoma patients.

AIMS: To study whether coexpression of the two hypoxia-related proteins hypoxia-inducible factor (HIF)-1α and glucose transporter (GLUT)-1 has prognostic relevance in oral squamous cell carcinomas (OSCCs). METHODS AND RESULTS: Eighty-two OSCC samples were analysed for expression levels of HIF-1α and GLUT-1 by immunohistochemistry. Protein expression was assessed with an immunoreactive score system, and the correlations between gene expression and both clinical and pathohistological parameters were examined. Overexpression of either GLUT-1 or HIF-1α was associated with poor disease-specific survival in OSCC patients. Multivariate Cox proportional-hazards regression analysis revealed that increased expression of HIF-1α was significantly associated with disease-specific survival (relative risk = 3.24, P = 0.024), as compared with the group with a low level of expression. Coexpression of HIF-1α and GLUT-1 was additively and significantly associated with adverse prognoses in patients with OSCC. Patients whose tumours had increased levels of expression of both HIF-1α and GLUT-1 were found to have a 5.13-fold increased risk of tumour-related death (P = 0.017). CONCLUSIONS: Coexpression of high levels of HIF-1α and GLUT-1 is significantly correlated with prognosis in OSCC patients, suggesting that the coexpression of these proteins can be used as both an early diagnostic and independent prognostic marker.

HIF-1alpha is a prognostic marker in oral squamous cell carcinomas.

The critical molecular regulator of hypoxia is the hypoxia-inducible factor 1 alpha (HIF-1alpha). The prognostic impact of this regulator protein in oral squamous cell carcinomas (OSCC) has not been comprehensively investigated. The aim of this study was to analyze the expression of HIF-1alpha in 82 patients with OSCC and to correlate it with their disease-specific survival. Immunohistochemical staining for HIF-1alpha was performed on 82 OSCC specimens using a standard immunoperoxidase technique. The expression of HIF-1alpha was correlated with poor disease-specific survival for OSCC patients. Patients with negatively or weakly HIF-1alpha-expressing tumors had a survival rate of 80%, whereas the survival decreased to only 33.6% in case of moderate or strong expression. In multivariate Cox regression analysis, we found a 3.5-fold increased risk of tumor-related death when HIF-1alpha was strongly expressed (p=0.016) compared to negative or weak expression of HIF-1alpha. We suggest HIF-1alpha is an independent prognostic marker in OSCC. Immunohistochemical detection of HIF-1alpha appears to be useful in the diagnosis of OSCC and to provide prognostic information in addition to TNM stage and histological grade.

P4HA1: A single-gene surrogate of hypoxia signatures in oral squamous cell carcinoma patients.

BACKGROUND AND PURPOSE: Hypoxia gene expression signatures are of high prognostic value for head and neck cancer patients. Recently, the prognostic information of a multiple-gene hypoxia signature was found to be provided by the mRNA level of P4HA1 alone (Tawk et al., 2016). Therefore, we studied the prognostic value of P4HA1 in an independent cohort of oral squamous cell carcinoma (OSCC) patients. MATERIAL AND METHODS: Frozen tumor samples of 118 adult OSCC patients were analysed for P4HA1 mRNA level by quantitative real-time TaqMan™ PCR analysis. Kaplan-Meier analysis and Cox's regression analysis were performed to characterize the prognostic impact of P4HA1 mRNA level in OSCC patients. RESULTS: The analyzed patient cohort was divided into four subgroups according to the quartiles of the P4HA1 mRNA levels. The highest intratumoral P4HA1 mRNA level was significantly correlated with a poor overall survival (RR = 2.2; P = 0.04) and an increased risk of locoregional recurrence (RR = 4.8; P = 0.02). In patients who received radiotherapy (n = 82) highest intratumoral P4HA1 mRNA level was significantly correlated with a poor overall survival (RR = 3.4; P = 0.01) and an increased risk of locoregional recurrence (RR = 10.3; P = 0.005). Moreover, significant correlations between the P4HA1 mRNA level and the mRNA level of several EMT and stem cell markers were found. CONCLUSIONS: A high P4HA1 mRNA level, as a single-gene surrogate of hypoxia, is an independent prognostic marker for the overall survival and locoregional recurrence of OSCC patients.

Long-term results and functional outcome after cervical evisceration in patients with thyroid cancer.

BACKGROUND: Surgical strategy in patients with thyroid cancer (TC) infiltrating the aerodigestive system is controversial. This study was undertaken to examine the long-term results of cervical evisceration (CE). PATIENTS AND METHODS: Since 1995, 14 consecutive patients with advanced TC underwent total laryngectomy (LE, n = 6) or esophagolaryngectomy (ELR, n = 8). Patients with unusual thyroid neoplasms or metastases to the thyroid (n = 3) were excluded. For esophageal reconstruction, free jejunal grafts (n = 6) and gastric tubes (n = 2) were used. RESULTS: Procedure-related morbidity and mortality were 42% and 14%, respectively. ELR was associated with a significant higher frequency of complications and reoperations compared with LE. Twelve-month and 30-month survival rates were 73% and 55%, respectively; 85% of the patients were satisfied with the surgical results. There were no long-term problems concerning food intake in the ELR patients. Two ELR patients were able to learn a substitutive voice. CONCLUSIONS: Cervical evisceration in patients with TC is associated with significant perioperative morbidity and mortality requiring careful patient selection. Regarding long-term survival, local tumor control, and patient's satisfaction, however, CE should be taken into account in suitable patients with advanced TC.

Detection of circulating tumor cells from renal carcinoma patients: experiences of a two-center study.

Approximately 30-40% of primary and localized renal cell carcinoma (RCC) will eventually become metastatic disease. Therefore, the detection and molecular characterization of circulating tumor cells (CTC) in RCC may have important prognostic and therapeutic implications. Venous blood samples were obtained from a total of 214 RCC patients before and after nephrectomy or during adjuvant immune chemotherapy in two urological centers. After density gradient centrifugation, the CD45-negative cell population was isolated from peripheral blood samples (BS) by a semi-automated immunomagnetic depletion procedure using the MACS technology. Enriched cell populations potentially containing CTC were stained for cytokeratin and evaluated by a trained pathologist. CTC were found in 105 out of 363 BS (29%) originating from 80 out of 214 patients (37%). The median tumor cell number was five (range 1-51) per BS, i.e. approximately one CTC was detectable per 2-3 ml peripheral blood after tumor cell enrichment. For a subpopulation, follow-up data indicate that 62% of the patients with CTC detection in the blood developed progressive disease with single or multiple distant metastases or died because of RCC within two years. Here we show that the standardized immunomagnetic depletion protocol is a powerful tool for detecting and isolating intact RCC-derived CTC. The occurrence and the quantity of CTC in RCC patients is an early disease event. Furthermore, the occurrence of CTC is correlated with an advanced tumor stage and seems to be associated with a more aggressive tumor phenotype.

Surgical treatment of bisphosphonate-associated osteonecrosis: Prognostic score and long-term results.

Over a century after the first synthesis of bisphosphonates (1897) and a decade (2003) since the initial report on bisphosphonate-related osteonecrosis of the jaw (ONJ), this osteopathy remains a serious clinical challenge. A single center longitudinal study (2005-2014) was carried out to prospectively characterize inpatients with manifest ONJ and to evaluate their outcomes. The data recorded were: medical history, bisphosphonate treatment, localization, imaging, treatment, histomorphological features, and complications. A prognostic score (modified UCONN-Score) was adopted to predict outcomes. Eighty patients were included (mean age 69.4 years; 40 male, 40 female). Breast cancer (n = 25), multiple myeloma (n = 16), and prostate cancer (n = 15) were the three most common malignancies; and cardiovascular disease (n = 31), diabetes mellitus (16), and renal disorders (6) were the most important comorbidities. The severity of ONJ was stage I in three patients, stage II in 37, and stage III in 40, being predominantly localized in the posterior mandible and needing gradual resection. The average duration of bisphosphonate treatment was 38.3 months. The typical histological aspects of ONJ were predominantly osteonecrosis, bone marrow fibrosis, and bacterial colonization (Actinomyces) with suppurative inflammation. Within the resected jawbone a primary malignancy was diagnosed in two cases. The overall success rate was 83.6% (follow-up 23.5 months), with a UCONN-Score ≥15 predicting unfavorable treatment results (OR = 5.2). The past decade has enhanced experience with ONJ treatment and knowledge about its pathogenesis, which seems to be a multistep process. This study demonstrates the importance of bone and multilayer soft tissue management, preferably as an early intervention. The UCONN-Score might help to assess individual prognosis in ONJ surgery and the potential benefit of an antiresorptive drug holiday. To our knowledge it is the first use of a prognostic score in ONJ surgery.

mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme.

Quantification of disseminated tumor cells in the bloodstream of patients with hormone-refractory prostate carcinoma undergoing cytotoxic chemotherapy.

Only very limited data are available on the presence of circulating tumor cells during cytotoxic chemotherapy for hormone-refractory prostate cancer. We analyzed 241 blood samples from 32 patients with hormone-refractory PCa under a chemotherapy schedule. The etoposide, estramustine phosphate and paclitaxel scheme as well as the mitoxantrone and prednisone schedule were used to treat patients with advanced prostate cancer. The pre-therapy serum PSA values were in the range from 1.4 ng/ml to 2,870.9 ng/ml (median 74.5 ng/ml). We isolated the CD45-negative cell population by immunomagnetic depletion from 16 ml of peripheral blood samples. These cells were stained for pan-cytokeratin and evaluated. Patients were observed for an average of 67 weeks (range 16-120). In 77 (32%) samples originating from 27 (84%) patients, tumor cells were detected at least once. Twenty of these patients had shown an initial response to therapy as indicated by a >/=50% decrease of the pre-therapy PSA value. Of these, 14 patients experienced a biochemical and/or a clinical progression. For 13 (93%) of them, circulating tumor cells were detectable during the time of PSA response, i.e. during the PSA decline and before a biochemical or clinical progression. However, we could not correlate the amount of circulating tumor cells with the observed PSA levels. This study demonstrates that circulating tumor cells are detectable during chemotherapy for hormone-refractory prostate cancer regardless of the degree of PSA response.

Isolation and enrichment of urologic tumor cells in blood samples by a semi-automated CD45 depletion autoMACS protocol.

The purpose of this study was to demonstrate the efficacy of an enrichment protocol for the detection of circulating carcinoma cells in the bloodstream of patients with various urologic cancers. Using 16-ml peripheral blood samples (BS) the mononuclear cells were isolated by density gradient centrifugation. The CD45 leukocyte depletion method based on a previous study was slightly modified and semi-automated by an immunomagnetic cell separation unit (autoMACS). Enriched tumor cells were analyzed on a single slide by cytokeratin (CK) immunocytochemistry. The number of recovered DU-145 prostate cancer cells in various spiking experiments was 70-88%. By the optimized tumor cell enrichment protocol 186 BS originated from 128 patients with different urologic cancers (60 prostate carcinoma, 34 bladder cancers, 24 renal cell carcinoma and 10 other tumors) were investigated before, during and after tumor surgery. In 59 BS from 52 patients on average 5 tumor cells were detected in each BS containing tumor cells. The median number of identified tumor cells was 8 cells per BS and patient. Tumor cells were found for the 3 tumor types with representative BS numbers in 29-39% of the investigated BS and in 38-53% of the affected patients. The detection rates increased in the order prostate carcinoma < renal cell carcinoma < bladder cancer. Surprisingly, in four bladder tumor cases with identified disseminated tumor cells in BS, the histopathological examination of the transurethral resection of bladder tumor specimens showed no evidence for tumor cells in situ but the affected patients had clinically known and histologically defined tumor residue or a bladder tumor recurrence during the follow-up. The semi-automated CD45 autoMACS depletion protocol for the enrichment and the detection of disseminated tumor cells in the peripheral bloodstream allows to study up to 20 BS per working day prospectively by one technician. The improved sensitivity and specificity might be of importance when applying the protocol to BS in future clinical studies.

Detection of circulating tumor cells in peripheral blood of patients with renal cell carcinoma correlates with prognosis.

PURPOSE: The aim of this study was to evaluate the clinical relevance of the presence of disseminated tumor cells in peripheral blood (so-called circulating tumor cells) for renal cell carcinoma patients. METHODS: Two hundred thirty-three peripheral blood samples from 154 renal cell carcinoma patients were investigated for the presence of disseminated tumor cells by autoMACS technique and immunocytochemical staining of cytokeratin. The frequency of circulating tumor cells was analyzed statistically for correlation with relevant clinical data. RESULTS: Two kinds of tumor cells were detected: those with expression of cytokeratin 8/18 (CK+) and cells without a detectable cytokeratin expression, which we called large blue-stained cells with a tumorlike morphology. After following the CD45 autoMACS depletion protocol, we identified circulating tumor cells in 96 (41%) of 233 peripheral blood samples, which originated from 81 (53%) of 154 renal cell carcinoma patients. A significant correlation between the detection of circulating tumor cells and positive lymph node status (P < 0.001; chi(2) test) and the presence of synchronous metastases at the time of primary tumor resection (P = 0.014; chi(2) test) was found. In a multivariate Cox's regression hazard model, presence of CK+ circulating tumor cells was significantly correlated with poor overall survival for renal cell carcinoma patients (relative risk, 2.3; P = 0.048). CONCLUSIONS: The presence of circulating tumor cells correlated to lymph node status and presence of synchronous metastases in renal cell carcinoma. It is important to evaluate CK+ and blue-stained tumor cells together to determine the role of circulating tumor cells in tumor behavior and disease progression. Detection of CK+ circulating tumor cells in peripheral blood is a significant and independent prognostic factor for renal cell carcinoma.

Single center experience in primary surgery for medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is a rare disease, and most studies are either based on small numbers or multicenter studies with their inherent difficulties. Since 1995, a total of 440 patients with MTC underwent surgery in our clinic. A primary operation was performed in 188 patients (43% of 440). In 60 patients, the primary operation was performed because of a germline RET mutation ("prophylactic surgery"). Most (84%, 158/188) of the patients had pathologic calcitonin levels. Notably, MTC was found in almost 10% (3/30) of patients with normal calcitonin levels. However, all patients with lymph node metastases (LNMs) had elevated calcitonin levels. Total thyroidectomy (TTx) was performed in all patients. Lymph node dissection (LND) was performed at various extensions: one-compartment LND in 35% (66/188), three-compartment LND in 31% (58/188), and four-compartment LND in 29% (22/188). In general, lymph node dissection increased the likelihood of complications. LNM and distant metastases (DM) correlated with the extent of the primary tumor (pT category). The presence of LNM ranged from 17% (pT1 tumor) to 100% (pT4 tumor), whereas the presence of DM ranged from 0% (pT1 tumor) to 81% (pT4 tumor). Biochemical cure (normal calcitonin levels) was obtained in 72% (137/188) of patients. All 60 patients undergoing prophylactic surgery (tumor stage pT0/pT1) were biochemically cured. In contrast, only 60% (77/128) of the remaining patients were cured. The data suggest that primary surgery should be scheduled as soon as possible to treat patients at a node-negative stage. In the case of normal basal and elevated stimulated calcitonin levels, TTx and cervicocentral LND is recommended. If the basal calcitonin level is elevated, LND should include the cervicolateral compartment.

Primary hyperparathyroidism, C-cell hyperplasia and papillary thyroid carcinoma in a patient with RET germline polymorphism S836S.

BACKGROUND: In most examined populations the RET germline polymorphism S836S is found in about 3.6% of the normal population but in about 9% of patients suffering from sporadic C-cell hyperplasia or medullary thyroid carcinoma. The polymorphism S836S is thought to be involved in the development of sporadic medullary thyroid carcinoma. CASE PRESENTATION: We report a 48-year-old woman suffering from primary hyperparathyroidism (parathormone 121-166 pg/ml, normal <72), bilateral diffuse and nodular C-cell hyperplasia (calcitonin after pentagastrin administration 156 pg/ml, normal <4.6), and papillary thyroid carcinoma. Two commercial analyses of RET did not reveal any germline mutation within the known hot spots. However, sequencing revealed the presence of the RET polymorphism S836S. Following total thyroidectomy and removal of two hyperplastic parathyroid glands parathormone decreased to 51 pg/ml and calcitonin was no longer detected. CONCLUSIONS: The pathogenetic importance of the RET polymorphism S836S is still obscure. However, according to the published overrepresentation of the RET polymorphism S836S in patients suffering from apparent sporadic medullary thyroid carcinoma, it is conceivable that it also plays a role in multiglandular endocrine disease.

Detection of disseminated tumor cells in peripheral blood of patients with breast cancer: correlation to nodal status and occurrence of metastases.

OBJECTIVE: Soon after breast cancer becomes invasive, it sheds cancer cells into the blood stream or the cancer cells are spread via lymphatic vessels. The early and unambiguous detection of these disseminated tumor cells (DTC) is of importance for the evaluation of the tumor process and for monitoring therapy response. The detection of disseminated tumor cells by immunocytochemistry (ICC) without previously performing tumor cell enrichment is time consuming and may miss a considerable part of these cells. Therefore, we have applied a negative immunomagnetic enrichment of disseminated tumor cells from the peripheral blood of patients with breast cancer by the depletion of CD45(+) leukocytes using automated magnetic activated cell separation (autoMACS). METHODS: One hundred twenty-five blood samples from 83 breast cancer patients were investigated for occurrence of disseminated tumor cells by autoMACS technique and immunocytochemical cytokeratin staining. Frequency of disseminated tumor cells was analyzed statistically for correlation to clinical data. RESULTS: Thirty-three of the 125 blood samples (26%) originating from 29 of 83 breast cancer patients (35%) carried cytokeratin positive (CK(+)) tumor cells. The occurrence of CK(+) tumor cells correlated significantly with the nodal status (P = 0.009) and with the occurrence of metastases at the time of primary tumor resection (P = 0.003). CONCLUSIONS: The finding that occurrence DTC detected in peripheral blood of breast cancer patients correlated with nodal stage and metastases is described for the first time. It suggests that disseminated tumor cells identified in peripheral blood by autoMACS are associated with tumor characteristics of breast cancer.