Tumour Recurrence, Depth of Invasion, and Temple Location as Independent Prognostic Parameters of Lymph Node Metastases of Head and Neck Cutaneous Squamous Cell Carcinomas.

The excellent survival rate of cutaneous squamous cell carcinoma (cSCC) exceeding 90% is reduced by the presence of nodal metastases by over 50%. We analysed various risk parameters of cSCC to predict the incidence of nodal metastases. A total of 118 patients with the head cSCC were included in a single-institution retrospective study covering the period from 2008 to 2020. Tumour recurrence, temple location, and tumour infiltration depth were found to be independent predictors of nodal metastases (increasing the probability of metastases by 8.0, 8.1, and 4.3 times, respectively). Furthermore, univariate analysis shows that the tumour size and T stage are significant factors increasing the risk of metastases. Several independent risk factors for the development of metastases in the head cSCC have been confirmed. These findings might help identify at-risk patients who require additional attention for adequate radical treatment and close follow-up. In contrast, elective treatment of lymph nodes is not recommended due to the low incidence of regional metastases.

Parotid Gland Metastases of Cutaneous Squamous Cell Carcinoma of the Head: Occult Metastases Occurrence and Their Late Manifestation.

Cutaneous squamous cell carcinomas (cSCC) are malignant tumours with excellent prognosis unless nodal metastases develop. The aim of our study is to determine the prognostic significance of the clinical stage of parotid gland metastases and the incidence of occult cervical lymph node involvement in cSCC of the head. Our retrospective analysis includes 39 patients with cSCC parotid gland metastases, 15 of whom had concurrent cervical node involvement. In 32 patients, the lymph nodes manifested at stage N3b. A total of 26 patients were treated with parotidectomy, 9 patients received radiotherapy alone, and 4 received symptomatic therapy. The surgical treatment included either total conservative (21 cases) or superficial parotidectomy (5 cases) and neck dissection (therapeutic neck dissections in 11 cases and elective in 14 cases). In all cases, surgery was performed with sufficient tumour-free resection margins. Adjuvant radiotherapy was administered postoperatively in 16 patients. Occult metastases were present in 21% of cases after an elective neck dissection, but not in any case in the deep lobe of the parotid gland. The five-year overall survival and recurrence-free interval were 52% and 55%, respectively. Patients with the cN3b stage and G3 histological grade tend to have a worse prognosis, but not at a statistically significant level. The prognosis was not worse in patients with concurrent parotid and cervical metastases compared to those with metastases limited to the parotid gland only. The addition of adjuvant irradiation, in comparison to a single modality surgical treatment, was the only statistically significant prognostic factor that reduced the risk of death from this diagnosis (p=0.013). The extent of parotidectomy (partial vs. total) had no impact on either the risk of recurrence or patient prognosis. The combination of surgery with irradiation provides the best results and should be applied to all patients who tolerate the treatment. A partial superficial parotidectomy should be sufficient, with a minimum risk of occult metastasis in the deep lobe. Conversely, the relatively high incidence of occult neck metastases indicates that patients could likely benefit from elective neck dissection.

DNA repair in head and neck tumor cells and possibilities of its monitoring to estimate individual tumor radioresistance and selection of optimal primary treatment.

In order to maximize post-therapeutic quality of life, radio(chemo)therapy becomes preferred over surgery in head-and-neck tumor (HNT) treatment. However, the therapy selection is only based on the clinical experience and patient's preferences as the radiosensitivity markers remain unknown. New possibilities of deciding on the best primary therapy, moving us towards personalized medicine based on quantifiable biomarkers, have been opened by studies on DNA radiation damage and repair in individual patients tumors. Together with the importance of radiotherapy in HNT oncology, we discuss here our preliminary results revealing the existence of several HNT groups with respect to genome stability and repair ability of tumor cells after irradiation. Monitoring of the formation and disappearance of γH2AX/53BP1 foci in tumor cell primo-cultures derived from individual patients suggests that DNA repair capacity of the identified groups correlates with the tumor cell radiosensitivity. Our findings thus improve understanding of HNT biology; nevertheless, the relationship between the repair groups and in vivo response of tumors to radiotherapy must be further studied. Since most HNTs do not suffer from repair defects, although their viability varies after irradiation, pre-therapeutic tests covering the full spectrum of HNT radiosensitivity causes will require the use of a combination of multiple, still undiscovered biomarkers.

Prognostic impact of combined immunoprofiles in oropharyngeal squamous cell carcinoma patients with respect to AJCC 8th edition.

OBJECTIVES: To examine combined immunoprofiles of epidermal growth factor receptor (EGFR), CD44, and p16 in oropharyngeal squamous cell carcinoma (OPSCC) and to correlate them with radiotherapy treatment outcomes and clinicopathological parameters. Prognostic impact of the American Joint Committee on Cancer (AJCC) 8th edition staging system in comparison with 7th edition was analyzed. METHODS: The study included 77 OPSCC patients treated by definitive intensity-modulated radiotherapy (IMRT). Clinical staging was assessed according to the AJCC, both 7th and 8th edition. Immunohistochemical (IHC) analysis of CD44 and EGFR was performed on primary biopsy tumor tissues. To evaluate the HPV status, IHC detection of p16 was employed. RESULTS: The AJCC 8th edition staging system revealed correlations between overall survival (OS), progression-free survival (PFS), locoregional control (LRC), and clinical stage. EGFR and CD44 positivity (+) and p16 negativity (-) were associated with clinical stage IV of the disease. CD44+ and EGFR+ OPSCC displayed worse OS and LRC, and these cases also showed the worst 3-year OS and LRC. Combined analysis of protein expressions identified an association between p16- and EGFR+, p16- and CD44+, EGFR+, and CD44+. Combined immunoprofiles CD44+/p16-, EGFR+/p16-, and EGFR+/CD44+ were associated with worst OS and LRC. CONCLUSIONS: Combined immunoprofiles of p16, EGFR, and CD44 might provide valuable prognostic and predictive information for the individual OPSCC patients, especially in terms of response to IMRT and prediction of treatment outcomes. Application of the AJCC 8th edition staging for HPV+ OPSCC proved to improve hazard discrimination and prognostication of OPSCC.

Differential expression of homologous recombination DNA repair genes in the early and advanced stages of myelodysplastic syndrome.

BACKGROUND: The high incidence of mutations and cytogenetic abnormalities in patients with myelodysplastic syndrome (MDS) suggests that defects in DNA repair mechanisms. We monitored DNA repair pathways in MDS and their alterations during disease progression. METHODS: Expression profiling of DNA repair genes was performed on CD34+ cells, and paired samples were used for monitoring of RAD51 and XRCC2 gene expression during disease progression. Immunohistochemical staining for RAD51 was done on histology samples. RESULTS: RAD51 and XRCC2 showed differential expression between low-risk and high-risk MDS (P<.0001), whereas RPA3 was generally decreased among the entire cohort (FC=-2.65, P<.0001). We demonstrated that RAD51 and XRCC2 expression gradually decreased during the progression of MDS. Down-regulation of XRCC2 and RAD51 expression was connected with abnormalities on chromosome 7 (P=.0858, P=.0457). Immunohistochemical staining revealed the presence of RAD51 only in the cytoplasm in low-risk MDS, while in both the cytoplasm and nucleus in high-risk MDS. The multivariate analysis identified RAD51 expression level (HR 0.49; P=.01) as significant prognostic factor for overall survival of patients with MDS. CONCLUSIONS: Our study demonstrates that the expression of DNA repair factors, primarily RAD51 and XRCC2, is deregulated in patients with MDS and presents a specific pattern with respect to prognostic categories.

Addictive drugs, arrhythmias, and cardiac inward rectifiers.

In many addictive drugs including alcohol and nicotine, proarrhythmic effects were reported. This review provides an overview of the current knowledge in this field (with a focus on the inward rectifier potassium currents) to promote the lacking data and appeal for their completion, thus, to improve understanding of the proarrhythmic potential of addictive drugs.

Expression profiles of miR-29c, miR-200b and miR-375 in tumour and tumour-adjacent tissues of head and neck cancers.

Altered expression of microRNAs (miRNAs) has been shown in many types of malignancies including the head and neck squamous cell carcinoma (HNSCC). Although there are many new and innovative approaches in the treatment of HNSCC, a clear marker of this disease is still missing. Three candidate miRNAs (miR-29c-3p, miR-200b-5p and miR-375-3p) were studied in connection with HNSCC using quantitative real-time PCR expression levels in 42 tissue samples of HNSCC patients and histologically normal tumour-adjacent tissue samples of these patients. Primary HNSCC carcinoma tissues can be distinguished from histologically normal-matched noncancerous tumour-adjacent tissues based on hsa-miR-375-3p expression (sensitivity 87.5 %, specificity 65 %). Additionally, a significant decrease of hsa-miR-200b-5p expression was revealed in tumour-adjacent tissue samples of patients with node positivity. Lower expression of hsa-miR-200b-5p and hsa-miR-29c-3p in HNSCC tumour tissue was associated with higher tumour grade. Consequently, survival analysis was performed. Lower expression of hsa-miR-29c-3p in tumour-adjacent tissue was associated with worse overall and disease-specific survivals. Lower expression of miR-29c-3p in tumourous tissue was associated with worse relapse-free survival. hsa-miR-375-3p seems to be a relatively promising diagnostic marker in HNSCC but is not suitable for prognosis of patients. Furthermore, this study highlighted the importance of histologically normal tumour-adjacent tissue in HNSCC progress (significant decrease of hsa-miR-200b-5p expression in tumour-adjacent tissue of patients with node positivity and low expression of hsa-miR-29c-3p in HNSCC tumour-adjacent tissue associated with worse prognosis).

Effect of HPV on tumor expression levels of the most commonly used markers in HNSCC.

Approximately 90 % of head and neck cancers are squamous cell carcinomas (HNSCC), and the overall 5-year survival rate is not higher than 50 %. There is much evidence that human papillomavirus (HPV) infection may influence the expression of commonly studied HNSCC markers. Our study was focused on the possible HPV-specificity of molecular markers that could be key players in important steps of cancerogenesis (MKI67, EGF, EGFR, BCL-2, BAX, FOS, JUN, TP53, MT1A, MT2A, VEGFA, FLT1, MMP2, MMP9, and POU5F). qRT-PCR analysis of these selected genes was performed on 74 biopsy samples of tumors from patients with histologically verified HNSCC (22 HPV-, 52 HPV+). Kaplan-Meier analysis was done to determine the relevance of these selected markers for HNSCC prognosis. In conclusion, our study confirms the impact of HPV infection on commonly studied HNSCC markers MT2A, MMP9, FLT1, VEGFA, and POU5F that were more highly expressed in HPV-negative HNSCC patients and also shows the relevance of studied markers in HPV-positive and HPV-negative HNSCC patients.

Prognostic significance of the tumour-adjacent tissue in head and neck cancers.

Even with significant advances in operative skills and adjuvant therapies, the overall survival of patients suffering with head and neck squamous cancers (HNSCC) is unsatisfactory. Accordingly, no clinically useful prognostic biomarkers have been found yet for HNSCC. Many studies analysed the expression of potential markers in tumour tissues compared to adjacent tissues. Nevertheless, due to the sharing of the same microenvironment, adjacent tissues show molecular similarity to tumour tissues. Thus, gene expression patterns of 94 HNSCC tumorous tissues were compared with 31 adjacent tissues and with 10 tonsillectomy specimens of non-cancer individuals. The genes analysed at RNA level using quantitative RT-PCR and correlated with clinico-pathological conditions were as follows: EGF, EGFR, MKI67, BCL2, BAX, FOS, JUN, TP53, VEGF, FLT1, MMP2, MMP9, MT1A and MT2A. The elevated MT2A, BAX, EGF and JUN expression was associated with the influence of tumour cells on the rearrangement of healthy tissues, as well as a significant shift in the BAX/BCL2 ratio. Our investigation also indicated that adjacent tissues play an important role in cancerogenesis by releasing several tumour-supporting factors such as EGF. A gradual increase in the metallothionein expression, from the lowest one in tonsillectomy samples to the highest ones in tumour samples, suggests that MT expression might be tissue reaction to the presence of tumour cells. The results of this study confirmed the significance of metallothionein in tumori-genesis and gave evidences for its use as a potential HNSCC biomarker. Furthermore, this study highlighted the importance of histologically normal tumour-adjacent tissue in prediction of HNSCC progress.

Elective parotidectomy and neck dissection are not beneficial in cutaneous squamous cell carcinoma of the head.

OBJECTIVE: Cutaneous Squamous Cell Carcinoma (cSCC), a tumor with a significantly increasing incidence, is mostly diagnosed in the head region, where tumors have a worse prognosis and a higher risk of metastases. The presence of metastases reduces specific five-year survival from 99% to 50%. As the risk of occult metastases does not exceed 10%, elective dissection of the tributary parotid and neck lymph nodes is not recommended. METHODS: We retrospectively analyzed a group of 12 patients with cSCC of the head after elective dissections of regional (parotid and cervical) nodes by means of superficial parotidectomy and selective neck dissection. RESULTS: We diagnosed occult metastases neither in the cervical nor parotid nodes in any patient. None were diagnosed as a regional recurrence during the follow-up period. CONCLUCION: Our negative opinion on elective parotidectomy and neck dissection in cSCC of the head is in agreement with the majority of published studies. These elective procedures are not indicated even for tumors showing the presence of known (clinical and histological) risk factors for lymphogenic spread, as their positive predictive value is too low. Elective parotidectomy is individually considered as safe deep surgical margin. If elective parotidectomy is planned it should include only the superficial lobe. Completion parotidectomy and elective neck dissection are done in rare cases of histologically confirmed parotid metastasis in the parotid specimen. Preoperatively diagnosed parotid metastases without neck involvement are sent for total parotidectomy and elective selective neck dissection. Cases of clinically evident neck metastasis with no parotid involvement, are referred for comprehensive neck dissection and elective superficial parotidectomy. The treatment of concurrent parotid and cervical metastases includes total conservative parotidectomy and comprehensive neck dissection. LEVEL OF EVIDENCE: How common is the problem? Step 4 (Case-series) Is this diagnostic or monitoring test accurate? (Diagnosis) Step 4 (poor or non-independent reference standard) What will happen if we do not add a therapy? (Prognosis) Step 4 (Case-series) Does this intervention help? (Treatment Benefits) Step 4 (Case-series) What are the COMMON harms? (Treatment Harms) Step 4 (Case-series) What are the RARE harms? (Treatment Harms) Step 4 (Case-series) Is this (early detection) test worthwhile? (Screening) Step 4 (Case-series).

Primary Squamous Cell Carcinoma of the Parotid Gland: Study and Review of the Literature.

BACKGROUND/AIM: Primary squamous cell carcinoma of the parotid gland (pPSCC) is a rare tumor, accounting for less than 3% of all parotid carcinomas. There is no general consensus on the management of this tumor, since clinical experience for pPSCC is minimal. Moreover, pPSCC is often misdiagnosed for metastatic cutaneous carcinoma. In our study, we focused on evaluating its biological and clinical characteristics, treatment results and prognosis. We proposed an update on diagnostic and therapeutic management of pPSCC. PATIENTS AND METHODS: The retrospective cohort included 18 patients diagnosed and treated for pPSCC in three tertiary head and neck centers between 2008 and 2022. We retrospectively evaluated their prognosis and established a therapeutic recommendation after analyzing our own and previously published data. RESULTS: Fourteen of 18 tumors were diagnosed in stage IV. Five-year overall survival was 36 months. Six patients received palliative therapy. Twelve patients underwent parotidectomy, neck dissection, and adjuvant radiotherapy. Remission was achieved in 8 patients (follow-up interval 3-56 months). One patient died with recurrent disease. The others are alive and in complete remission. CONCLUSION: The definitive diagnosis of pPSCC must meet the histological and clinical criteria. First of all, the metastatic origin of the tumor must be excluded. Five-year survival of this very aggressive tumor does not exceed 50%. Without surgery, the prognosis is poor. The best results, irrespective of tumor stage, are achieved with surgery. Therefore, a total parotidectomy, neck dissection (therapeutic or elective) and adjuvant radiotherapy are indicated for all resectable tumors.

Current status and future perspectives of oral HPV testing in the diagnosis and monitoring of oropharyngeal cancer. A review.

HPV16 status in oropharyngeal cancer (OPC) is an important prognostic factor. Its determination, based on immunistochemical analysis of p16 oncoprotein requires an invasive biopsy. Thus, alternative methods are being sought. Determining oral HPV16 status appears to be a promising alternative. However, it is not used routinely. This prompted us to perform a systematic literature review enabling us to evaluate the diagnostic and predictive ability of this approach. Thirty-four relevant studies were finally selected. For determination of HPV status in OPC, the calculated average sensitivity and specificity for oral sampling was 74% and 91%, respectively, with p16 tumour tissue marker being the gold standard. The method appears to be valuable in monitoring treatment response as well as the biological activity of the tumour, enabling early detection of persistent or relapsing carcinoma sufficiently long before its clinical and/or radiological manifestation. It can also contribute to identification of the primary tumour in cases of metastases of unknown origin. Last but not least, the screening HPV oral testing would help to identify individuals with persistent HPV oral infection who are at increased risk of development of OPC.

High perioperative level of oxidative stress as a prognostic tool for identifying patients with a high risk of recurrence of head and neck squamous cell carcinoma.

BACKGROUND: The aim of the study was to investigate the relationships among (a) glutathione peroxidase (GPx) and malondialdehyde (MDA); (b) oncological characteristics (i.e., TNM classification, tumor grade), and; (c) prognosis of head and neck squamous cell carcinoma. METHODS: In a prospective cohort study, we followed 88 patients for 67.4 months (median 40.3) after surgery for head and neck squamous cell carcinoma. Activity of GPx was determined by ELISA and plasma MDA concentration by liquid chromatography. RESULTS: Lower GPx activity was observed in the T3/4 patients than in the T1/2 group. Tumor grade was significantly correlated with both GPx (P = 0.001) and MDA (P = 0.05, both Spearman). The perioperative level of MDA was higher in patients who later recurred during the follow-up period (n = 15) than in the complete remission group (P = 0.01, Mann-Whitney). Median disease-free interval and overall survival in the group with MDA > median were 29.5 and 32.0 months, respectively, and 38.4 and 40.3 months in the patient group with MDA ≤ median (P = 0.10 and P = 0.08, respectively; Kaplan-Meier). Patients with MDA levels higher than the median had a more than twofold greater risk of recurrence than patients with MDA levels smaller than the median (31.3 vs. 15.2%, P = 0.06, logrank). CONCLUSION: Our results suggest that an increased MDA level at the time of initial surgery is found in patients with a high risk of recurrence, which suggests that each patient can be categorized according to risk of recurrence based on their MDA level at the time of initial surgery.

Salivary microRNAs identified by small RNA sequencing as potential predictors of response to intensity-modulated radiotherapy in head and neck cancer patients.

PURPOSE: Progress in radiation therapy of head and neck squamous cell carcinomas (HNSCCs) is logically linked to the development of molecular predictors that would help to enhance individually tailored treatment. MicroRNA (miRNA) expression profiles in tumors have repeatedly been tested to optimize the molecular diagnostics of HNSCC. In addition to tumor tissues, miRNAs are stably present in body fluids, including saliva, and can thus be collected non-invasively. The aim of our current study was to evaluate whether salivary miRNAs have potential as response predictors in HNSCC patients treated with intensity modulated radiation therapy (IMRT). METHODS: In total 48 HNSCC patients treated by definitive IMRT were enrolled in our prospective study. To identify predictive salivary miRNAs, we used small RNA sequencing in 14 saliva samples of HNSCC patients and qRT-PCR validation of selected miRNA candidates in an independent set of 34 patients. RESULTS: We found that salivary miR-15a-5p and miR-15b-5p exhibited differential levels between patients with and without complete remission (p = 0.025 and p = 0.028, respectively). Subsequent Kaplan-Meier analysis confirmed that patients with higher levels of miR-15a-5p reached a significantly longer locoregional progression-free survival (LPFS) than those with low levels (p = 0.024). Finally, multivariate Cox regression analysis revealed that miR-15a-5p may serve as an independent predictive biomarker of LPFS in HNSCC patients treated with IMRT (HR 0.104; 95% CI 0.004-0.911; p = 0.04). CONCLUSIONS: We conclude that salivary miR-15a-5p may represent a potential biomarker for individualized treatment decision-making in HNSCC patients.

Elective parotidectomy and neck dissection are not beneficial in cutaneous squamous cell carcinoma of the head

Abstract Objective Cutaneous Squamous Cell Carcinoma (cSCC), a tumor with a significantly increasing incidence, is mostly diagnosed in the head region, where tumors have a worse prognosis and a higher risk of metastases. The presence of metastases reduces specific five-year survival from 99% to 50%. As the risk of occult metastases does not exceed 10%, elective dissection of the tributary parotid and neck lymph nodes is not recommended. Methods We retrospectively analyzed a group of 12 patients with cSCC of the head after elective dissections of regional (parotid and cervical) nodes by means of superficial parotidectomy and selective neck dissection. Results We diagnosed occult metastases neither in the cervical nor parotid nodes in any patient. None were diagnosed as a regional recurrence during the follow-up period. Conclucion Our negative opinion on elective parotidectomy and neck dissection in cSCC of the head is in agreement with the majority of published studies. These elective procedures are not indicated even for tumors showing the presence of known (clinical and histological) risk factors for lymphogenic spread, as their positive predictive value is too low. Elective parotidectomy is individually considered as safe deep surgical margin. If elective parotidectomy is planned it should include only the superficial lobe. Completion parotidectomy and elective neck dissection are done in rare cases of histologically confirmed parotid metastasis in the parotid specimen. Preoperatively diagnosed parotid metastases without neck involvement are sent for total parotidectomy and elective selective neck dissection. Cases of clinically evident neck metastasis with no parotid involvement, are referred for comprehensive neck dissection and elective superficial parotidectomy. The treatment of concurrent parotid and cervical metastases includes total conservative parotidectomy and comprehensive neck dissection. Level of evidence How common is the problem? Step 4 (Case-series) Is this diagnostic or monitoring test accurate? (Diagnosis) Step 4 (poor or non-independent reference standard) What will happen if we do not add a therapy? (Prognosis) Step 4 (Case-series) Does this intervention help? (Treatment Benefits) Step 4 (Case-series) What are the COMMON harms? (Treatment Harms) Step 4 (Case-series) What are the RARE harms? (Treatment Harms) Step 4 (Case-series) Is this (early detection) test worthwhile? (Screening) Step 4 (Case-series)

MicroRNA-15b-5p Predicts Locoregional Relapse in Head and Neck Carcinoma Patients Treated With Intensity-modulated Radiotherapy.

BACKGROUND/AIM: Head and neck cancers are a heterogenous group of epithelial tumors represented mainly by squamous cell carcinomas (HNSCC), which are the sixth most common type of cancer worldwide. Surgery together with radiotherapy (RT) is among the basic treatment modalities for most HNSCC patients. Various biomarkers aiming to predict patients' response to RT are currently investigated. The reason behind this effort is, on one hand, to distinguish radioresistant patients that show weak benefit from RT and, on the other hand, reduce the ionizing radiation dose in less aggressive radiosensitive HNSCC with possibly less acute or late toxicity. MATERIALS AND METHODS: A total of 94 HNSCC patients treated by definitive intensity-modulated radiotherapy were included in our retrospective study. We used a global expression analysis of microRNAs (miRNAs) in 43 tumor samples and validated a series of selected miRNAs in an independent set of 51 tumors. RESULTS: We identified miR-15b-5p to be differentially expressed between patients with short and long time of locoregional control (LRC). Kaplan-Meier analysis confirmed that HNSCC patients with higher expression of miR-15b-5p reach a significantly longer locoregional relapse-free survival compared to patients expressing low levels. Finally, multivariable Cox regression analysis revealed that miR-15b-5p is an independent predictive biomarker of LRC in HNSCC patients (HR=0.25; 95% CI=0.05-0.78; p<0.016). CONCLUSION: miR-15b-5p represents a potentially helpful biomarker for individualized treatment decisions concerning the management of HNSCC patients.

One-step detection of human papilloma viral infection using quantum dot-nucleotide interaction specificity.

Due to the close relationship between carcinogenesis and human papillomavirus (HPV), and since they are transmitted via huge number of asymptomatic carriers, the detection of HPV is really needed to reduce the risk of developing cancer. According to the best of our knowledge, our study provides the very first method for one-step detection of viral infection and if it has initiated the subsequent cancer proliferation. The proposed novel nanosystem consists of magnetic glass particles (MGPs), which were attached with DNA probe on their surface to hybridize with target DNAs. The MGP-probe-DNA hybrid was finally conjugated with CdTe/ZnSe core/shell quantum dots (QDs). The proposed detection system is based on a novel mechanism in which the MGPs separate out the target DNAs from different biological samples using external magnetic field for better and clear detection and the QDs give different fluorescent maxima for different target DNAs due to their ability to interact differently with different nucleotides. Firstly, the method was optimized using HPV genes cloned into synthetic plasmids. Then it was applied directly on the samples from normal and cancerous cells. After that, the real hospital samples of head and neck squamous cell carcinoma (HNSCC) with or without the infection of HPV were also analyzed. Our novel nano-system is proved successful in detecting and distinguishing between the patients suffering by HPV infection with or without subsequent cancer having detection limit estimated as 1.0 x 109 (GEq/mL). The proposed methodology is faster and cost-effective, which can be applied at the clinical level to help the doctors to decide the strategy of medication that may save the life of the patients with an early treatment.

Expression of CD44, EGFR, p16, and their mutual combinations in patients with head and neck cancer: Impact on outcomes of intensity-modulated radiation therapy.

BACKGROUND: Progress in radiation treatment of head and neck squamous cell carcinoma (HNSCC) deserves the studies focused on molecular predictors that would help to enhance individually tailored treatment. METHODS: p16/epidermal growth factor receptor (EGFR)/cluster of differentiation-44 (CD44) was immunohistochemically analyzed in 165 HNSCC patients. RESULTS: In the entire group and the p16 negative cohort, better 3-year overall survival and locoregional control correlated with p16 positivity, CD44, and EGFR negativity were observed. Combined analysis revealed the worst results in the CD44+/p16-, EGFR+/p16-, and EGFR+/CD44+ groups and in the EGFR+/CD44+ within p16 negative cohort. Multivariate analysis found tumor stage, Karnofsky index, p16, and CD44 as prognostic factors of overall survival and clinical stage, and p16 as a prognostic factor for locoregional control. Clinical stage and Karnofsky index affected overall survival and tumor stage. EGFR affected locoregional control in the p16 negative subgroup. CONCLUSION: Our study confirmed the negative effect of CD44 and EGFR and the positive effect of p16 on radiotherapy results.

Establishment of oral squamous cell carcinoma cell line and magnetic bead-based isolation and characterization of its CD90/CD44 subpopulations.

In this study, we describe the establishment of the human papillomavirus 18-positive, stage II, grade 1, T2N0M0 head and neck tumor primary cell line derived from oral squamous cell carcinoma of a non-smoking patient by using two different protocols. Furthermore, a preparation of subpopulations derived from this primary cell line according to the cluster of differentiation molecules CD44/CD90 status using magnetic bead-based separation and their characterization was performed. Impedance-based real-time cell analysis, enzyme-linked immunsorbant assay (ELISA), wound-healing assay, flow-cytometry, gene expression analysis, and MTT assay were used to characterize these four subpopulations (CD44+/CD90-, CD44-/CD90-, CD44+/CD90+, CD44-/CD90-). We optimised methodics for establishement of primary cell lines derived from oral squamous cell carcinoma tissue samples and subsequent separation of mesenchymal (CD90+) and epithelial (CD90-) types of tumorous cells. Primary cell line prepared by using trypsin proteolysis was more viable than the one prepared by using collagenase. According to our results, CD90 separation is a necessary step in preparation of permanent tumor-tissue derived cell lines. Based on the wound-healing assay, CD44+ cells exhibited stronger migratory capacity than CD44- subpopulations. CD44+ subpopulations had also significantly higher expression of BIRC5 and SOX2, lower expression of FLT1 and IL6, and higher levels of basal autophagy compared to CD44- subpopulations. Furthermore, co-cultivation experiments revealed that CD44-/CD90+ cells supported growth of epithelial tumor cells (CD44+/CD90-). On the contrary, factors released by CD44+/CD90+ type of cells seem to have rather inhibiting effect. The most cisplatin-resistant subpopulation with the shortest doubling time was CD44-/CD90+, but this subpopulation had a low migratory capacity.