BRCA1/BRCA2 variants of uncertain significance in clinical practice: A case report.

The influence of BRCA1/2 variants of uncertain significance (VUSs) on the cancer risk and their association with the response to treatment is uncertain. The aim of the present study was to evaluate the role of BRCA VUS in patients with breast cancer. A total of two cases of breast cancer patients with the BRCA VUS were described. The complete coding sequence of BRCA1/2 genes was analyzed from the genomic DNA material by next generation sequencing on the Ion Torrent platform. The presence of c.3454G>A (p.Asp1152Asn) VUS in the BRCA1 gene was reported in a 64-year-old woman with invasive breast carcinoma. The characteristics of the breast tumors were the following: moderately differentiated-intermediate grade (NG-2 G-2), HER2 (+), estrogen receptor (ER) (+++), progesterone receptor (PR) (+++), luminal A subtype and pT2 N1a Mx. The second detected VUS was the c.2374T>C (p.Tyr792His) variant in the BRCA2 gene. This variant was reported in a 33-year-old woman who was diagnosed with right breast cancer (cT2N1M0). The invasive breast carcinoma was characterized as follows: NG-2 G-2, ER (+++), PR (+++), Ki-67 10%, HER2 (+++) and luminal B subtype. The data demonstrated that patients with VUSs should be managed based on their family history of cancer and clinicopathological characteristics. The clinical significance of the VUS in BRCA1/2 may change over time and reclassification of the variant to 'pathogenic' or 'benign' should be undertaken. Patients with VUS should be followed up regularly.

Circulating HPV16 DNA may complement imaging assessment of early treatment efficacy in patients with HPV-positive oropharyngeal cancer.

BACKGROUND: Early detection of treatment failure may improve clinical outcome and overall survival in patients with head and neck cancer after first-line treatment. Circulating cell-free HPV16 DNA (cfHPV16 DNA) was evaluated as a possible complementary marker to radiological assessment of early response in patients with HPV-related oropharyngeal cancer (OPC) after radiotherapy alone or combined with chemotherapy. METHODS: The study included 66 patients with HPV-related OPC receiving radical radiotherapy alone or in combination with chemotherapy. cfHPV16 DNA was assessed in the blood of all patients before treatment using TaqMan-based qPCR. Subsequent analysis of cfHPV16 DNA was performed 12 weeks after treatment completion, along with radiological assessment of early treatment results. RESULTS: Complete (CRR) and incomplete radiological response (IRR) was found in 43 (65%) and 23 (35%) patients respectively. cfHPV16 DNA was present in 5 (28%) patients with IRR, while only in 1 (4%) with CRR. Three of five patients with IRR that were positive for cfHPV16 DNA exhibited histopathologically confirmed local or regional treatment failure, and other two developed distant metastases. None of the patients with negative cfHPV16 DNA presented disease failure. CONCLUSION: The post-treatment assessment of cfHPV16 DNA in patients with HPV-related OPC may be used as a complementary biomarker to conventional imaging-based examinations for early identification of treatment failure.

Genetic Profiling of Advanced Melanoma: Candidate Mutations for Predicting Sensitivity and Resistance to Targeted Therapy.

BACKGROUND: Molecularly targeted therapy has revolutionized the treatment of advanced melanoma. However, despite its high efficiency, a majority of patients experience relapse within 1 year of treatment because of acquired resistance, and approximately 10-25% patients gain no benefit from these agents owing to intrinsic resistance. This is mainly caused by the genetic heterogeneity of melanoma cells. OBJECTIVE: We aimed to validate the predictive significance of selected genes in advanced melanoma patients before treatment with BRAF/MEK inhibitors. PATIENTS AND METHODS: Archival DNA derived from 37 formalin-fixed paraffin-embedded pre-treatment advanced melanoma samples of patients treated with targeted therapy was used for next-generation sequencing analysis using the Ion Torrent platform. The AmpliSeq Custom Panel comprised coding sequences or hot spots of 23 melanoma genes: ATM, BRAF, CDK4, CDKN2A, CTNNB1, EGFR, HOXD8, HRAS, IDH1, KIT, KRAS, MAP3K8, MAP2K1, MAP2K2, MITF, MYC, NF1, NRAS, PAX5, PIK3R1, PTEN, RAC1, and RB1. The sequences were evaluated for genomic alterations and further validated using Sanger sequencing. RESULTS: Our analysis revealed non-BRAF genetic alterations in 28 out of 37 samples (75.7%). Genetic changes were identified in PTEN, CDK4, CDKN2A, CTNNB1, EGFR, HOXD8, HRAS, KIT, MAP2K1, MAP2K2, MITF, MYC, NF1, PAX5, RAC1, and RB1. Fifteen known pathogenic mutations (single nucleotide variants or indels) and 11 variants of unknown significance were detected. Statistical analysis revealed an association between the presence of pathogenic mutations and time to progression during treatment with combination therapy. CONCLUSIONS: Pathogenic mutations identified by gene panel sequencing have potential predictive value for targeted therapy of melanoma and are worth further validation in a larger series of cases. The role of some known mutations (e.g. CDK4R24, PTEN c.801 + 1G > A, CTNNB1S45F) as well as variants of unknown significance identified in this study (e.g. MITFR316K, KITG498S) in the generation of resistance to BRAF/MEK inhibitors should be further investigated.

BRCA2 genemutation, c.2808_2811delACAA (p.Ala938Profs), in malebreastcancer - clinicopathologicalanalysisbased on a case report.

Male breast cancer (MBC) is a rare disease that occurs in ~0.2% of all neoplasms among men. The risk of developing MBC is higher in men with a BRCA2 genetic mutation (7%). The aim of this study was to evaluate the association between c.2808_2811del ACAA (p.Ala938Profs) BRCA2 mutation in MBC and clinicopathological factors. A 75-year-old patient was admitted to the Genetic Outpatient Clinic with a diagnosis of right breast cancer and with a family history of cancer (two daughters who were diagnosed with breast cancer at ages 46 and 38 years). Postoperative histopathological examination revealed tumor type pT2 N1a Mx, NST NG-3 G-3, Ki-67 (75%), HER2 (+), ER (+++), PR (+++), invasive carcinoma and luminal B subtype. The complete coding sequences of the BRCA1 and BRCA2 genes were analyzed for genomic DNA material using next generation sequencing on the Ion Torrent platform. The c.2808_2811delACAA (p.Ala938Profs) mutation was observed in the BRCA2 gene. The presence of the c.2808_2811delACAA (p.Ala938Profs) mutation in the BRCA2 gene was confirmed using the Sanger method. The same BRCA2 gene mutation was reported in one of the patient's daughters. The detected mutation causes the frameshift mutation, resulting in the creation of an additional translation termination codon and the synthesis of an abnormal protein. This mutation was associated with a later age of disease, a higher histological degree, a higher mitotic index, a positive steroid receptor status and the luminal B subtype HER2- breast cancer.

Detection of circulating HPV16 DNA as a biomarker in the blood of patients with human papillomavirus-positive oropharyngeal squamous cell carcinoma.

BACKGROUND: Development of biomarker analysis using the circulating cell-free DNA (cfDNA) methodology is a challenge for noninvasive cancer diagnosis. In this study, a comparison between the plasma and tumor tissue HPV16 DNA viral loads (VLs) has been presented. METHODS: Real-time polymerase chain reaction was performed for quantitating of HPV16 DNA in the plasma and tumor samples of patients with oropharyngeal cancer. RESULTS: Among the tissues, HPV16-positive patients with oropharyngeal squamous cell carcinoma, nonsmoking patients, displayed significantly higher HPV16 DNA VLs in their tissue. No smoking and advanced N disease were the most important predictors for cHPV16 DNA (circulating HPV16 DNA) detection. The cHPV16-positive women displayed significantly higher VLs in their tumor tissues compared to the men, although without notable impact on the blood detection. CONCLUSIONS: Many factors were responsible for human papillomavirus DNA circulation in blood. As a result of the small size of the analyzed group, some observed discrepancies need to be proven on a larger cohort.

Changes in Liver Gene Expression and Plasma Concentration of Rbp4, Fetuin-A, and Fgf21 in Sprague-Dawley Rats Subjected to Different Dietary Interventions and Bariatric Surgery.

PURPOSE: To study the effect of duodenal-jejunal omega switch (DJOS) in combination with different dietary patterns on the retinol-binding protein (RBP4), fetuin-A, and fibroblast growth factor 21 (FGF21) plasma levels and their hepatic gene expressions in rats. METHODS: A high-fat diet (HF) was given to 28 rats and 28 more were fed with a control diet (CD) for 2 months. After that, half of each group underwent either DJOS or SHAM surgery. For the next 2 months, half of the animals in each operation group were kept on the same diet as before and half of them had the diet changed. After 16 weeks of the experiment RBP4, fetuin-A, and FGF21 plasma levels as well as liver Rbp4, Ahsg, and Fgf21 gene expressions were measured. RESULTS: DJOS had a reductive impact on plasma levels of RBP4, fetuin-A, and FGF21 and Rbp4, Ahsg, and Fgf21 relative gene expression in the liver when compared to SHAM. The HF/HF group expressed significantly higher RBP4 and fetuin-A plasma levels in comparison to the control. The HF diet used before and/or after surgery led to upregulation of Rbp4, Ahsg, and Fgf21 relative gene expression. The lowest levels of analyzed parameters were observed in the CD/CD group. CONCLUSIONS: The efficiency of DJOS surgery, measured by hepatokines' plasma levels and their gene expressions in the liver, depends on the type of diet applied before and after surgery. Manipulation of dietary patterns can lead to marked improvements in metabolic profile after DJOS surgery.

Establishment and Characterization of the Novel High-Grade Serous Ovarian Cancer Cell Line OVPA8.

High-grade serous ovarian carcinoma (HGSOC) is the most frequent histological type of ovarian cancer and the one with worst prognosis. Unfortunately, the majority of established ovarian cancer cell lines which are used in the research have unclear histological origin and probably do not represent HGSOC. Thus, new and reliable models of HGSOC are needed. Ascitic fluid from a patient with recurrent HGSOC was used to establish a stable cancer cell line. Cells were characterized by cytogenetic karyotyping and short tandem repeat (STR) profiling. New generation sequencing was applied to test for hot-spot mutations in 50 cancer-associated genes and fluorescence in situ hybridization (FISH) analysis was used to check for TP53 status. Cells were analyzed for expression of several marker genes/proteins by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS), and immunocytochemistry (ICC). Functional tests were performed to compare OVPA8 cells with five commercially available and frequently used ovarian cancer cell lines: SKOV3, A2780, OVCAR3, ES2, and OAW42. Our newly-established OVPA8 cell line shows morphologic and genetic features consistent with HGSOC, such as epithelial morphology, multiple chromosomal aberrations, TP53 mutation, BRCA1 mutation, and loss of one copy of BRCA2. The OVPA8 line has a stable STR profile. Cells are positive for EpCAM, CK19, and CD44; they have relatively low plating efficiency/ability to form spheroids, a low migration rate, and intermediate invasiveness in matrigel, as compared to other ovarian cancer lines. OVPA8 is sensitive to paclitaxel and resistant to cisplatin. We also tested two FGFR inhibitors; OVPA8 cells were resistant to AZD4547 (AstraZeneca, London, UK), but sensitive to the new inhibitor CPL304-110-01 (Celon Pharma, Lomianki/Kielpin, Poland). We have established and characterized a novel cell line, OVPA8, which can be a valuable preclinical model for studies on high-grade serous ovarian cancer.

Vertically transmitted HPV-dependent squamous cell carcinoma of the external auditory canal : Case report of a child.

BACKGROUND: There is much evidence that high-risk human papillomavirus (HPV) plays a causative role in a subset of head and neck squamous cell cancer (HNSCC) in adults. HPV-positive tumors behave differently even in their response to treatment and are therefore a distinct subset. Both HPV-positive and HPV-negative tumors of the head and neck region are usually in the domain of adults and cases in children are rare; thus when a 2­year-old child was diagnosed with this cancer in the external auditory canal, an in-depth assessment of the tumor was considered necessary. CASE REPORT: A 2­year-old girl was born to a HPV-positive mother who was diagnosed with cervical cancer during pregnancy. The child was delivered by caesarean section and the mother died of her cancer 7 months after delivery. After the diagnosis of locally invasive HPV-positive squamous cell cancer of the external auditory canal, the child was treated surgically, and with chemotherapy and radiotherapy. Full remission was obtained lasting up to 325 weeks since treatment was started, resulting in over 6 years of disease-free survival. CONCLUSION: This is the first case of advanced, HPV-related HNSCC in a 2­year-old child, in whom the tumor was located in the external auditory canal and who made a dramatic recovery after treatment with nonradical surgery, chemotherapy and radiotherapy. The child has currently been disease free for 6 years. This case supports the observation that HPV-related HNSCC tumors appear to respond favorably to treatment despite the patient's young age and the clinically advanced stage of the tumor.

BRAFV600E-Associated Gene Expression Profile: Early Changes in the Transcriptome, Based on a Transgenic Mouse Model of Papillary Thyroid Carcinoma.

BACKGROUND: The molecular mechanisms driving the papillary thyroid carcinoma (PTC) are still poorly understood. The most frequent genetic alteration in PTC is the BRAFV600E mutation--its impact may extend even beyond PTC genomic profile and influence the tumor characteristics and even clinical behavior. METHODS: In order to identify BRAF-dependent signature of early carcinogenesis in PTC, a transgenic mouse model with BRAFV600E-induced PTC was developed. Mice thyroid samples were used in microarray analysis and the data were referred to a human thyroid dataset. RESULTS: Most of BRAF(+) mice developed malignant lesions. Nevertheless, 16% of BRAF(+) mice displayed only benign hyperplastic lesions or apparently asymptomatic thyroids. After comparison of non-malignant BRAF(+) thyroids to BRAF(-) ones, we selected 862 significantly deregulated genes. When the mouse BRAF-dependent signature was transposed to the human HG-U133A microarray, we identified 532 genes, potentially indicating the BRAF signature (representing early changes, not related to developed malignant tumor). Comparing BRAF(+) PTCs to healthy human thyroids, PTCs without BRAF and RET alterations and RET(+), RAS(+) PTCs, 18 of these 532 genes displayed significantly deregulated expression in all subgroups. All 18 genes, among them 7 novel and previously not reported, were validated as BRAFV600E-specific in the dataset of independent PTC samples, made available by The Cancer Genome Atlas Project. CONCLUSION: The study identified 7 BRAF-induced genes that are specific for BRAF V600E-driven PTC and not previously reported as related to BRAF mutation or thyroid carcinoma: MMD, ITPR3, AACS, LAD1, PVRL3, ALDH3B1, and RASA1. The full signature of BRAF-related 532 genes may encompass other BRAF-related important transcripts and require further study.

Human papillomavirus DNA in pharyngeal scrapes as a marker of HPV-related squamous cell cancer of the oropharynx.

BACKGROUND: Recent studies have indicated that human papillomavirus is an etiologic agent for a subset of head and neck cancers associated with better prognosis, therefore, prompt confirmation of such etiology seems to be crucial for choosing the optimal therapeutic option. Standard HPV diagnosis is currently based on histopathological material. In the present study, the novel diagnostic method based on pharyngeal brush biopsy is proposed. OBJECTIVES: The aim of this study was to evaluate the usefulness of the Real-Time PCR-based (RT-PCR) test in detecting HPV-related cancer of the oropharynx using superficial scraps taken from the oropharyngeal region. STUDY DESIGN: Ninety patients with head and neck squamous cell cancer were enrolled in the study. The presence of HPV DNA in pharyngeal superficial scrapes assessed by RT-PCR was compared to the HPV status in the tumor tissue samples determined by a combined RT-PCR/P16(INK4A) expression algorithm. Analytical sensitivity and specificity were calculated and the clinical outcome was analyzed in correlation to the HPV status. RESULTS: HR-HPV DNA in pharyngeal swabs was revealed in 25 cases (28.4%) and simultaneously confirmed in all corresponding tissue samples. Sensitivity and specificity of the viral status assessment in the brush biopsies in respect to the RT-PCR/P16(INK4A) 20 were 100% and 96.2%, respectively. HR-HPV positive status was associated with an excellent clinical outcome and reduced hazard ratio of recurrence and disease-related death. CONCLUSIONS: The proposed novel method of HPV status assessment using RT-PCR and superficial scraps appeared to be highly sensitive, specific, and useful in predicting the clinical outcome.

Potential role of human papilloma virus in the pathogenesis of gastric cancer.

AIM: To demonstrate the presence and biological activity of human papilloma virus (HPV) in gastric cancer (GAC) tissues. METHODS: The study involved 84 surgically treated patients with gastric adenocarcinoma, regardless of the clinical stage of the disease. The presence of HPV DNA of high oncogenic risk types in formalin-fixed, paraffin-embedded tumor samples was determined using quantitative polymerase chain reaction analysis. A stringent protocol of prevention of cross- and environmental contamination was applied during DNA isolation, and amplification, as well as confirmation of the biological activity of the virus in tumor cells, was implemented. The study utilized the Real-time High Risk HPV test, which detects the DNA of 14 HPV subtypes that are considered to have high oncogenic potential. The overexpression of the p16(INK4a) protein assessed immunohistochemically was considered confirmation of the HPV infection. RESULTS: Among the 89 patients initially included in the study group, diagnostic results were obtained for 84 individuals. In five cases, either the histopathological material was too scant to isolate the necessary amount of DNA, or the isolated DNA was significantly degraded, resulting in the failure of internal control amplification within the predefined number of 35 cycles. Those patients were excluded from further analysis. The amplification of HPV DNA was demonstrated in none of the 84 tissue samples; thus, all cases were considered to have a negative DNA status of highly oncogenic HPV subtypes. Immunohistochemical staining provided diagnostic results for all of the examined tissue samples, and excluded the accumulation of the p16(INK4a) protein in tumor cells, thus confirming the lack of active HPV infection in all of the individuals. CONCLUSION: The study does not confirm the presence or biological activity of HPV in tumor tissues. Thus, the relationship between GAC and HPV infection, in the Central European population seems doubtful.

PTEN expression profiles in colorectal adenocarcinoma and its precancerous lesions.

The aim of the study was to determine expression of the PTEN suppressor gene in colorectal adenocarcinoma and its precancerous lesions (adenomatous polyps) in correlation with common clinical and histopathological features. Forty-four patients with adenomatous polyps and 32 with primary adenocarcinoma of the colon or rectum were enrolled in the study. They underwent endoscopic removal of polyps or major surgery and postoperative adjuvant chemo- and radiotherapy depending on staging of the disease. No patient had received chemotherapy and/or radiotherapy before the surgery. PTEN expression was evaluated using immunohistochemical staining on paraffin-embedded specimens and compared to clinicopathological features of tumors. In colorectal cancers, PTEN expression was found to be significantly lower than in normal intestinal mucosa and adenomatous polyps. That was associated with complete loss of PTEN expression observed more frequently in colorectal cancer, contrary to reduction of PTEN expression occurring mostly in polyps. A correlation between polyp diameter and loss of PTEN was demonstrated as well as between tumor size and TNM advanced stage and PTEN expression. The obtained results suggest that the PTEN/PI3K/Akt pathway may play an important role in early stages of sporadic colorectal carcinogenesis and reduced PTEN expression in late oncogenesis is associated with some adverse clinical and pathological features.

HSPA2 overexpression protects V79 fibroblasts against bortezomib-induced apoptosis.

Human HSPA2 is a member of the HSPA (HSP70) family of heat-shock proteins, encoded by the gene originally described as testis-specific. Recently, it has been reported that HSPA2 can be also expressed in human somatic tissues in a cell-type specific manner. The aim of the present study was to find out whether HSPA2 can increase the resistance of somatic cells to the toxic effect of heat shock, proteasome inhibitors, and several anticancer cytostatics. We used a Chinese hamster fibroblast V79 cell line because these cells do not express the HSPA2 and cytoprotective HSPA1 proteins under normal culture conditions and show limited ability to express HSPA1 in response to heat shock and proteasome inhibitors. We established, by retroviral gene transfer, a stable V79/HSPA2 cell line, which constitutively overexpressed HSPA2 protein. The major observation of our study was that HSPA2 increased long-term survival of cells subjected to heat shock and proteasome inhibitors. We found, that HSPA2 confers resistance to bortezomib-induced apoptosis. Thus, we showed for the first time that in somatic cells HSPA2 can be a part of a system protecting cells against cytotoxic stimuli inducing proteotoxic stress.

PTEN as a prognostic and predictive marker in postoperative radiotherapy for squamous cell cancer of the head and neck.

BACKGROUND: Tumor suppressor PTEN is known to control a variety of processes related to cell survival, proliferation, and growth. PTEN expression is considered as a prognostic factor in some human neoplasms like breast, prostate, and thyroid cancer. METHODOLOGY/PRINCIPAL FINDINGS: In this study we analyzed the influence of PTEN expression on the outcome of a randomized clinical trial of conventional versus 7-days-a-week postoperative radiotherapy for squamous cell cancer of the head and neck. The patients with cancer of the oral cavity, oropharynx, and larynx were randomized to receive 63 Gy in fractions of 1.8 Gy given 5 days a week (CF) or 7 days a week (p-CAIR). Out of 279 patients enrolled in the study, 147 paraffin blocks were available for an immunohistochemical assessment of PTEN. To evaluate the prognostic value of PTEN expression and the effect of fractionation relative to PTEN, the data on the outcome of a randomized clinical trial were analyzed. Tumors with a high intensity of PTEN staining had significant gain in the loco-regional control (LRC) from p-CAIR (5-year LRC 92.7% vs. 70.8%, for p-CAIR vs. CF, p = 0.016, RR = 0.26). By contrast, tumors with low intensity of PTEN did not gain from p-CAIR (5-year LRC 56.2% vs. 47.2%, p = 0.49, RR = 0.94). The intensity of PTEN highly affected the LRC in a whole group of 147 patients (5-year LRC 80.9% vs. 52.3% for high vs. low PTEN, p = 0.0007, RR = 0.32). In multivariate Cox analysis, including neck node involvement, EGFR, nm23, Ki-67, p53, cyclin D1, tumor site and margins, PTEN remained an independent predictor of LRC (RR = 2.8 p = 0.004). CONCLUSIONS/SIGNIFICANCE: These results suggest that PTEN may serve as a potent prognostic and predictive marker in postoperative radiotherapy for high-risk squamous cell cancer of the head and neck.

Analysis of PTEN expression in large intestine polyps and its relation to the recognized histopathological and clinical risk factors for cancer development in this location.

AIM OF THE STUDY: PTEN is an important gene whose protein product is double specific phosphatase holding key regulatory functions in sending signals from membrane receptors for growth factors into the cell downstreams. Its participation, mainly by PI3K/AKT signaling pathway in the pathomechanism of many malignant cancers was unambiguously confirmed. The PTEN function gets disturbed on many levels and for various reasons. Disorders of PTEN protein expression seem to be even more common in many carcinomas. The aim of the study is to enquire the meaning of PTEN expression in the cancer transformation process in large intestine glandular polyps. MATERIAL AND METHODS: The group includes 40 patients, 21 men and 19 women, age median 64 years (51-83) qualified to endoscopic removal of large intestine polyp. Tissue material obtained during polyp removal endoscopy was immediately fixed in 4% buffered formalin solution with the mixture of phosphatase activity inhibitors (PhosStop Roche). Time of fixation 24-48 h. After fixation, the material was embedded in paraffin. PTEN visualization was based on specific rabbit monoclonal antibodies (Cell Signaling). The expression of PTEN protein in large intestine and rectum polyps was marked by a semi-quantitative method and an attempt to correlate the results with the acknowledged clinical and histopathological malignancy risk factors was undertaken. RESULTS: Loss or weakening of protein expression was found in 45% cases. Moreover, the relationship between polyp diameter and a loss of PTEN expression was proved. The received results can indicate a significant participation of PTEN gene in early oncogenesis stages of large intestine cancer.

NF-κB signaling pathway is inhibited by heat shock independently of active transcription factor HSF1 and increased levels of inducible heat shock proteins.

NF-κB transcription factor regulates numerous genes important for inflammation, immune responses and cell survival. HSF1 is the primary transcription factor activated under stress conditions that is responsible for induction of genes encoding heat shock proteins. Previous studies have shown that the NF-κB activation pathway is blocked by heat shock possibly involving heat shock proteins. Here, we investigate whether active HSF1 inhibited this pathway in the absence of stress conditions. Activation of the NF-κB pathway and expression of NF-κB-dependent genes were analyzed in TNFα-stimulated U-2 OS human osteosarcoma cells that were either heat-shocked or engineered to express a constitutively active form of HSF1 in the absence of heat shock. As expected, heat shock resulted in a general blockade in the degradation of the IκBα inhibitor, nuclear translocation of NF-κB and expression of NF-κB-dependent target genes. In marked contrast, the presence of constitutively active HSF1 did not block TNFα-induced activation of the NF-κB pathway or expression of a set of the NF-κB-dependent genes. We conclude that in the absence of heat shock, the NF-κB activation pathway is inhibited by neither active HSF1 transcription factor nor by increased levels of HSF1-induced heat shock proteins.

Differential expression of HSPA1 and HSPA2 proteins in human tissues; tissue microarray-based immunohistochemical study.

In the present study we determined the expression pattern of HSPA1 and HSPA2 proteins in various normal human tissues by tissue-microarray based immunohistochemical analysis. Both proteins belong to the HSPA (HSP70) family of heat shock proteins. The HSPA2 is encoded by the gene originally defined as testis-specific, while HSPA1 is encoded by the stress-inducible genes (HSPA1A and HSPA1B). Our study revealed that both proteins are expressed only in some tissues from the 24 ones examined. HSPA2 was detected in adrenal gland, bronchus, cerebellum, cerebrum, colon, esophagus, kidney, skin, small intestine, stomach and testis, but not in adipose tissue, bladder, breast, cardiac muscle, diaphragm, liver, lung, lymph node, pancreas, prostate, skeletal muscle, spleen, thyroid. Expression of HSPA1 was detected in adrenal gland, bladder, breast, bronchus, cardiac muscle, esophagus, kidney, prostate, skin, but not in other tissues examined. Moreover, HSPA2 and HSPA1 proteins were found to be expressed in a cell-type-specific manner. The most pronounced cell-type expression pattern was found for HSPA2 protein. In the case of stratified squamous epithelia of the skin and esophagus, as well as in ciliated pseudostratified columnar epithelium lining respiratory tract, the HSPA2 positive cells were located in the basal layer. In the colon, small intestine and bronchus epithelia HSPA2 was detected in goblet cells. In adrenal gland cortex HSPA2 expression was limited to cells of zona reticularis. The presented results clearly show that certain human tissues constitutively express varying levels of HSPA1 and HSPA2 proteins in a highly differentiated way. Thus, our study can help designing experimental models suitable for cell- and tissue-type-specific functional differences between HSPA2 and HSPA1 proteins in human tissues.

Impact of HPV infection on the clinical outcome of p-CAIR trial in head and neck cancer.

The purpose of the study was to analyse the influence of HPV infection on the outcome of a randomized clinical trial of conventional (CF) versus 7-days-a-week postoperative radiotherapy (p-CAIR) for squamous cell cancer of the head and neck (SCCHN). Between 2001 and 2004, 279 patients with high-risk SCC of the larynx or cancer of the oral cavity/oropharynx were randomized to receive 63 Gy in fractions of 1.8 Gy given 5 days a week or 7 days a week (Radiother Oncol 87:155-163, 2008). The presence of HPV DNA in 131 archival paraffin blocks was assessed with multiplex quantitative real-time PCR using five consensus primers for the conservative L1 region and molecular beacon probes targeting 14 high-risk HPV subtypes. Following the RT-PCR procedure, we could determine the presence and type of HPV16, HPV18 and the other 12 less frequent oncogenic subtypes. Out of 131 samples, 9 were positive for HPV infection (6.9%), all of them with HPV16 subtype. None of the 65 laryngeal tumours was HPV positive. The 5-year LRC in HPV-positive patients was 100%, compared to 58% in the HPV-negative group (p = 0.02, log-rank test). Amongst 122 patients with HPV-negative tumours, 5-year LRC was 50.3% in p-CF versus 65.2 in p-CAIR (p = 0.37). HPV infection was associated with low expression of EGFR and cyclin D. This study demonstrates a favourable outcome for HPV-positive patients with SCCHN treated with postoperative radiotherapy. While considering the small number of HPV+ tumours, the data set can be considered as hypothesis generating only, the outcome raises new questions on the necessity of aggressive postoperative treatment in HPV+ patients.

High-risk HPV DNA status and p16 (INK4a) expression as prognostic markers in patients with squamous cell cancer of oral cavity and oropharynx.

AIM OF THE STUDY: To determine and compare the influence of the HPV DNA status and p16(INK4a) expression on the outcome of postoperative radiotherapy for squamous cell cancer of the oral cavity or oropharynx. MATERIAL AND METHODS: 59 patients with high-risk squamous cell cancer of the oral cavity or oropharynx were enrolled. They underwent major surgery and postoperative radiotherapy. The HPV DNA status and p16(INK4a) expression were assessed with QPCR and immunohistochemistry and correlated with loco-regional control and overall survival. RESULTS: 15.3% of tissue samples were HPV positive. All positive patients were identified with HPV16 subtype infection, and no other subtypes of high-risk HPV were detected. 5-year LRC in HPV(+) patients was 100%, compared to 50% in the HPV(-) group. 17.9% of all samples had evident p16(INK4a) expression. Among HPV(+) cases, 55.6% showed p16(INK4a) expression. 5-year LRC in patients with p16(INK4a) expression was 89%, compared to 51% in the p16(INK4a) negative group but this tendency was not significant (p = 0.055). CONCLUSION: These data show that the HPV status is a good predictor of loco-regional control and overall survival in patients treated with radical surgery and adjuvant radiotherapy. The study shows a strong correlation between high-risk HPV infection and p16(INK4a) expression, but detection of viral DNA with QPCR has stronger prognostic potential.

The expression pattern of the 70-kDa heat shock protein Hspa2 in mouse tissues.

The highest expression level of a 70-kDa heat shock protein family member Hspa2 is detected specifically in meiotic and post-meiotic male germ cells, which is reflected by original name of this protein, i.e., testis-specific Hsp70. However, this chaperon protein could be also detected in certain somatic tissues. Here, the extra-testicular expression pattern of mouse Hspa2 was analyzed. We found expression of Hspa2 in various epithelial cells including lining of bronchioles and oviduct, columnar epithelium of endometrium, epithelial reticular cells of thymus, transitional epithelium of the urinary bladder, or ependymal cells covering walls of the ventricular system of the brain. Surprisingly, Hspa2 was a putative secretory protein in intestine, endometrial glands and subcommissural organ. Hspa2 was detected in central and peripheral nervous system: in neuron's bodies and fiber tracts, in the subventricular zone of the lateral ventricles, in the dentate gyrus of the hippocampus, in enteric ganglia of the gastrointestinal tract. Hspa2 was also expressed in smooth muscles and at low level in immune system (in germinal centers associated with B-lymphocyte production). In addition to somatic tissues listed above, Hspa2 was detected in oocytes arrested at diplotene of the first meiotic division.