Circulating HPV16 DNA in Blood Plasma as Prognosticator and Early Indicator of Cancer Recurrence in Radio-Chemotherapy for Anal Cancer.

BACKGROUND: Implementation of anal squamous cell carcinoma (ASCC) treatment modifications requires reliable patient risk stratification. The circulating tumor-related human papillomavirus type 16 (ctHPV16) may play a role in predicting survival or assessing treatment response. METHODS: The study included 62 ASCC patients treated with chemoradiotherapy. A threshold of 2.5 was used to determine the maximum standardized uptake value (SUVmax). The ctHPV16 viral load (VL) was quantified by qPCR. RESULTS: In the multivariate Cox analysis, lower SUVmax (p = 0.047) and ctHPV16-positive (p = 0.054) proved to be independent prognostic factors for favorable overall survival (OS). In the subgroup with the higher SUVmax, ctHPV16 and nodal (N) status were independent prognostic factors with p = 0.022 for ctHPV16 and p = 0.053 for N. The best survival rate (95%) presented ctHPV16-positive/N-negative patients. High ctHPV16 VL tended to be slightly specific for patients younger than 63 years (p = 0.152). The decrease in ctHPV16 VL to undetectable level after the end of treatment correlated with the overall clinical response. CONCLUSIONS: A prognostic stratification by SUVmax, ctHPV16 and N-positive status allows consideration of more aggressive treatment in high-risk patients (those with high SUVmax, ctHPV16-negative, and N-positive) or de-intensification of therapy in low-risk patients (those with low SUVmax, ctHPV16-positive and N-negative). However, prospective clinical trials on a large group are needed.

Quantitative analysis of plasma DNA in anal cancer patients.

Introduction: The availability and non-invasiveness of circulating cell-free DNA (cfDNA) opens up new possibilities for real-time serial testing. The relationship between cfDNA concentration, clinical factors and suitability for monitoring was analyzed in patients with newly diagnosed anal squamous cell carcinoma (ASCC). Material and methods: Blood samples were collected at several points during and after treatment. Patients were homogeneously treated with chemoradiotherapy. Results: The concentration of cfDNA strongly correlated with the tumor volume (r = 0.9, p = 0.00006) and number of neutrophils (r = 0.706, p = 0.0069). Monitoring of cfDNA levels during treatment showed an increase after initiation of therapy, a peak at the end of treatment with significantly higher values for advanced than in T1/T2 tumors, and a decrease (T3/T4) during follow-up. However, neither the concentration of cfDNA before treatment nor its changes correlated with the response to chemoradiotherapy. There was no association between baseline cfDNA levels and T, N, age and gender. Conclusions: Substantial changes in plasma cfDNA content can be observed after chemoradiotherapy for ASCC. However, the small number of cases studied makes it difficult to assess the usefulness of the cfDNA test.

Significance of HPV16 Viral Load Testing in Anal Cancer.

Human papilloma virus (HPV) is highly frequent among patients with anal squamous cell carcinoma, but the viral load (VL) differs between patients. This study aimed to compare the rate of HPV positivity, HPV16VL, p16INK4A and p53 expression between treatment naive and recurrent anal cancer patients. HPV was genotyped via AmpliSens® HPV HCR-genotype-titre-FRT kit. HPV16 VL was determined via quantitative polymerase chain reaction-based in-house test. p16INK4A and p53 expression was tested via immunohistochemistry. The cohort comprised 13 treatment-naive and 17 recurrent anal SCC patients. High-risk HPV was detected in 87% of cases, and HPV16 (73%) was the predominant genotype. The rate of HPV positivity was higher among women and nonsmokers, and majority of HPV-positive cases were also p16INK4A-positive. All p53-negative tumors were HPV16-positive. The most predominant p53 staining pattern in the HPV-positive group was scattered type, whereas it was diffuse type in the HPV-negative group. The HPV16 VL was higher in the treatment-naive group. Further, in the treatment-naive group, cases with scattered staining pattern of p53 had higher HPV16 VL than cases with diffuse staining pattern. The opposite result was noted in the recurrent cancer group. Moreover, p16-positive cases with scattered p53 staining pattern in the treatment naive group had higher HPV16 VL than their counterparts in the recurrent cancer group. In conclusion, the HPV VL, as is the association between VL and p16INK4A /p53, is in an inversed trend in treatment naive and recurrent cancer patients, highlighting the importance of HPV VL measurement in anal SCC.

Assessment of the total cfDNA and HPV16/18 detection in plasma samples of head and neck squamous cell carcinoma patients.

OBJECTIVES: The advantages of the circulating cell-free DNA (cfDNA) methodology are quick results and the possibility of repeated analysis. The main aim of our study was to establish the relationship of the total cfDNA with patients' clinical characteristics and circulating HPV DNA detection in the blood of patients with head and neck squamous cell carcinoma (HNSCC). METHODS: The cfDNA level of 200 HNSCC patients in plasma was quantified using TaqMan-based TERT amplification. TaqMan technology was also used for HPV16/18 detection. Additionally, mutations in KRAS and EGFR were investigated. RESULTS: A higher level (p=0.011) of the total cfDNA was found in patients with oropharyngeal squamous cell carcinoma (OPSCC) (9.60 ± 6.23 ng/ml) in comparison with other HNSCC (7.67 ± 4.44 ng/ml). The level of cfDNA in patients with clinical N2-N3 disease (9.28 ± 6.34 ng/ml) was (p=0.015) higher than in patients with a clinical N0-N1 disease (7.50 ± 3.69 ng/ml). It was also higher in patients with stage IV (9.16 ± 6.04 ng/ml) compared with stages I-III of cancer (7.26 ± 3.63 ng/ml) (p=0.011). Analysis of HPV16/18 in plasma revealed that 14% of patients were HPV-positive, the majority of whom had the type HPV16 (96.4%). CfDNA level was comparable in HPV-positive and HPV-negative HNSCC patients, as well in the OPSCC subgroup. Somatic mutations in EGFR and KRAS were not found. CONCLUSIONS: A high level of cfDNA is specific for patients with OPSCC. HPV detection in cfDNA does not depend on the cfDNA concentration. Our results prove the diagnostic potential of plasma-based HPV cfDNA tests for the early detection and monitoring of HPV-positive HNSCC.

HSPA2 is expressed in human tumors and correlates with clinical features in non-small cell lung carcinoma patients.

BACKGROUND/AIM: It has been shown that HSPA2 protein, a testis-enriched member of HSPA/HSP70 family, is important for cancer cell growth and metastasis. However, the status of HSPA2 expression in tumors and its clinical/prognostic significance are obscure. Herein we aimed to investigate the expression of HSPA2 in various types of tumors and to determine the possible clinical and prognostic significance of HSPA2 in non-small cell lung carcinoma (NSCLC). MATERIALS AND METHODS: Tissue microarrays and postoperative NSCLC tumors were tested for HSPA2 by immunohistochemistry. RESULTS: HSPA2 is expressed in the majority of tumor histotypes. In NSCLC patients (n=85), nuclear HSPA2 expression was associated with histology, TNM staging and prognosis. High HSPA2 expression was significantly related to shorter overall survival (OS) in stage I-II patients. In multivariate analysis, high HSPA2, together with stage IIIA and male sex, were associated with shorter OS in the whole group. CONCLUSIONS: As exemplified in NSCLC the status of HSPA2 in human tumors may have certain prognostic significance.

The HspA2 protein localizes in nucleoli and centrosomes of heat shocked cancer cells.

The human HSPA2 gene, which belongs to the HSP70 family of heat shock genes, is a counterpart of rodent testis-specific HspA2 gene. Rodent genes are expressed mainly in pachytene spermatocytes, while transcripts of human HSPA2 gene have been detected in various normal somatic tissues, albeit translation of the messenger RNA into corresponding protein has not been yet unambiguously demonstrated, except for several cancer cell lines. The aim of our work, a first step in search for HspA2 function in cancer cells, was to establish its intracellular localization at physiological temperature and during heat shock. First, we used qRT-PCR and a highly specific antibody to select cell lines with the highest expression of the HspA2 protein, which turned out to be A549 and NCI-H1299 lines originating from non-small cell lung carcinoma (NSCLC). Significant expression of the HspA2 was also detected by immunohistochemistry in primary NSCLC specimens. Intracellular localization of the HspA2 was studied using both the specific anti-HspA2 polyclonal antibody and transfection of cells with fusion proteins HspA2-EGFP and mRFP-HspA2. We found that, at physiological temperature, the HspA2 was localized primarily in cytoplasm whereas, during heat shock, localization shifted to nucleus and nucleoli. Moreover, we demonstrate that in heat-shocked cells HspA2 accumulated in centrosomes. Our results suggest that the HspA2, like Hsp70 protein, can be involved in protecting nucleoli and centrosomes integrity in cancer cells subjected to heat shock and, possibly, other cellular stressors.

Stress proteins HSP27 and HSP70i predict survival in non-small cell lung carcinoma.

BACKGROUND: The expression pattern of stress (heat shock) proteins (HSPs) in cancer cells is frequently different from that observed in normal cells; most often some stress-inducible HSPs are constitutively and highly expressed. The objective of this study was to determine the prognostic significance of stress proteins HSP70i and HSP27 in non-small cell lung carcinoma (NSCLC). MATERIALS AND METHODS: An immunohistochemical procedure that enables unambiguous detection of HSP70i protein was used. RESULTS: Strong HSP70i staining showed a survival advantage, although multivariate analysis did not confirm this result. There was an evident correlation between HSP27 overexpression and survival of patients and the results were confirmed by multivariate analysis: 70% of patients with HSP27-negative tumors died within one year after the surgery. CONCLUSION: Our data suggest that HSP27 and HSP70i positivity may represent a favorable prognostic factor in NSCLC.

Heat shock transcription factor 1 down-regulates spermatocyte-specific 70 kDa heat shock protein expression prior to the induction of apoptosis in mouse testes.

Expression of constitutively active heat shock transcription factor 1 (HSF1) in mouse spermatocytes induces apoptosis and leads to male infertility. We report here that prior to the onset of massive apoptosis caused by expression of active HSF1 in spermatocytes a marked reduction in spermatocyte-specific Hsp70.2 mRNA and protein levels occurs. In addition, HSP70.2 protein relocalizes from a predominant cytoplasmic to a nuclear position in developing spermatocytes that express active HSF1. Later in the developmental stages, cells undergoing HSF1-induced apoptosis essentially lack the HSP70.2 protein. The down-regulation of Hsp70.2 gene expression by HSF1 is paradoxical because HSF1 is the prototypical activator of HSP genes. Furthermore, HSF1-mediated repression neither involved a heat shock element (HSE)-like sequence adjacent to the Hsp70.2 gene nor were Hsp70.2 promoter sequences associated directly with HSF1. Interestingly, other spermatocyte- and spermatid-specific transcripts are also down-regulated in testes of transgenic mice expressing active HSF1, suggesting involvement of a putative HSF1-dependent block of development of spermatogenic cells. Importantly however, transcription of the Hsp70.2 gene is down-regulated in testes of wild-type mice subjected to a hyperthermia that induces transient activation of HSF1, indicating that the spermatocyte-specific activity of HSF1 might misdirect a network of transcription factors required for proper regulation of Hsp70.2.

Inducible 70 kDa heat shock protein does not protect spermatogenic cells from damage induced by cryptorchidism.

Accumulation of inducible heat shock proteins (e.g. Hsp70i) during cellular stress confers thermotolerance, reduces the consequences of damage and facilitates cellular recovery, while abrogation of Hsp70i expression renders sensitivity to apoptosis. Testis translocation into abdominal cavity, which results in temperature elevation, does not induce expression of the Hsp70i proteins. Despite constitutive expression of testis-specific Hsp70 proteins, spermatocytes are very sensitive to damage at elevated temperatures. To test whether Hsp70i protein could protect testes from heat-induced damage, we have engineered transgenic mice that over-express this protein selectively in spermatocytes and spermatids. We demonstrate that the testes of cryptorchid transgenic mice, like those of wild-type mice, exhibit reduced weight and smaller sizes of their seminiferous tubules, disorganization of their germinal epithelium structures, appearance of multinucleated giant cells, and reduced populations of germ cells. The data show that constitutive expression of Hsp70i does not protect the seminiferous epithelium against cryptorchidism-induced damage.

Immunohistochemical detection of the inducible heat shock protein hsp70: a methodological study.

Stress-inducible Hsp70i and constitutively expressed Hsc70 are highly related heat shock proteins. Aberrant expression levels and intracellular localization of these proteins has been suggested as a potential marker in certain tumors. The aim of our study was to work out a reliable, immunohistochemical detection of the stress-inducible Hsp70i protein and enabling discrimination between Hsp70i and Hsc70 proteins in paraffin-embedded human tissues. We tested the effect of several fixative procedures and antigen retrieval on the effectiveness of the Hsp70i detection in murine cells cultured in vitro and in liver of rats subjected to heat shock. For cells grown in vitro, specific Hsp70i immunoreactivity was obtained with all fixatives used. However, samples fixed in 10% formalin and 4% paraformaldehyde required antigen retrieval. In liver tissue embedded in paraffin, regardless the fixative used, positive Hsp70i staining could be visible only if antigen retrieval was applied. We applied this procedure for detection of Hsp70i in routine sections of breast and lung cancers fixed with 10% formalin and found that the application of thermal antigen retrieval significantly enhanced the SPA810 immunoreactivity and reduced background staining. This procedure enabled also the differential detection of Hsp70i and Hsc70 in routine histopathological preparations.

Intronic polymorphism (1541-1542delGT) of the constitutive heat shock protein 70 gene has functional significance and shows evidence of association with lung cancer risk.

Somatic mutations of 11q23.3-linked constitutive heat shock protein 70 gene (HSPA8 alias HSC70) are detected by others in breast carcinomas. To examine whether intragenic, somatic mutations of HSPA8 occur in lung carcinomas, we sequenced its exons 2-8, with adjacent intronic sequences, in a series of DNA samples from non-small-cell lung cancers (NSCLC). Twenty-one polymorphisms were detected, but no somatic mutation. However, we observed an association between the HSC70 1541-1542delGT genotype and the immunohistochemical staining pattern of HSC70 protein. Tumors with weak (+) HSC70 protein staining were more frequent in the carriers of the polymorphic 1541-1542delGT allele than in the homozygotes of the major allele (20% vs. 6%, P=0.05 by Fisher's exact test). This statistically significant association prompted us to test the polymorphism functionally. The method we developed for the functional evaluation of intronic sequence alterations showed that the HSPA8 intron 2 with the deleted GT dinucleotide was associated with noticeable (approximately 20%) and statistically significant (P=0.005) reduction of the reporter gene activity. Our case-control analysis showed that the 1541-1542delGT heterozygous genotype was associated with significantly decreased risk for lung cancer (crude odds ratio (OR)=0.44; 95% confidence interval (CI): 0.23-0.84). To the best of our knowledge, this is the first report on the association between a polymorphism of a gene coding for the chaperone protein and lung cancer risk. Moreover, the simple method reported here, based on the dual-luciferase reporter assay system, can be useful for testing functional significance of polymorphisms located in introns of other genes.

Expression of a constitutively active mutant of heat shock factor 1 under the control of testis-specific hst70 gene promoter in transgenic mice induces degeneration of seminiferous epithelium.

Heat shock activates in somatic cells a set of genes encoding heat shock proteins which function as molecular chaperones. The basic mechanism by which these genes are activated is the interaction of the specific transcription factor HSF1 with a regulatory DNA sequence called heat shock element (HSE). In higher eukaryotes HSF1 is present in unstressed cells as inactive monomers which, in response to cellular stress, aggregate into transcriptionally competent homotrimers. In the present paper we showed that the expression of a transgene encoding mutated constitutively active HSF1 placed under the control of a spermatocyte-specific promoter derived from the hst70 gene severely affects spermatogenesis. We found the testes of transgenic mice to be significantly smaller than those of wild-type males and histological analysis showed massive degeneration of the seminiferous epithelium. The lumen of tubules was devoid of spermatids and spermatozoa and using the TUNEL method we demonstrated a high rate of spermatocyte apoptosis. The molecular mechanism by which constitutively active HSF1 arrests spermatogenesis is not known so far. One can assume that HSF1 can either induce or repress so far unknown target genes involved in germ cell apoptosis.

In vivo electroporation of the testis versus transgenic mice model in functional studies of spermatocyte-specific hst70 gene promoter: A comparative study.

To determine whether DNA transfer to mouse testes by in vivo electroporation could be useful method for studying regulatory elements of genes specifically active in spermatocytes first we compared the expression pattern of a construct containing the EGFP reporter gene ligated to a fragment of the heat shock testis-specific hst70 gene promoter, both in testis of transgenic mice and in testis electroporated in vivo. While in transgenic mice the EGFP was expressed in all seminiferous tubules in a cell- and stage-specific manner, in the testes electroporated in vivo only small fraction of cells expressed this marker protein. In order to make a quantitative comparison between the specificity of these two experimental systems we used several vectors containing the CAT gene ligated to fragments of the hst70 gene 5' upstream of DNA sequences which either promoted or did not activate expression of the reporter gene in the testes of transgenic mice. Also, as a reference opposite to spermatogenic cells we examined the expression pattern of the same set of vectors in the rat hepatoma FTO 2B cells. Although electroporated testes retain some spermatocyte-specific features such as the ability to repress promoters which do not contain regulatory elements responsible for testis-specific transcription, several important drawbacks of the method are evident. They include basal activity of constructs which are not transcribed in testes of transgenic mice and low overall transfection efficiency. This may hamper studies in which subtle changes in the expression pattern are under investigation. However, the in vivo electroporation of the testis can be useful for preliminary screening of constructs aimed to study in transgenic mice.

Immunohistochemical studies on the expression pattern of molecular chaperones HSC70 and HSP25 and cell cycle-related proteins cyclin D1 and PCNA in rat liver after thioacetamide intoxication.

Intoxication of rats with thioacetamide (TAA) is a model system to investigate mechanisms involved in liver cell death and tissue reconstitution. Our study was undertaken to determine by immunohistochemistry the expression pattern of the cytoprotective chaperone proteins HSC70 and HSP25 and proliferation markers cyclin D1 and PCNA in livers of Wistar rats intraperitoneally injected with TAA at a single dose of 50 mg/kg. For each protein studied we observed distinct dynamic changes in appearance and localization in liver lobules. During 24-36 h after TAA injection the HSC70 cytoplasmic immunoreaction gradually disappeared from hepatocytes localized around central veins and a shift of immunostaining to cell nuclei took place. Then, 36-48 h after TAA injection the HSC70 cytoplasmic immunoreaction reappeared with the highest intensity in hepatocytes surrounding the areas of inflammatory cells. HSP25, undetectable in control hepatocytes began to appear at approximately 36 h after TAA injection and HSP25-immunopositive cells formed a characteristic ring around areas of inflammation. Of the proteins studied, the most rapid reaction to TAA was observed for cyclin D1. As early as 15 min after TAA administration cyclin D1-positive hepatocytes appeared in intermediate and periportal areas of liver lobules and a subsequent shift of staining to centrilobular hepatocytes took place at 36 and 48 h. There was no correlation of cyclin D1 localization either with PCNA-positive cells or mitotic cells. Our observations suggest that in TAA-treated livers HSP25 and HSC70 proteins can play an anti-inflammatory role, and the early and distinct cyclin D1 expression is not related to proliferation of hepatocytes.