Lack of type-specific concordance between human papillomavirus (HPV) serology and HPV DNA detection in the uterine cervix and oral mucosa.

There is limited knowledge about longitudinal genotype-specific concordance between human papillomavirus (HPV) serology and co-existent presence of HPV DNA in the uterine cervix. The role of oral HPV infections in inducing serological response is unclear, as is the effect of HPV antibodies on the outcome of oral HPV infections. The present study is part of the Finnish Family HPV Study designed to evaluate dynamics of HPV infections within families. Here, we correlated the point prevalence of HPV6, 11, 16, 18 and 45 antibodies and concomitant genotype-specific HPV DNA detection in cervical and oral samples of 323 mothers during their 3 year (mean 37.5 months) follow-up. The mean age of these pregnant mothers at enrolment (third trimester) was 25.5 years. HPV antibodies were analysed with multiplex HPV serology and HPV genotyping was performed using a Multimetrix kit (Progen Biotechnik). There was no concordance between cervical DNA detection and co-existent seropositivity, and the same was true even in samples taken 12 months apart. Women who cleared their cervical HPV16 infection had the highest HPV16 antibody levels, whereas those who acquired incident HPV16 infections had the lowest antibody levels. Neither the presence nor the dynamics of oral HPV DNA had any correlation with HPV serology.

Prepolymerized vs. in situ-polymerized fiber-reinforced composite implants--a pilot study.

The aim of this study was to investigate bone response to bioactive fiber-reinforced composite (FRC) implants under two polymerization conditions. Glass-fiber-dimethacrylate composite was tested as prepolymerized cylinder-shaped FRC implants and as cylindrical FRC implants polymerized in situ with blue light transmitted and scattered by the glass fibers. Ten FRC implants (6 prepolymerized and 4 in situ-polymerized implants) were placed in the right tibias of 3 pigs by means of a press-fit technique. After 12 weeks, light microscopy revealed only mild foreign-body reaction, with no accumulation of inflammatory cells on both the prepolymerized and the in situ-polymerized implants. The prepolymerized implants appeared to be fully integrated, whereas the in situ-polymerized implants were almost completely surrounded by a fibrous capsule. The present study suggests that in situ polymerization of FRC implants results in fibrous capsule formation and prevents integration with bone.

Early integration of high copy HPV16 detectable in women with normal and low grade cervical cytology and histology.

BACKGROUND: Integration of human papillomavirus (HPV) DNA has been considered a late event in cervical carcinogenesis. However, integrated forms of HPV were recently detected in cancer precursor lesions using a new real time polymerase chain reaction (PCR) to detect the deletions at the 3362-3443 region of HPV16 E2 OBJECTIVE: To study the frequency of HPV16 DNA integration in cervical lesions and compare the sensitivity of an additional upstream region of the E2 ORF (2962-3138) in detecting HPV integration. METHODS: Using the TaqMan based PCR, HPV16 positive DNA samples were analysed in 164 cervical scrapings from women participating in a multicentre screening trial. Biopsy confirmation was available in 62 cases. RESULTS: Primers targeting the 3362-3443 region detected the majority of E2 deletions. In only 23% of the samples was the E2 upstream region equal or better target than the 3362-3443 region. Mixed (episomal/integrated) pattern was the most prevalent physical state of HPV16, also present in PAP smears with normal morphology. Pure integrated form was most prevalent in HSIL and cancer lesions, but also detectable in low grade abnormalities (NSIL, ASC-US, LSIL). Women with only integrated HPV16 were almost 10 years older than those with episomal HPV16. Viral load of integrated HPV16 was related to cytological abnormality (p = 0.003) but not to histology. CONCLUSIONS: Integrated HPV16 is present in low grade cervical lesions, mostly mixed with the episomal form. Women with the pure integrated form of HPV16 are older than those with the other forms.

Detection of high-risk HPV DNA in semen and its association with the quality of semen.

The effects of seminal high-risk human papillomavirus (HPV) DNA were assessed on the quality of semen. Semen samples of 65 men participating in the ongoing Finnish HPV Family Study were collected. Semen analyses were done by the guidelines of the Nordic Association for Andrology. HPV DNA was detected by nested polymerase chain reaction and confirmed by Southern blot hybridization for high-risk types. Altogether, 10/65 men (15.4%) had high-risk HPV DNA positive semen sample. Seminal high-risk HPV DNA did not affect semen volume, sperm concentration, motility and vitality of spermatozoa. However, semen pH was borderline lower in HPV DNA positive than negative samples (7.4 vs 7.5). Neither oligo- nor asthenozoospermia was associated with seminal HPV DNA. In conclusion, seminal high-risk HPV DNA was detected in 15% of men. It did not affect the semen analysis, except semen pH by borderline significance. Sperm donors have not been tested for HPV infections, sperm washing does not seem to eliminate the risk of HPV transmission and the consequences of HPV in the semen are at present unknown.

Systemic and local effects of long-term exposure to alkaline drinking water in rats.

Alkaline conditions in the oral cavity may be caused by a variety of stimuli, including tobacco products, antacids, alkaline drinking water or bicarbonate toothpaste. The effects of alkaline pH on oral mucosa have not been systematically studied. To assess the systemic (organ) and local (oral mucosal) effects of alkalinity, drinking water supplemented with Ca(OH)2 or NaOH, with pH 11.2 or 12 was administered to rats (n = 36) for 52 weeks. Tissues were subjected to histopathological examination; oral mucosal biopsy samples were also subjected to immunohistochemical (IHC) analyses for pankeratin, CK19, CK5, CK4, PCNA, ICAM-1, CD44, CD68, S-100, HSP 60, HSP70, and HSP90. At completion of the study, animals in the study groups had lower body weights (up to 29% less) than controls despite equal food and water intake, suggesting a systemic response to the alkaline treatment. The lowest body weight was found in rats exposed to water with the highest pH value and starting the experiment when young (6 weeks). No histological changes attributable to alkaline exposure occurred in the oral mucosa or other tissues studied. Alkaline exposure did not affect cell proliferation in the oral epithelium, as shown by the equal expression of PCNA in groups. The up-regulation of HSP70 protein expression in the oral mucosa of rats exposed to alkaline water, especially Ca(OH)2 treated rats, may indicate a protective response. Intercellular adhesion molecule-1 (ICAM-1) positivity was lost in 6/12 rats treated with Ca(OH)2 with pH 11.2, and loss of CD44 expression was seen in 3/6 rats in both study groups exposed to alkaline water with pH 12. The results suggest that the oral mucosa in rats is resistant to the effects of highly alkaline drinking water. However, high alkalinity may have some unknown systemic effects leading to growth retardation, the cause of which remains to be determined.

New concepts on the role of human papillomavirus in cell cycle regulation.

Human papillomaviruses (HPVs) are strictly host-specific and also show a distinct tropism to squamous epithelial cells. Upon HPV infection, only a portion of the virus reaching the nucleus seems to undergo replication, suggesting that HPV replication remains confined to a small number of cells. HPVs critically depend on the cellular machinery for the replication of their genome. Viral replication is restricted to differentiated keratinocytes that are normally growth arrested. Hence, HPVs have developed strategies to subvert cellular growth regulatory pathways and are able to uncouple cellular proliferation and differentiation. Endogenous growth factors and cellular oncogenes modify HPV E (early) and L (late) gene expression and influence on the pathogenesis of HPV infections. HPV oncoproteins (E5, E6, E7) are important proteins not only in cell transformation but also in the regulation of the mitotic cycle of the cell, thus allowing the continuous proliferation of the host cells. Cyclins are important regulators of cell cycle transitions through their ability to bind cyclin-dependent kinases (cdks). Cdks have no kinase activity unless they are associated with a cyclin. Several classes of cyclins exist which are thought to coordinate the timing of different events necessary for cell cycle progression. Each cdk catalytic subunit can associate with different cyclins, and the associated cyclin determines which proteins are phosphorylated by the cdk-cyclin complex. The effects of HPVs on the cell cycle are mediated through the inhibition of antioncogens (mostly p53 and retinoblastoma) and through interference with the cyclins and cdks, resulting in target cell proliferation, their delayed differentiation, and as a side-effect, in malignant transformation.

Snuff use and smoking among senior high school students: effects of a snuff sales ban.

OBJECTIVES: The popularity of snuff especially among adolescents is rising. The association between long-term snuff use and oral cancer discovered in epidemiological studies has prompted a variety of preventive measures to be taken to reduce snuff use and prevent adoption of the habit. In this study, the effect of a recent (1 March, 1995) snuff sales ban introduced in Finland was investigated. Further, the rates of smoking, snuff use, alcohol use and drug experimenting were investigated before the introduction of the ban to characterize the study population. DESIGN AND SUBJECTS: Two questionnaire studies were carried out. The first was carried out 3 months prior to the ban in 1994 and the second 9 months after the ban in 1995 in a senior high school population in southwestern Finland. The participants were 793 students (aged 15-22 years) in the first survey and 545 students (aged 16-23) in the second. Associations between variables were analyzed using cross-tabulation and step-wise logistic regression. The effects of the ban were determined on the basis of direct questions in the second questionnaire relating to the snuff sales ban. RESULTS: Snuff was used by 9% of the students participating in the first study. The results of the second questionnaire indicate that the implementation of the snuff sales ban reduced the rate of snuff use by 1% in the study population. The majority of the snuff users (76%) reported that they had maintained their snuff habit. Of those reporting that they were snuff users before implementation of the snuff sales ban, 12% had switched to smoking and 5% to drugs. CONCLUSIONS: The results of the present study suggest that the snuff sales ban in this population with a high rate of snuff use had little effect on snuff use rates and may have some short-term negative consequences as some snuff users switch to other substitutes, such as smoking, with known adverse health effects.

Expression and mutations of p53 in salivary gland tumours.

A series of 219 salivary gland tumours (103 carcinomas and 116 benign tumours) were analysed for p53 protein expression using immunohistochemistry, and for mutations in p53 gene using non-radioactive single strand conformation polymorphism (SSCP). p53 expression was present in 36% (42/116) of the benign tumours and in 54% (56/103) of the carcinomas. The highest prevalence of p53 expression was found in adenoid cystic carcinomas (69%), followed by mucoepidermoid carcinomas (67%). Of the benign tumours, pleomorphic adenomas showed the highest prevalence of p53 positivity (41%). In malignant tumours, expression of p53 bore no correlation to local recurrence, metastatic disease or survival of the patients. Exons 5 through 9 were analysed and four mutations were found in 20 cases of p53-immunopositive tumours and two in 20 p53-negative tumours. Each of the exons 5, 6 and 8/9 had two mutations, whereas no mutations were detected in exon 7.

Exposure of an infant to cervical human papillomavirus infection of the mother is common.

OBJECTIVE: The purpose of this study was to determine the potential of exposure of an infant to cervical human papillomavirus infection of the mother. STUDY DESIGN: Cervical scrapes of the mothers and nasopharyngeal aspirate fluids of their infants were analyzed at the time of delivery. The study included 106 infants born by vaginal delivery or by cesarean section and their 105 mothers. Positive results were confirmed and typed by direct deoxyribonucleic acid sequencing or single-strand conformation polymorphism of the polymerase chain reaction product. RESULTS: Both the mother's and her infant's samples were positive for the same type of human papillomavirus in 29 mother-infant pairs. Interestingly, five infants born by cesarean section were found to be human papillomavirus deoxyribonucleic acid positive for the same human papillomavirus type as their mother. The overall concordance between human papillomavirus types in the mother and her newborn was 69% (29/42). Regardless of match in types found in the mother's and her infant's sample, human papillomavirus deoxyribonucleic acid positivity was found in 39 of all the 106 infants (37%). CONCLUSIONS: Our results indicate that the infant of the human papillomavirus-infected mother is exposed to infection even when the cervical infection of the mother is subclinical. The possibility of transplacental exposure has to be considered as well.

Correlation of MIB-1 antigen expression with transcription factors Skn-1, Oct-1, AP-2, and HPV type in cervical intraepithelial neoplasia.

Recently, interest in transcription factor coding genes has emerged in many human diseases. Transcription factors, responding both to extracellular and to intracellular signals, exercise an important regulatory control over the proliferation and differentiation of cells. During the development of CIN (cervical intraepithelial neoplasia) lesions, normal regulation of the cell cycle seems to be disturbed. Transcription factors have been shown in vitro to be intimately involved in the expression of HPV (human papillomavirus) early genes, which affect the development of cervical precancer lesions. To test the relevance of in vitro findings in clinical samples, the expression of transcription factors Skn-1, Oct-1, and AP-2 was analysed in 31 normal cervical epithelial samples and in 55 CIN lesions. The results were correlated with the HPV status and cell proliferation activity of the squamous epithelium as measured by MIB-1 antibody. MIB-1 staining is an applicable marker of CIN, correlating strongly with the CIN grade (P < 0.001). The presence of HPV DNA did not accelerate the cell proliferation rate or change significantly the immunoreactivity of Skn-1, Oct-1, or AP-2 antibodies. The staining patterns of these transcription factors were significantly influenced only by the CIN grade. Transcription factors generally showed weaker expression in the dysplastic samples, although the nuclear staining of AP-2 increased markedly (P = 0.046) in the superficial layer of the CIN III samples. These findings suggest that changes in the expression of transcription factors may be important in studying the proliferative activity of CIN lesions.

Detection of Epstein-Barr virus in salivary gland specimens from Sjögren's syndrome patients.

The cause of Sjögren's syndrome remains unclear, but several environmental and genetic factors have been implicated. The Epstein-Barr virus (EBV), among others (e.g., cytomegalovirus, human herpesvirus 6, and retroviruses), has been widely studied in connection with Sjögren's syndrome without conclusive results. To determine the role of EBV infection in patients with Sjögren's syndrome, the presence of EBV deoxyribonucleic acid (DNA) in major and minor salivary gland biopsy specimens was investigated by means of sulfur 35 in situ hybridization and polymerase chain reaction. Additionally, the presence of latent virus proteins EBV latent membrane protein and Epstein-Barr nuclear antigen 2 was analyzed by immunohistochemical methods. Viral DNA, detected by in situ hybridization, was found in 19% of patients with a diagnosis of Sjögren's syndrome and in 3% of controls. All tissues studied were found to be negative for EBV DNA by polymerase chain reaction. EBV latent membrane protein-positive staining was seen in 17% of patients and 22% of control subjects, while Epstein-Barr-positive staining was found in 25% of patients and 39% of controls. The low frequency of EBV DNA detected in the biopsy specimens does not indicate that the virus itself is the cause of Sjögren's syndrome. However, the possibility that the virus acts as a cofactor cannot be ruled out.

Immunocompetent cells in benign and malignant salivary gland tumors.

IgA-, IgG, and IgM-producing plasma cells as well as 3- and T-lymphocytes were immunophenotyped and quantitated in a series of 216 benign and malignant salivary gland tumors, with special emphasis placed on the clinical behavior of the tumors. Highest number of plasma cells were found in mucoepidermoid carcinomas, where IgG-plasma cells were the sole Ig-class secreted. No IgA-immunoreactivity was found in adenoid cystic, undifferentiated, acinic cell, carcinoma in pleomorphic adenoma, and mucoepidermoid carcinomas. In squamous cell carcinomas, the number of IgM-plasma cells was higher than that in other salivary gland tumors. Basal cell adenomas contained only IgM-positive plasma cells. In logistic regression analysis, IgG- and IgM-producing plasma cells in malignant salivary gland tumors were related to an increased tumor diameter (p = 0.022 and 0.046, respectively). In benign tumors, neither clinical nor prognostic value could be attributed to the distribution of plasma cells. T-cells and B-cells were present in 63.9% and 33.8% of all tumors, found in 63.8% and 26.7% (p = 0.0048) of the benign tumors, and in 64.1% and 41.7% (not significant) of the malignant tumors, respectively. The presence of T- of B-lymphocytes was of no prognostic significance in malignant tumors. In benign tumors, however, the mean age of the patients was significantly higher (p = 0.010) and the mean time to recurrence significantly shorter (p = 0.018) in patients with tumors containing T-cells than in those devoid of these cells. In conclusion, the cell-mediated immunity (T-cells and their subsets) seems to play a more important role in pathogenesis and prognostication of salivary gland neoplasms than do the cells of the B-cell lineage, and, clearly, further studies are needed to elucidate these issues.

Detection of p53 protein and Ki-67 proliferation antigen in human papillomavirus (HPV)-positive and HPV-negative cervical lesions by immunohistochemical double-staining.

In HPV-associated genital lesions, low or absent expression of p53 has been attributed to the rapid degradation of p53 through its binding with HPV E6 protein. In this study, we examined p53 protein expression with two antibodies (CM1 polyclonal and PAb 1801 monoclonal antibodies), and Ki-67 proliferation antigen (monoclonal antibody) using an immunohistochemical (IHC) double-staining technique in 77 HPV-positive cervical lesions (HPV6, HPV11, HPV16, HPV18, HPV31, and HPV33) and in 15 HPV-negative cases. p53 protein expression was detected in 36/92 (39.1%) of the specimens. Of the p53-positive cases, 80.6% (29/36) were HPV-positive samples, including 10/23 (43.5%) of HPV16- and 3/10 (30%) of HPV18-positive biopsies. In 52.8% of the p53-positive samples, the expression was found in less than 5% of the basal cells which were also positive for Ki-67. Ki-67 proliferation marker was found in 91/92 specimens, most intensely in those infected by HPV16. p53 was more abundant in progressive or persistent lesions, but no differences were found between HPV-positive and HPV-negative samples. The positive IHC double-staining of both p53 and Ki-67 proliferation antigen in the same basal (and parabasal) cells indicates that these two normal cell-cycle proteins are being expressed while the cells are entering from the G1 to the S phase of the cell cycle. Since the latter property is only attributed to the wild-type p53 (but not to mutated p53), the p53 protein detected in HPV lesions by IHC is likely to be the wild-type p53 rather than mutated p53, and the result was also confirmed by using p53 mutant specific antibody PAb 240. Accordingly, the concept of HPV inactivating the wild-type p53 protein should be re-examined, and other mechanisms for HPV-mediated carcinogenesis should be considered.

Human papillomavirus in laryngeal papillomas and in adjacent normal epithelium.

Eleven adults with laryngeal papillomas were studied for the presence of human papillomavirus (HPV) DNA by in situ hybridization. As well as from the papillomas, three additional biopsies were taken from the normal-appearing mucosa as follows: the involved vocal cord, the opposite vocal cord (when the papilloma was unilateral), and from the ventricular fold on the side of the lesion. These normal tissues were analysed by polymerase chain reaction (PCR) to detect HPV DNA. All except one of the 11 papillomas contained HPV DNA; nine were HPV 6/11 DNA positive and one positive for HPV 16 DNA. The normal-appearing laryngeal mucosa harboured HPV DNA in eight out of 11 patients. The present results strongly support the concept that the adult-type laryngeal papilloma is an HPV-induced lesion, mostly due to HPV types 6 and 11. The persistence of HPV DNA in the adjacent normal epithelium is consistent with the frequent recurrence of these lesions.

Lesions of the oral mucosa in lymphoma patients receiving cytostatic drugs.

The 1-year incidence of oral mucosal lesions during cytostatic therapy was investigated in 67 patients [34 men and 33 women (mean age 49 years)] out of 79 original patients, being treated for non-Hodgkin lymphoma or Hodgkin's disease. The incidence of lesions during examinations was 43.4%. Recurrent lesions were observed in 19.4% of cases. Mean leukocyte counts were statistically significantly lower (P < 0.01) during lesion periods than before cytostatic therapy in all lesion groups. Leukocytopenia was found in 85.4% of patients with hairy leukoplakia-like lesions (HLL), and in 81.8% of the patients with angular cheilitis. 5 out of 14 patients with oral ulcers (35.7%) had episodes of septicaemia. Mean thrombocyte counts of patients in various lesion groups were normal (< 140 x 10/1). However, low thrombocyte counts were more statistically significant (P < 0.05), when haemorrhages or HLL were present. Clinical candidiasis was diagnosed in 28.4% of patients during the treatment. However, cultivation revealed that 62.3% of salivary yeast cultures were positive. The study reported here shows a correlation between mucosal ulcers and septicemia, and between leukocytopenia, angular cheilitis and HLL. The disparity between clinically diagnosed candidiasis and the occurrence of salivary yeast counts suggests that antifungal drugs might be of prophylactic value during cytostatic therapy.

Expression of p53 protein related to the presence of human papillomavirus (HPV) DNA in genital carcinomas and precancer lesions.

The E6 protein of the high-risk human papillomavirus (HPV) types 16 and 18 is capable of complexing with the wild-type p53 tumor suppressor gene product, leading to loss of the normal p53 function as an anti-oncogene, whereas the low-risk HPV types 6 and 11 lack this binding property. The malignant potential of HPV 16 and 18 has been ascribed to this complexing of E6 with p53, which regularly leads to undetectable expression of the latter in HPV-positive lesions. To assess the role of p53 in HPV-associated genital carcinogenesis, the expression of p53 protein was studied immunohistochemically in 22 genital carcinomas and precancer lesions; 8 vulvar carcinomas, 1 VIN (vulvar intraepithelial neoplasia), 5 cervical carcinomas and 8 CIN (cervical intraepithelial neoplasia) using monoclonal antibody PAb 1801. Presence of HPV was demonstrated by PCR using HPV consensus primers, and amplified HPV-DNA was digested with the restriction enzymes giving distinct patterns for various HPV-types in gel electrophoresis. HPV-typing was confirmed by in situ hybridization with biotinylated DNA probes. Altogether, 17 of the 22 specimens (77%) showed p53 expression: 67% of the precancer lesions and 83% of carcinomas. Expression was more frequent (89%) in the vulvar than (70%) in cervical lesions. Using PCR,HPV DNA was detected in 19/22(86%) of the samples. The following HPV types were identified: HPV 6 (2 samples), HPV 11 (3 cases), HPV 16 (5 cases), HPV 33 (3 cases), and 6 contained unidentified HPV types. All HPV DNA-negative specimens showed p53 expression. Of the 19 HPV DNA-positive lesions, 5 were p53-negative, three of these being HPV 16 positive CIN lesions. The remaining two HPV 16 lesions were invasive carcinomas with a weak p53 expression. HPV 6 and 11-positive lesions showed a weak p53 expression more frequently than HPV-negative cases and HPV 33 lesions. The results indicate that p53 expression is detectable, but it is less frequent and less intense in HPV DNA-positive genital precancer lesions and carcinomas (particularly those with HPV 16 DNA) as compared with HPV DNA-negative lesions.

Southern blot hybridization and PCR in detection of oral human papillomavirus (HPV) infections in women with genital HPV infections.

The presence of human papillomavirus (HPV) in biopsies taken from clinically normal buccal mucosa (n = 212) and clinical lesions (n = 60) was examined by Southern blot hybridization (SBH) using 32P-labelled HPV DNA probes. Furthermore, one hundred formalin-fixed, paraffin-embedded biopsies were analyzed by using polymerase chain reaction (PCR), combined with dot blot hybridization and biotinylated HPV DNA probes. With SBH and PCR, 15.4% and 29.4% of the biopsies, respectively, contained HPV DNA. In clinically normal epithelium, 15.6% and 23.1% of the samples were HPV-positive with SBH and PCR, respectively. The HPV types detected in the genital and oral mucosa of index patients differed in all except two cases. Histology could not be relied on distinguishing HPV DNA positive and HPV DNA negative samples. Hand warts were encountered significantly more frequently in patients with a concomitant oral HPV infection. To conclude, oral HPV infections as detected by SBH and PCR are surprisingly common, but similar to the genital tract, the virus seems to exist in a latent form in the vast majority of cases. The frequent concomitant finding of skin warts and oral HPV infection may suggest some kind of HPV-specific immunosuppression.

Immunohistochemistry, in situ hybridization and polymerase chain reaction (PCR) in detecting c-myc expression in human malignancies.

The assessment of oncogene expression at cellular level is important in understanding the role of those genes in carcinogenesis. Using in situ hybridization and immunohistochemistry, the expression of oncogenes can be visualized in topographic relation to tissue morphology. In the present study, c-myc overexpression was studied in ten carcinomas of different origin (6 mammary adenocarcinomas, 2 vulvar and 2 bronchial squamous cell carcinomas) by in situ hybridization (ISH) with 35S-labeled RNA probes and by immunohistochemistry (IHC). DNA amplification and transcription of c-myc oncogene were also studied with polymerase chain reaction (PCR) using beta-globin as an intrinsic standard for DNA amplification. The effect of formalin fixation of c-myc expression was simultaneously studied. Half of the tumours (5/10) demonstrated c-myc mRNA overexpression by ISH performed on frozen sections and two of the samples were shown to over-express c-myc protein by IHC. Only two samples fixed in formalin showed positive signals for c-myc mRNA. None of the biopsies showed DNA amplifications either with ISH or PCR. The present results suggest that ISH with RNA probes is a useful method for detecting the transcription of activated oncogenes in malignant tissues, especially when applied on frozen sections. The results also indicate that in some cases, c-myc gene may be adequately transcribed to mRNA but the latter is not translated to the appropriate oncoprotein.