Casein Protein Block to Optimize Immunohistochemistry Detection of Surface Immunoglobulin Heavy Chain Expression in Lymphoid Cells.

We describe our recent experience of studying expression of immunoglobulin (Ig) heavy chain (IgG, IgM, and IgA) in lymphoid cells comprising a research set of formalin-fixed, paraffin-embedded human diffuse large B-cell lymphoma samples. We found that using typical clinical automated immunohistochemistry protocols and usual buffers as blocking agents, the extent of undesirable staining was extreme and impaired our ability to interpret heavy chain Ig expression by individual lymphoid cells. We were not able to optimize this with serial dilutions in antibody concentration or time of primary antibody exposure. We therefore developed an added step of casein protein block, which solved the problem. We are not aware of other such reports in clinical or human research tissue sets and believe this solution may be useful when clinical pathologists or researchers encounter similar technical issues.

Evaluation of osteopontin expression in chronic wounds: a potential prognostic and therapeutic biomarker.

OBJECTIVE: Osteopontin (OPN) is abundantly expressed during tissue repair, acting as a powerful chemokine that recruits inflammatory cells such as neutrophils, macrophages, and Langerhans cells. The role of OPN in chronic wounds has not been explored. In this study, we assess the expression levels of OPN in chronic wounds to assess its potential contribution to the exacerbated inflammation seen in chronic ulcers, which is thought to contribute to poor healing. METHODS: This retrospective study included archived biopsies of chronic wounds from several aetiologies. Immunohistochemical staining and blind analysis of OPN expression were carried out. RESULTS: We assessed biopsies from venous leg ulcers (n=5), diabetic foot ulcers (n=5), pyoderma gangrenosum (n=5), squamous cell carcinoma ulcers (n=4), and calciphylaxis ulcers (n=3). The data revealed that all these sets of chronic ulcers expressed high levels of OPN. CONCLUSION: This study provides strong histopathologic evidence that OPN expression is significantly increased in chronic wounds, suggesting that its upregulation could contribute to the exacerbated inflammation. Furthermore, further characterisation of the role of OPN in wound healing could aid the development of specific and efficient anti-OPN therapies for the treatment of chronic wounds.

Facial Nerve Function and Quality of Resection in Large and Giant Vestibular Schwannomas Surgery Operated By Retrosigmoid Transmeatal Approach in Semi-sitting Position with Intraoperative Facial Nerve Monitoring.

INTRODUCTION: Large and giant vestibular schwannomas pose a real problem in their management. The preservation of facial nerve function may limit tumor resection despite the use of intraoperative monitoring of the facial nerve. In Algeria, vestibular schwannomas represent 5% of all intracranial tumors operated on, 80.5% of which are large or giant. METHODS: From January 2010 to December 2015, 151 large and giant vestibular schwannomas were operated in our department. Tumor diameter was between 30 and 60 mm. The most common presenting symptom was hearing loss, which was observed in 41.66% of all our patients. All patients were operated in the semi-sitting position with opening of the posterior wall of the internal auditory canal and under continuous intraoperative facial nerve function monitoring. RESULTS: Tumor resection was total in 126 patients. Anatomic preservation of the facial nerve was the reason for nontotal resection in 25 patients. The facial nerve was anatomically preserved in 149 patients. Two years after surgery, the facial nerve function was grade I-II House-Brackmann (H-B) score in 124 cases (82%), grade III-IV H-B score in 21 cases (14%), and grade V-VI H-B score in 06 cases (04%). The status and the improvement of postoperative facial nerve function depend on 4 factors: anatomic preservation of nerve, stimulation threshold, cystic form, and the presence of train activity. CONCLUSIONS: The development of anesthesia techniques and microsurgery and the systematic use of intraoperative monitoring of the facial nerve have allowed us to move from a life preservation era to another era of preservation of function.

Immunohistochemistry of tissue prepared by a molecular-friendly fixation and processing system.

A recently introduced histologic fixative (Universal Molecular Fixative [UMFIX]) has been shown to preserve macromolecules in tissue at ambient temperature. When UMFIX-exposed tissues are processed by a formalin-free, microwave-assisted rapid processing system, the resulting paraffin blocks retain good histomorphology and intact nucleic acids suitable for expression microarray analysis. Because UMFIX may be used as an alternative to formalin, the authors set out to study the effect of this new fixation and processing system on immunohistochemistry (IHC) by analyzing a range of human neoplastic and non-neoplastic specimens. Parallel slices from surgically removed specimens were fixed in formalin and UMFIX and processed in a rapid microwave-assisted tissue processor. IHC was performed following routine procedures. The staining for those antibodies that normally required antigen retrieval was carried out with and without that step. The intensity and pattern of reactions were compared in 144 tissue samples fixed by the two methods using 70 monoclonal and polyclonal antibodies. The intensity of IHC reactions for most cytoplasmic antigens was generally equal or stronger in UMFIX tissues. This was particularly true with intermediate filaments and HercepTest, where the antigen retrieval step became unnecessary. Conversely, there was a decrease in the intensity of reactions for HepPar1, bcl-2, and three nuclear antigens (Ki-67, TTF-1, and estrogen receptor). Increasing their exposure times optimized the sensitivity of the latter four antibodies. The study shows that IHC staining results of tissues fixed in UMFIX and processed by the microwave-assisted system are comparable to those obtained on formalin-fixed, similarly processed specimens. There is an enhancement of the sensitivity of few antibodies in UMFIX-exposed tissue, rendering antigen retrieval unnecessary. This increased sensitivity may be due to the effect of eliminating formalin from fixation and processing or the microwave energy.

Antimicrobial activity of UMFix tissue fixative.

AIMS: The aim of this study was to determine the antimicrobial effects of UMFix, an alcohol based tissue fixative, on various microorganisms. The UMFix solution was compared with 10% neutral buffered formalin. METHODS: Standard methods to determine microorganism colony counts were performed after exposure of the microorganisms to UMFix and 10% neutral buffered formalin. RESULTS: After a short exposure, UMFix rapidly killed vegetative bacteria, yeasts, moulds, and viruses. Bacterial spores were resistant to killing by UMFix. All organisms were killed by the 10% neutral buffered formalin preparation. CONCLUSIONS: UMFix was microbicidal for vegetative bacteria, yeasts, and aspergillus species after a short exposure, although it was not active against spore forming bacillus species. The methanol content of the fixative was responsible for the killing effect of this fixative. No killing was seen when polyethylene glycol was used alone.

Infliximab monotherapy in psoriasis: a case of rapid clinical and histological response.

BACKGROUND: Psoriasis is a common chronic relapsing, inflammatory, hyperproliferative skin disorder with genetic predisposition. There is currently no experimental model for psoriasis and the pathogenesis is not fully understood. Psoriatic plaques have been shown to contain increased levels of cytokines, including tumor necrosis factor alpha (TNF-alpha). Anti-tumor necrosis factor therapy with infliximab has been shown to be highly effective in recalcitrant psoriasis. METHODS: We evaluated the efficacy and timeline of histological changes in a psoriatic plaque following infliximab infusion. A patient with severe recalcitrant plaque psoriasis was clinically and histologically assessed for improvement. RESULTS: We found rapid clinical improvement with infliximab accompanied by histopathological changes. The earliest effects were seen on neutrophils and lymphocytes whereas keratinocyte normalization was not evident at the early stages. CONCLUSION: Infliximab is not only an effective agent in the treatment of psoriasis but appears to have a very rapid onset of action.

Expression of Fas-receptor on basal cell carcinomas after treatment with imiquimod 5% cream or vehicle.

Treatment with imiquimod 5% cream, capable of inducing interferon (IFN)-alpha, effectively cures basal cell carcinoma (BCC), both clinically and histologically. IFN-alpha induces expression of CD95-receptor (FasR) on BCC cells, which normally fail to express Fas receptor (FasR). Expression of the FasR is postulated to lead to apoptosis via CD95 receptor-CD95 ligand (FasL) interaction. Absence of this receptor may be responsible for the longevity of the cells of BCCs by preventing them undergoing 'suicidal' apoptosis, as well as apoptosis induced by neighbouring BCC cells and/or by infiltrating T-lymphocytes. We examined the expression of FasR on BCC after very short-term exposure to imiquimod 5% cream or vehicle. In a double-blind study, 10 patients with BCC applied either imiquimod (n = 5) or vehicle (n = 5) five times per week for up to 2 weeks. At the end of treatment, the treated area was excised and examined for the presence or absence of FasR by immunoperoxidase staining of rat antihuman FasR with haematoxylin and eosin counterstaining. Histologically, BCC cells were present in all (5/5) of the vehicle-treated BCCs and in 4/5 of the imiquimod-treated BCCs. BCC cells expressed FasR in 3/4 imiquimod-treated BCCs but in none (0/5) of the vehicle-treated tumours. T-lymphocytes apposed to BCC cells were evident in all three imiquimod-treated BCCs expressing FasR and in none of the FasR-negative, vehicle-treated BCCs. Imiquimod-induced FasR-mediated apoptosis may contribute to the effectiveness of imiquimod 5% cream for the treatment of BCC.

The Prevalence of Human Papillomavirus in Cervical Cancer in Iran.

Background: The human papiloma virus (HPV), which is sexually transmitted, and most commonly causes genital warts, has been linked to cervical intraepithelial neoplasia and invasive carcinoma. Of ninety plus types of HPV, HPV-16 is the most prevalent in cervical cancer, followed by HPV-18, and HPV-33. As HPV's implication has not been assessed in the Middle East the main focus of this retrospective study was to determine the prevalence of HPV -16,18, and 33 in cases of cervical cancer from Iran. Material and Methods: This retrospective study covered 100 patients with uterine cervical carcinomas who were referred to two referral centers for cancer in Tehran-Iran. Pathological blocks were collected for these cases and initial review of the blocks showed poor specimens in 18 cases, which left 82 cases for the study. These samples were histologically examined to verify the presence and the type of carcinoma. The next step was in situ hybridzation for the detection of HPV common DNA. In Situ hybridization was preformed on all samples. Finally, Polymerase Chain Reaction (PCR) was preformed for the HPV types 16, 18, and 33. PCR amplification of exon 5 of the p53 gene was used as an internal control for the integrity of DNA. Takara PCR Human papilloma Detection method was used which includes primer for HPV 16, 18, and 33. Three primers were used alone, or in combination, in order to increase the sensitivity of the detection. Results: The majority of tumors were squamous cell carcinomas (87%). The rest were adenosquamous carcinoma and adenocarcinomas. None of the 82 different cervical carcinoma tissue samples were found to be positive by in situ hybridization. In the PCR samples, amplification of DNA was observed for 69 tumor specimens. In the remainning13 cases, the DNA in fixed tissue was degraded, as verified by the absence of an internal control band (p53). Out of the total 69 tumors (85.5%) with adequate DNA contained HPV band on PCR. The majority (73.9%) of HPV positive tumors contained HPV-16; the rest (11.6%) demonstrated type 18 and 33. There was no correlation between the histology of carcinoma and presence of types of HPV. Conclusion: The prevalence of HPV in carcinomas of uterine cervix in Iran is similar to those reported in other regions of the world. Similarly, it appears that HPV-16 is the most common type associated with cervical cancer in Iran. Further studies on larger samples of patients, particularly in those with pre-invasive forms of the disease, are needed to elucidate the carcinogenic role of HPV types in cervical cancer in Iranian women.

Pathophysiology of apolipoprotein E deficiency in mice: relevance to apo E-related disorders in humans.

Apolipoprotein E (apo E) deficiency (or its abnormalities in humans) is associated with a series of pathological conditions including dyslipidemia, atherosclerosis, Alzheimer's disease, and shorter life span. The purpose of this study was to characterize these conditions in apo E-deficient C57BL/6J mice and relate them to human disorders. Deletion of apo E gene in mice is associated with changes in lipoprotein metabolism [plasma total cholesterol (TC) (>+400%), HDL cholesterol (-80%), HDL/TC, and HDL/LDL ratios (-93% and -96%, respectively), esterification rate in apo B-depleted plasma (+100%), plasma triglyceride (+200%), hepatic HMG-CoA reductase activity (-50%), hepatic cholesterol content (+30%)], decreased plasma homocyst(e)ine and glucose levels, and severe atherosclerosis and cutaneous xanthomatosis. Hepatic and lipoprotein lipase activities, hepatic LDL receptor function, and organ antioxidant capacity remain unchanged. Several histological/immunohistological stainings failed to detect potential markers for neurodegenerative disease in the brain of 37-wk-old male apo E-KO mice. Apo E-KO mice may have normal growth and development, but advanced atherosclerosis and xanthomatosis may indirectly reduce their life span. Apo E plays a crucial role in regulation of lipid metabolism and atherogenesis without affecting lipase activities, endogenous antioxidant capacity, or appearance of neurodegenerative markers in 37-wk-old male mice.

GLUT-1 expression in ovarian carcinoma: association with survival and response to chemotherapy.

BACKGROUND: Cancer cell growth is an energy-related process supported by an increased glucose metabolism. The objective of this study was to investigate the association of GLUT-1 with response to chemotherapy and outcome in patients with ovarian carcinoma. METHODS: Histologic sections of formalin fixed, paraffin embedded specimens from 113 primary ovarian carcinomas were stained for GLUT-1 by using polyclonal GLUT-1 antibody (Dako Co., Carpinteria, CA) and the labeled streptavidin biotin procedure. Intensity of GLUT-1 staining was compared with disease free survival (DFS), chemotherapy response, and other clinicopathologic characteristics. RESULTS: GLUT-1 cytoplasmic membrane staining was observed in 89 of 104 (85.6%) malignant tumors. Poorly differentiated tumors showed a trend to overexpress the GLUT-1 protein compared with the more differentiated counterparts (27.6% vs. 8.7%; P = 0.08). Patients who experienced a complete clinical response to chemotherapy were more frequently GLUT-1 positive than GLUT-1 negative (80% vs. 51.5%; P = 0.036). In multivariate analysis of advanced stage disease, residual tumor (P = 0.0001) and high GLUT-1 expression levels (P = 0.028) were the only independent variables that maintained a significant association with response to chemotherapy (P = 0.0001; chi-square = 38.13). In the subgroup of Stage III-IV (International Federation of Gynecology and Obstetrics patients showing a complete clinical response, GLUT-1 overexpression was associated with a shorter DFS. The median time to progression was 30 months in GLUT-1 strongly positive cases (> 50% of cancer cells positive) versus 60 months in GLUT-1 weakly positive cases (< or = 50% of cancer cells positive; P = 0.024). CONCLUSIONS: GLUT-1 status is an independent prognostic factor of response to chemotherapy in advanced stage ovarian carcinoma. Moreover, patients overexpressing GLUT-1 show a significantly shorter DFS. These results suggest that the assessment of GLUT-1 status may provide clinically useful prognostic information in patients with ovarian carcinoma.

A description of parasite-harbouring cells in localized lymphadenitis in dry type cutaneous leishmaniasis.

Lymphadenitis with or without dry-type cutaneous leishmaniasis is rare. The lesion might self heal or show excellent response to antimonial therapy. Routine histopathological changes of localized leishmaniasis lymphadenitis are non-caseating to suppurative granulomata mostly in paracortical areas, some with extension to germinal centres, medullary cords and/or pericapsular spaces which have to be distinguished from other causes of lymphadenitis such as tuberculosis, cat-scratch disease and toxoplasmosis. Dense lymphoplasmocytic infiltrate was observed surrounding the necrotizing granuloma together with dense capsular fibrosis with multiple granulomata in subcapsular and pericapsular areas. Immunostaining of lymph nodes showed that a few macrophages were harbouring Leishman bodies. Dispersed Langerhans cells were also harbouring Leishman bodies in the parasitophorous vacuoles between their cytoplasmic pseudopods. In conclusion multiple noncaseating to suppurative granulomata with dense pericapsular and capsular granulomo-sclerotic changes should be considered in the differential diagnosis of leishmaniasis lymphadenitis.

Stromal and epithelial expression of tumor markers hyaluronic acid and HYAL1 hyaluronidase in prostate cancer.

Hyaluronic acid (HA), a glycosaminoglycan, regulates cell adhesion and migration. Hyaluronidase (HAase), an endoglycosidase, degrades HA into small angiogenic fragments. Using an enzyme-linked immunosorbent assay-like assay, we found increased HA levels (3-8-fold) in prostate cancer (CaP) tissues when compared with normal (NAP) and benign (BPH) tissues. The majority ( approximately 75-80%) of HA in prostate tissues was found to exist in the free form. Primary CaP fibroblast and epithelial cells secreted 3-8-fold more HA than respective NAP and BPH cultures. Only CaP epithelial cells and established CaP lines secreted HAase and the secretion increased with tumor grade and metastasis. The pH activity profile and optimum (4.2; range 4.0-4.3) of CaP HAase was identical to the HYAL1-type HAase present in human serum and urine. Full-length HYAL1 transcript and splice variants were detected in CaP cells by reverse transcriptase-polymerase chain reaction, cloning, and sequencing. Immunoblotting confirmed secretion of a approximately 60-kDa HYAL1-related protein by CaP cells. Immunohistochemistry showed minimal HA and HYAL1 staining in NAP and BPH tissues. However, a stromal and epithelial pattern of HA and HYAL1 expression was observed in CaP tissues. While high HA staining was observed in tumor-associated stroma, HYAL1 staining in tumor cells increased with tumor grade and metastasis. The gel-filtration column profiles of HA species in NAP, BPH, and CaP tissues were different. While the higher molecular mass and intermediate size HA was found in all tissues, the HA fragments were found only in CaP tissues. In particular, the high-grade CaP tissues, which showed both elevated HA and HYAL1 levels, contained angiogenic HA fragments. The stromal-epithelial HA and HYAL1 expression may promote angiogenesis in CaP and may serve as prognostic markers for CaP.

Normal endometrial cells in Papanicolaou smears: prevalence in women with and without endometrial disease.

OBJECTIVE: To determine whether the prevalence of normal endometrial cells in Papanicolaou smears of women with and those without endometrial carcinoma or hyperplasia differs significantly. METHODS: Papanicolaou smears of women with biopsy-proved endometrial hyperplasia or carcinoma diagnosed between 1990 and 1998 were reviewed for the presence of normal endometrial cells. Chi-square and a power analysis were used to compare these smears with results of smears from women older than 35 years of age with tissue diagnoses other than hyperplasia or carcinoma. All Papanicolaou smears obtained within the 5 years before endometrial sampling were reviewed. Each patient had at least one smear done within the previous 12 months. Clinical information was available for all patients. RESULTS: Of the 201 women in whom endometrial hyperplasia (n = 103) or carcinoma (n = 98) was diagnosed, 4 (2%) had normal endometrial cells in otherwise negative Papanicolaou smears. Of the 289 women in the comparison group, 15 (5%) had normal endometrial cells in their Papanicolaou smears. The prevalence of normal endometrial cells did not differ significantly between the two groups (P =.071). The study had 80% power to detect a 5% or greater difference between groups. CONCLUSION: The prevalence of normal endometrial cells in Papanicolaou smears of women with endometrial carcinoma or hyperplasia does not significantly differ from that in women without these conditions. Reporting normal endometrial cells in Papanicolaou smears according to the recommendations of the Bethesda System may lead to unnecessary procedures and patient anxiety.

Expression of GLUT-1 glucose transporter in borderline and malignant epithelial tumors of the ovary.

OBJECTIVE: Cancer cells have increased rates of glucose metabolism when compared to normal cells. One of the mechanisms proposed for the accelerated glucose use in malignant cells is the overexpression of glucose transporters. In this study we evaluated the expression of the GLUT-1 glucose transporter in borderline and malignant epithelial neoplasms of the ovary. METHODS: Histologic sections of tumor tissues from 21 borderline and 82 malignant epithelial neoplasms of the ovary were stained for GLUT-1 using polyclonal GLUT-1 antibody (Dako, Carpinteria, CA) and the labeled streptavidin biotin procedure. DAB was used as chromagen and tissues were counterstained with hematoxylin. RESULTS: Normal ovarian surface epithelial cells were either negative or weakly positive. Of the 82 carcinomas, 81 (98.8%) were positive for GLUT-1. The staining intensity was significantly associated with the grade of tumor (P = 0.001). Of the 21 borderline neoplasms, 20 (95.2%) were positive for GLUT-1. Carcinomas had a significantly stronger stain than borderline tumors (P = 0.0001). The intensity of the stain was also stronger in serous carcinomas compared to other subtypes (P = 0. 0001). Positive cells demonstrated a cytoplasmic membrane staining that was more intense in tumor cells farther away from blood supply. CONCLUSION: Overexpression of the GLUT-1 transporter is associated with the histology and grade of the tumors. Our findings show a progressive increase in the expression of the GLUT-1 transporter from the borderline tumor to the high-grade carcinomas. These data suggest that the expression of this transporter may be closely related to the malignant transformation of epithelial ovarian tumors.

Analysis of thyroid transcription factor-1 and cytokeratin 20 separates merkel cell carcinoma from small cell carcinoma of lung.

Merkel cell carcinoma needs to be separated from small cell carcinoma metastatic from visceral sites to skin. Pulmonary small cell carcinoma is the most common primary site of small cell carcinoma. We evaluated the immunophenotypic characteristics of 21 Merkel cell carcinomas and 33 small cell carcinomas of lung using thyroid transcription factor-1 and cytokeratin 20. Thyroid transcription factor-1 was 100% specific for the diagnosis of small cell carcinoma of lung associated with a diagnostic sensitivity of 85%. Cytokeratin 20 was present in 95% of Merkel cell carcinomas; however, 33% of small cell carcinoma of lung were also positive. Both antibodies typically demonstrate diffuse and intense staining of their respective tumor cells. We conclude that thyroid transcription factor-1 is a sensitive and specific marker for small cell carcinomas of lung and that a combination of thyroid transcription factor-1 and cytokeratin 20 is indicated to assist in the differentiation of metastatic small cell carcinoma of lung from merkel cell carcinoma.

Mapping of genetic deletions on the long arm of chromosome 22 in human pancreatic adenocarcinomas.

The molecular mechanisms of carcinogenesis in pancreatic cancer are still poorly understood, although the inactivation of tumor suppressor genes at multiple loci is suspected. We investigated the loss of heterozygosity (LOH) on chromosome 22 in pancreatic cancer by means of a PCR-based microsatellite analysis of archival paraffin-embedded histological sections in order to better define deleted region(s) and to test whether the NF-2 gene is involved. Using a panel of thirteen markers that spanned the long arm of chromosome 22, loss of heterozygosity was identified for at least one locus in 37% of investigated pancreatic adenocarcinomas. These deletions are clustered into two separate areas of the chromosome 22--one proximal to the NF-2 gene and one distal. The NF-2 gene itself is not involved. These regions are likely locations of tumor suppressor genes that may contribute to the development of pancreatic cancer.

Multiple mutations of the p53 gene in human mammary carcinoma.

Alteration of the p53 tumor suppressor gene is the most common genetic abnormality in human cancer. In breast cancer, depending on the stage of disease and method of detection, mutation rates of 25-60% have been observed. Multiple mutations of p53 gene in the same tumor however, are rarely reported. In this study we explored the frequency of multiple mutations of p53 gene in mammary carcinoma in a cohort of south Florida patients. Three hundred eighty-four cases of primary breast cancer diagnosed between 1984 and 1986 at the University of Miami, Jackson Medical Center were subjects of this study. Sequence analysis of exons 5 through 8 of p53 was performed on cloned PCR-amplified DNA of formalin-fixed, paraffin-embedded tumors. Two hundred thirty-four of 384 breast cancers (61%) had p53 mutation. Of those, 36 tumors showed more than one mutation; 31 tumors had two mutations, three showed three, one tumor had five mutations, and one case carried six mutations. The majority of mutations were missense (43) followed by silent (35); and most occurred within a single exon. Our study suggests that multiple mutations of p53 suppressor gene in breast cancer are more common than currently believed.

Reporting normal endometrial cells in Pap smears: an outcome appraisal.

OBJECTIVE: The purpose of this study was to determine the clinical relevance of reporting the presence of normal endometrial cells in the Pap smears of women over the age of 35 years and the significance of this practice as it relates to patient management. METHODS: From January 1992 to December 1995, normal endometrial cells were reported in 206 consecutive Pap smears of women over the age of 35 years. Clinical follow-up was available for all patients, including the results of diagnostic procedures whenever performed. RESULTS: Of the 206 women with normal endometrial cells in their Pap smears, 162 presented with the chief complaint of abnormal vaginal bleeding. They were all evaluated by direct endometrial sampling, resulting in detection of 10 endometrial hyperplasias and 7 endometrial carcinomas. The remaining 44 women who were clinically asymptomatic were followed up with only routine annual gynecologic examinations for a minimum of 3 years. All had negative clinical courses. CONCLUSION: Reporting the presence of normal endometrial cells in Pap smears has little, if any, impact on subsequent patient management. Women who present with abnormal uterine bleeding are worked up for endometrial disease regardless of their Pap smear findings. In clinically asymptomatic patients, practitioners may, and in our experience often do, choose to disregard normal endometrial cells in Pap smear reports. The negative follow-up for the asymptomatic women in our study supports this practice. Therefore, reporting the presence of normal endometrial cells in Pap smears is of no clinical relevance and may, in fact, create a management dilemma for clinicians.

Frequency and pattern of p53 gene mutation in a cohort of Spanish women with node-negative breast cancer.

Ethnic, racial and regional differences in the frequency and pattern of p53 gene mutations have been well documented. Some of these differences have been shown to have an impact on the survival of patients with breast cancer. In this study we explored the frequency and pattern of p53 abnormality in a cohort of Spanish women with node-negative breast cancer using PCR, subcloning and DNA sequencing of archival tumors. One hundred and seventy-eight cases of breast cancer diagnosed between 1981 and 1986 at the University of Oviedo Hospital in Oviedo, Spain were subjects of this study. Sequence analysis of exons 5 through 8 of p53 was performed on subcloned PCR-amplified DNA, extracted from formalin-fixed, paraffin-embedded tumors. Appropriate positive, negative, PCR, and polymerase controls were utilized and evaluated. Duplicate samples of the genomic DNA were re-evaluated on all cases showing more than one mutation. One hundred and five out of 178 breast cases (59%) carried one or more p53 gene mutations. Mutations were distributed randomly from codon 128 to 305. There were 123 (88%) transition, 10 (7%) transversion, 5 (3.5%) splice junction mutations, and 2 (1.5%) deletions. Eighty-three cases (61.5%) had missense mutation, 45 (33.5%) silent, 5 (3.5%) nonsense and 2 (1.5%) frameshifts. Eighty (75%) of 120 transitions were G:C to A:T, 11 (25%) of which occurred at CpG sites. Sixteen mutations were in novel codons not reported in breast cancers previously. Codons with the highest frequency of mutations in this group were 278, 273, 213 and 227. We also detected 27 tumors with more than one mutation within a single exon or in different exons in the same patient. These findings suggest that the frequency and pattern of p53 mutations in this group of Spanish women with breast cancer is different than those reported in the United States and Northern Europe.