Co-overexpression of bcl-2 and c-myc in uterine cervix carcinomas and premalignant lesions

To establish the role of co-overexpression of bcl-2 and c-myc protooncogenes in uterine cervix carcinogenesis, we examined 138 tissue samples of low grade cervical squamous intraepithelial lesions (SIL), high grade SIL, portio vaginalis uteri (PVU) carcinoma in situ and PVU carcinoma invasive, stage IA-IIA (study group) and 36 samples without SIL or malignancy (control group). The expression of bcl-2 and c-myc was detected immunohistochemically using a monoclonal antibody. Fisher’s exact test (P<0.05) was used to assess statistical significance. Overexpression of bcl-2 was found to increase in direct relation to the grade of the cervical lesions. High sensitivity was of great diagnostic significance for the detection of these types of changes in the uterine cervix. On the basis of high predictive values it can be said that in patients with bcl-2 overexpression there is a great possibility that they have premalignant or malignant changes in the uterine cervix. Co-overexpression of bcl-2 and c-myc oncogenes was found only in patients with PVU invasive carcinoma (6/26-23.0%). Statistically significant difference was not found in the frequency of co-overexpression in patients with PVU invasive carcinoma in relation to the control group (Fisher’s test; P=0.064). The method's sensitivity of determining these oncogenes with the aim of detecting PVU invasive carcinoma was 23%, while specificity was 72.2%. On the basis of high predictive values (100%), speaking in statistical terms, it can be concluded that all patients with co-overexpression of bcl-2 and c-myc oncogenes will have PVU invasive carcinoma. We confirmed in our research that co-overexpression of bcl-2 and c-myc oncogenes was increased only in PVU invasive carcinoma. However, a more extensive series of samples and additional tests are required to establish the prognostic significance of bcl-2 and c-myc co-overexpression in cervical carcinogenesis.     

Digital image analysis of AgNORs in cervical smears of women with premalignant and malignant lesions of the uterine cervix

We performed a hospital-based, unmatched case-control study to investigate the association between progressive stages of cervical neoplasia and digital analysis of cell proliferation by silver stained nucleolus organizer region associated proteins (AgNORs). We measured cell proliferation levels in the cervical epithelial cells of 10 women with low grade squamous intraepithelial lesions (LG-SIL), eight with high grade squamous intraepithelial lesions (HG-SIL), 11 with cervical cancer (CC) and eight with no cervical lesions (controls) using the AgNORs technique. Cell proliferation was measured by digital image analysis (DIA). DIA revealed increased total areas of AgNORs in HG-SIL and CC compared to LG-SIL and control patients. AgNORs with a kidney or cluster shape exhibited greater areas than those with a spherical or long shape. We propose a cut-off of 118 pixels to differentiate benign (control and LG-SIL) from malignant (HG-SIL and CC) lesions. DIA of AgNORs is a simple and inexpensive method for studying proliferation. The increased total area of AgNORs in malignant lesions provides information regarding cell behavior and may be related to cervical carcinogenesis; however, further validation studies are required to establish its usefulness in cytological analysis.     

The role of mast cell tryptase in neoangiogenesis of premalignant and malignant lesions of the uterine cervix.

Recently, mast cell tryptase has been identified as another potent proangiogenic factor in tumors, along with fibroblast and vascular endothelial growth factors. Its role has been studied in a number of cancers, including carcinoma of the uterine cervix, with discordant results. Our aim was to study the expression of tryptase and bFGF in mast cells (MCs) during development of neoangiogenesis in premalignant and malignant lesions of the cervix. Biopsy specimens from 21 patients without cancer and from 63 patients with dysplasias and squamous cell carcinomas were used. They were stained with Alcian blue-safranin O (ABSO) and immunostained with specific antibodies against factor VIII, CD105, tryptase, and bFGF. Tryptase-positive mast cells increased with tumor progression and were close to newly formed blood vessels. Vascularization showed a linear increase from dysplasia to invasive cancer. We suggest that MC tryptase may upregulate neoangiogenesis in carcinogenesis of the uterine cervix.     

Inactivation of SLIT2-ROBO1/2 pathway in premalignant lesions of uterine cervix: clinical and prognostic significances

The SLIT2-ROBO1/2 pathways control diverse biological processes, including growth regulation. To understand the role of SLIT2 and ROBO1/2 in cervical carcinogenesis, firstly their RNA expression profiles were screened in 21 primary uterine cervical carcinoma (CACX) samples and two CACX cell lines. Highly reduced expressions of these genes were evident. Concomitant alterations [deletion/methylation] of the genes were then analyzed in 23 cervical intraepithelial neoplasia (CIN) and 110 CACX samples. In CIN, SLIT2 was deleted in 22% samples compared to 9% for ROBO1 and none for ROBO2, whereas comparable methylation was observed for both SLIT2 (30%) and ROBO1 (22%) followed by ROBO2 (9%). In CACX, alteration of the genes were in the following order: Deletion:ROBO1 (48%) > SLIT2 (35%) > ROBO2 (33%), Methylation:SLIT2 (34%) > ROBO1 (29%) > ROBO2 (26%). Overall alterations of SLIT2 and/or ROBO1 (44%) and SLIT2 and/or ROBO2 (39%) were high in CIN followed by significant increase in stage I/II tumors, suggesting deregulation of these interactions in premalignant lesions and early invasive tumors. Immunohistochemical analysis of SLIT2 and ROBO1/2 in CACX also showed reduced expression concordant with molecular alterations. Alteration of all these genes predicted poor patient outcome. Multiparous (≥ 5) women with altered SLIT2 and ROBO1 along with advanced tumor stage (III/IV) and early sexual debut (<19 years) had worst prognosis. Our data suggests the importance of abrogation of SLIT2-ROBO1 and SLIT2-ROBO2 interactions in the initiation and progression of CACX and also for early diagnosis and prognosis of the disease.     

Nomenclature of precancerous lesions of the uterine cervix. A position paper

There is universal and almost unanimous belief that there is a need for a radical revision of the terminology relating to the epithelial abnormalities of the uterine cervix that precede invasive carcinoma. Although this belief has been held for some years, no generally accepted alternative system has, as yet, been devised. This position paper examines (1) the origin of the existing system of terminology, (2) the problems associated with this system and (3) the requirements of any new, alternative nomenclature. The various alternatives that have been suggested are considered with particular reference to their advantages and disadvantages. Only two such alternatives are considered to be acceptable, and it is suggested that neither is likely to gain universal acceptance at this time. However, it is possible that new diagnostic techniques, with their potential for a more accurate evaluation of cell abnormalities, may facilitate the development or application of an acceptable system of terminology.     

Discrimination between precancerous and cancerous lesions of the uterine cervix by DNA measurements on tissue sections

Morphologically typical uterine cervical biopsies were separated into normal cervices, condylomas and cervical intraepithelial neoplasias (CIN) grades I, II and III. At least 100 nuclei per lesion were measured on 4 micron Feulgen-stained sections using a Zeiss microspectrophotometer, with a variant of the plug method used to compute the nuclear DNA content. DNA distribution histograms were then decomposed into subsets of diploid, tetraploid, octoploid and aneuploid cells. The decomposition, which assumed a log-normal model of polydiploidy distribution, led to the identification of six indices: (1) the percentage of diploid cells, (2) the percentage of tetraploid cells, (3) the percentage of octoploid cells, (4) the percentage of aneuploid cells with DNA contents less than tetraploidy, (5) the percentage of aneuploid cells with DNA contents between tetraploidy and octaploidy and (6) the percentage of aneuploid cells with DNA contents greater than octoploidy. These indices, along with the mean nuclear radius, the 5c exceeding rate and the 2c deviation index, generated a nine-dimensional space. Two methods of discriminant analysis on this space showed discriminating powers of 78.22% and 87.13%, respectively, as compared to the original diagnoses. The most discriminating variable in both analyses appeared to be the percentage of octoploid cells.     

Natural history of precancerous and early cancerous lesions of the uterine cervix

A prospective study of cervical dysplasia cases and control cases matched for age and parity was undertaken in search of factors related to cervical carcinogenesis. Cytologic examination of 66,736 women revealed negative findings in 28.5%, inflammation in 70.3%, dysplasia in 1.4% and carcinoma in 0.1% of the cases. Data on epidemiologic features, cytomorphologic characteristics and serologic findings of antibodies to herpes simplex virus (HSV) were collected for proven cancer patients, dysplasia cases and control subjects. Cancer patients revealed significantly elevated antibodies to HSV as compared to the controls. The analysis revealed a higher proportion of dysplasia cases with an age at consummation of marriage of less than or equal to 15 years as compared to controls, with a relative risk of 1.5 (P less than .05). Similarly, a higher proportion of women with dysplasia had HSV-II-specific antibodies as compared to control women. The relative risk was found to be 1.3, which was not statistically significant (P greater than .05). The Mantel-Haenszel summary relative risk between antibodies to HSV and the two groups (dysplasia cases and controls), adjusted for the age at consummation of marriage, worked out to be 1.38, which was also statistically not significant (P greater than .05). The overall progression rate of dysplasia to malignancy was found to be 11.7% at the end of 54 months (during a total follow-up period of 84 months). Progression to cancer was highest in severe dysplasia cases and less in mild dysplasia cases. The progression rates were also significantly higher in the group of women who revealed antibodies to HSV II. Similar differences in the progression rates were observed with regard to the age at consummation of marriage.     

DNA sequences of human papillomavirus types 11, 16, and 18 in lesions of the uterine cervix in the west of Scotland.

Punch biopsy specimens of the cervix were examined both histologically and for the presence of human papillomavirus (HPV) DNA sequences. The presence of HPV DNA sequences was sought with the Southern blot technique using radioactively labelled HPV-6, 11, 16, and 18 DNA probes, both together and separately. Twenty six biopsy specimens were examined. Histological examination showed cervical intraepithelial neoplasia grade 2 or 3 in 16 specimens, viral changes (koilocytosis) in four, and inflammation or a normal appearance in three. Eleven specimens were negative for HPV DNA sequences, 10 contained HPV-16 DNA, four contained HPV-18 DNA, and one contained both HPV-18 and HPV-11 DNA. Episomal HPV-16 DNA was detected in one case of cervical intraepithelial neoplasia grade 3 and in five cases of cervical intraepithelial neoplasia grade 2/3 with koilocytosis; and episomal HPV-18 DNA was found in two specimens classed as cervical intraepithelial neoplasia grade 2/3, one of which also contained HPV-11 DNA, and in one specimen that showed viral changes alone. Integrated HPV DNA was found in six specimens (four with HPV-16 DNA and two with HPV-18 DNA), including two cases of chronically inflamed cervix with no histological evidence of viral infection or cervical intraepithelial neoplasia. Detection of viral DNA in early lesions may identify patients at risk of malignant progression. This is the first report of HPV-18 DNA in cervical intraepithelial neoplasia in Scotland.     

Two-mutation model for carcinogenesis: joint analysis of premalignant and malignant lesions.

A model for carcinogenesis that postulates two rate-limiting events for malignant transformation is a generalization of the recessive oncogenesis hypothesis, according to which inactivation of homologous tumor suppressor genes leads to cancer. This model has been shown to be consistent with a large body of epidemiologic and experimental data and has recently been used for the analysis of altered hepatic foci in rodents. These foci are considered to be premalignant lesions. In this paper the necessary mathematics for the joint analysis of premalignant and malignant lesions are developed within the framework of this model.     

DNA-cytometric diagnosis of prospective malignancy in borderline lesions of the uterine cervix

Interactive DNA cytometry was used for the diagnosis of prospective malignancy in 48 smears with borderline lesions (mild and moderate dysplasias) of the uterine cervix. In addition, 183 smears with benign squamous epithelia, 38 with carcinoma in situ and 7 with invasive squamous carcinoma were also measured. Nuclear Feulgen-DNA measurements were performed using various methods, and the resulting data were analyzed by an algorithm for a DNA-cytophotometric diagnosis of malignancy. The results were compared with the data on follow-up and subsequent histologic studies in these cases. There was no false-positive diagnosis in the 183 benign smears and only 1 false-negative diagnosis in the 76 histologically proven squamous-cell carcinomas, which yields a specificity of 100% and a sensitivity of 98.6%. The sensitivity for the detection of subsequent histologically proven malignancy in cases with cytologically mild or moderate dysplasia amounted to 97%. In 13 borderline cases, there was a mean interval of 21 months between the taking of the cytologic smear on which the DNA diagnosis of malignancy was made and the date on which the histologic confirmation of malignancy was made. In 17% of the cytologically dysplastic cases, the DNA diagnosis of malignancy was not verified by subsequent histologic investigation. These results indicate that interactive DNA cytometry is able to detect prospective malignancy in smears from borderline lesions of the uterine cervix with a high sensitivity.     

Adenoid basal lesions of the uterine cervix: evolving terminology and clinicopathological concepts

The epithelial proliferations that are designated adenoid basal carcinoma (ABC) in the current classification from the World Health Organization represent <1% of all cervical malignancies. These lesions may be associated, and occasionally show morphologic transitions with, conventional cervical malignancies. The determination of the precise frequency with which these so-called ABCs show this association is hampered by the inherent selection bias in the reported cases. However, this frequency appears to be substantial (>15%). The biologic course of ABCs that are associated with separate malignancies is largely dependent on the clinicopathologic parameters of the associated malignancies. Morphologically pure lesions, in contrast, have largely been associated with favorable patient outcomes, as none of the 66 reported patients have experienced tumor recurrence, metastases or tumor-associated death, irrespective of the modality of treatment. Although the finding of genome integrated high-risk human papillomavirus (HPV) types and p53 alterations in adenoid basal lesions (ABL) argue in support of their neoplastic nature, we identified no lines evidence that suggest an inherent malignancy for morphologically pure lesions. The finding of morphologic transitions between ABLs and conventional malignancies and shared HPV types in these areas, suggest that ABLs have some malignant potential. However, the precise magnitude of this potential is not readily quantifiable and should not dictate the management of morphologically pure lesions that are entirely evaluable. ABLs continue to occupy a unique position in human oncology in which the term carcinoma (without an in-situ suffix) is applied to a tumor that has not been shown to recur, metastasize or cause death. We concur with a previous proposal that the term ABC should be discarded and replaced with Adenoid Basal Epithelioma (ABE). In our opinion, there is insufficient evidence at present time to expose patients with morphologically pure lesions to the ominous implications – social, psychological, medical, financial – of a "carcinoma" diagnosis. Morphologically impure lesions should not be designated ABC or ABE. Furthermore, given the uncertainties regarding the frequency with which ABE are associated with separate malignancies, we suggest that the ABE designation only be applied when the tumor in question is entirely evaluable e.g in a hysterectomy specimen or in an excisional biopsy with negative margins. Otherwise, the generic designation Adenoid Basal Tumor is preferable. This approach strikes an appropriate balance between the need to prevent over-treatment of pure lesions on one hand, and the need to ensure that the lesions are indeed pure on the other.     

False-negative Papanicolaou smears from women with cancerous and precancerous lesions of the uterine cervix

The occurrence from 1980 to 1989 of false-negative Papanicolaou smears in women with cancerous and precancerous lesions of the uterine cervix was studied. The 4,781 cases of cancer (2,814 invasive carcinomas and 593 carcinomas in situ) and precancerous lesions (418 severe dysplasias, 748 moderate dysplasias and 208 mild dysplasias) included 70 cases (1.5%) with false-negative smears. These 70 cases included 43 invasive carcinomas (61.4%), 17 carcinomas in situ and adenocarcinomas in situ (24.2%), and 10 dysplasias (14.4%); all were diagnosed histologically. The mean age of women with false-negative smears was 44.1 +/- 13.7 years. Review of the original cytologic samples showed a screening error in 41 cases (58.5%), an interpretation error in 2 cases (2.9%) and a sampling error in 27 cases (38.6%). Methods for eliminating false-negative smears are discussed.     

P-cadherin expression in glandular lesions of the uterine cervix detected by liquid-based cytology

OBJECTIVE: To study P-cadherin aberrant expression as a possible marker for cervical adenocarcinomas in cytological samples. METHODS: We studied P-cadherin immunoexpression in liquid-based cervical cytology samples of biopsy-proven cervical lesions. RESULTS: We found a statistically significant correlation between P-cadherin expression and a cytological diagnosis of malignancy, either glandular or squamous (P < 0.0001). Twenty-two of 33 malignant cases showed P-cadherin membrane staining. None of the 30 benign cases tested showed membrane staining, but three of them displayed an aberrant nuclear P-cadherin expression. CONCLUSIONS: We concluded that P-cadherin can be used to discriminate between malignant and benign cervical cytological specimens, but not to discriminate glandular from squamous lesions.     

Immunohistochemical localization of glutathione S-transferase in preneoplastic and neoplastic lesions of the human uterine cervix

A mouse monoclonal antibody and a rabbit polyclonal antibody prepared against the placental form of the enzyme glutathione S-transferase (GST-pi) were used to immunohistochemically stain normal and neoplastic human uterine cervical tissues from 88 cases. Of 65 cases of preneoplastic squamous lesions and invasive carcinomas of the cervix, 94% stained with the monoclonal antibody and 100% with the polyclonal antibody. In the 23 benign tissues, staining of ectocervical squamous epithelium was generally not observed; however, areas of reserve-cell hyperplasia, immature squamous metaplasia and adjacent endocervical cells did show staining (68% with the monoclonal antibody and 95% with the polyclonal antibody). Many of the positive tissue types showed a variety of staining patterns and intensities. These findings do not support the concept that GST-pi staining can be used to distinguish preneoplastic lesions of the cervix from benign reactive or proliferative processes. These results are of interest in the investigation of cervical carcinogenesis since GST-pi may be involved in an early stage of neoplastic transformation of the cervical epithelium. The correlation of these findings with the results of human papillomavirus testing and DNA content analysis should be of interest in determining the relationship of this enzyme to cervical neoplasia.     

Prognostic significance of DNA cytometry in carcinoma of the uterine cervix FIGO stage IB and II.

OBJECTIVE: To assess the prognostic value of DNA-image cytometry in cervical carcinoma of the uterus and its relation to other established prognostic factors. STUDY DESIGN: The study included 116 cases of cervical carcinoma FIGO stages IB and II which were treated with radical abdominal hysterectomy. The median follow-up was 55 months (range 1-162 months). DNA image cytometry was performed on cytologic specimens prepared by enzymatic cell separation from formalin-fixed, paraffin-embedded tissues. DNA stemline ploidy, DNA stemline aneuploidy, 5c exceeding rate, 9c exceeding rate, 2c deviation index, and DNA malignancy grade were computed. DNA-variables as well as various clinical and histological variables were related to survival rates. RESULTS: In multivariate statistical analysis DNA stemline ploidy using 2.2c as a cut-off value and FIGO stage showed to be statistically significant available presurgery predictors of survival, whereas the postsurgical parameters lymphonodal status, tumor size and parametrial involvement were significantly correlated with survival. The synopsis of all parameters in a multivariate Cox model indicated that - with declining relevance - the number of positive pelvic lymph nodes, DNA stemline ploidy using a cut-off level at a modal value of 2.2c, largest pelvic lymph node, 5c exceeding rate, and ratio of carcinoma area to cervix area, were of predictive value for survival. CONCLUSIONS: Our results suggest that prognostic information deducted from classical staging parameters is successfully complemented by DNA image cytometry which can be applied pretherapeutically.     

Expression analysis of PMP22/Gas3 in premalignant and malignant pancreatic lesions.

PMP22 is a structural protein of Schwann cells, but it also influences cell proliferation. In the present study, quantitative RT-PCR (QRT-PCR) and immunohistochemistry were used to determine PMP22 mRNA levels and to localize PMP22 in the normal pancreas (n=20), chronic pancreatitis (CP) (n=22), pancreatic ductal adenocarcinoma (PDAC) (n=31), intraductal papillary mucinous neoplasms (IPMN) (n=9), mucinous cystic tumors (MCN) (n=4), and in a panel of PanIN lesions (n=29). PMP22 mRNA levels were significantly higher in CP (3-fold) and PDAC (2.5-fold), compared to normal pancreatic tissues. PMP22 expression was restricted to nerves in the normal pancreas, while in CP and PDAC PMP22 was also expressed in PanIN lesions and in a small percentage of pancreatic cancer cells. PMP22 was weak to absent in the tumor cells of IPMNs and MCNs. PMP22 mRNA was present at different levels in cultured pancreatic cancer cells and up-regulated by transforming growth factor (TGF)-beta1 in 2 of 8 of these cell lines. In conclusion, PMP22 expression is present in both CP and PDAC tissues. Its expression in PanIN lesions and some pancreatic cancer cells in vitro and in vivo suggests a role of PMP22 in the neoplastic transformation process from the normal pancreas to pre-malignant lesions to pancreatic cancer.