Apparent improvement in survival for carcinoma of the cervix following the introduction of chemoradiation--a Will Rogers phenomenon.

The improved survival for bulky cervical cancers (> 4cm) reported with combination platinum based chemoradiation (1999) prompted a move away from surgery as these cases frequently received adjuvant radiotherapy and were exposed to the morbidity of multimodality treatment. The period pre-1999 (Group 1) was compared with post-1999 (Group 2) when chemoradiation was the preferred treatment for bulky operable cervical cancer. Significantly more cases were treated surgically among Group 1 compared with Group 2 (79% vs. 62%; P < 0.001). Switching from surgery to radiotherapy improved survival in both treatment categories (73% vs. 78% and 37% vs. 44%, respectively) but with no improvement in overall survival (70%/ov.s 70%). Survival (86%) was similar in both groups among surgically treated women with tumors < 4 cm, but significantly more in Group 2 with negative nodes received postoperative adjuvant chemoradiotherapy (Groups 1 vs. 2; 16% vs.37.5%: P < 0.001) and overall the surgically treated patients received more not less multimodality treatment (46.5% vs. 59%; P = 0.7).

A detailed morphologic and immunohistochemical comparison of pre- and postmenopausal endometriosis.

BACKGROUND: Endometriosis depends on oestrogenic stimulation for its continued growth, accounting for its prevalence during reproductive years. There is doubt among clinicians regarding its existence in postmenopausal women in the absence of exogenous or endogenous sex hormones. We postulated that endometriosis occurring in postmenopausal women would show a different morphologic and immunohistochemical profile to the disease occurring in premenopausal women. METHODS: We reviewed the most recent 100 cases of endometriosis in our department plus all cases occurring in women aged > or =50 dating back to 1999. Cases were divided into <50 (n = 91), 50-59 (8) and > or =60 (6), and analysed for extent of disease, proportions of epithelium and stroma, and amount of haemorrhage. Immunohistochemistry for oestrogen receptor (OR), progesterone receptor (PR) and CD10 was performed and analysed on all cases aged > or =50 and on controls <50. RESULTS: There was statistically less disease in older women when cases occurring in the cervix and in scars were excluded (p = 0.0191). There was no statistical difference in the proportions of epithelium or stroma, but there was a statistical difference in the amount of haemorrhage (p = 0.0154) with older women showing less haemorrhage. There was no significant difference in immunohistochemical profile. CONCLUSIONS: Endometriosis does occur in postmenopausal women but is less common, is present in smaller volumes, and is less active. It has the same immunohistochemical profile as the disease occurring in premenopausal women and we infer from this that it has the potential to reactivate given the appropriate stimulation.

Unsuspected endometrial pathology in the subfertile woman.

Routine sampling of the endometrium is not considered necessary in the investigation of female infertility in the presence of normal menstruation. We present the cases of five women diagnosed with endometrial pathology during the course of fertility investigations. Three women had atypical polypoid adenomyoma, one had complex endometrial hyperplasia and one had stage 1 endometrial adenocarcinoma. Only the latter described any abnormality in menstruation. No woman had polycystic ovarian syndrome nor any other reason in her history to suspect endometrial pathology. Two women had abnormal transvaginal ultrasound findings. Atypical polypoid adenomyoma is frequently associated with subfertility. Although usually biologically benign, malignant transformation has been reported. With current trends of increasing obesity and later age at attempted conception, the possibility of discovering endometrial pathology during fertility investigation is likely to increase. We believe that a thorough menstrual history and careful assessment of the endometrium is warranted in all women with fertility problems. A transvaginal pelvic ultrasound should be performed in the follicular (early) phase of the cycle. If this ultrasound examination and the woman's menstrual history are both normal, no further evaluation of the endometrial cavity is routinely required. Sonohysterography is superior to pelvic ultrasound in detecting intracavitary pathology and is thus recommended prior to IVF treatment. Hysteroscopy is the gold standard in the detection of intrauterine pathology and is well tolerated in the office setting. Where abnormality is suspected or detected at screening, futher investigation and concomitant treatment is essential. This is ideally performed via hysteroscopy with endometrial sampling or excision of focally abnormal areas.

Histological grading of epithelial ovarian carcinomas.

The grade of an ovarian epithelial neoplasm provides useful information. However, different approaches to grading exist and many ovarian cancers are not graded. We examined primary ovarian cancers from patients treated at our hospital and applied the 'universal' grading system. We found a significant association between grade and clinical stage, with a survival difference between grades for low-stage tumours. The application of grade is discussed in the light of developments in the grading of other gynaecological cancers.

Vibrational spectroscopy for cervical cancer pathology, from biochemical analysis to diagnostic tool.

Cervical cancer is the second most common cancer in women worldwide with 80% of cases arising in the developing world. The mortality associated with cervical cancer can be reduced if this disease is detected at the early stages of development or at the pre-malignant state (cervical intraepithelial neoplasia, CIN). The aim of this study was to investigate the potential of Raman spectroscopy as a diagnostic tool to detect biochemical changes accompanying cervical cancer progression. Raman spectra were acquired from proteins, nucleic acids, lipids and carbohydrates in order to gain an insight into the biochemical composition of cells and tissues. Spectra were also obtained from histological samples of normal, CIN and invasive carcinoma tissue from 40 patients. Multivariate analysis of the spectra was carried out to develop a classification model to discriminate normal from abnormal tissue. The results show that Raman spectroscopy displays a high sensitivity to biochemical changes in tissue during disease progression resulting in an exceptional prediction accuracy when discriminating between normal cervical tissue, invasive carcinoma and cervical intraepithelial neoplasia (CIN). Raman spectroscopy shows enormous clinical potential as a rapid non-invasive diagnostic tool for cervical and other cancers.

Early stage endometrial carcinoma--a study of management and outcome.

The surgical management of early stage endometrial carcinoma is controversial. The benefits of pelvic lymphadenectomy and administration of radiotherapy in this group have been disputed. We aimed to document the experience of stage 1 endometrial carcinoma at the National Maternity Hospital during the 10 year period 1989-1998 and to evaluate and compare clinical outcomes between retrospectively-assigned low and high-risk tumour groups. Seventy seven women were diagnosed with Stage 1 endometrial carcinoma in this period. Thirty-nine women had low-risk and 38 had high-risk tumours. Women with high-risk tumours were older and had a higher rate of lymph-vascular space invasion by tumour on histological examination. Three women (3.9%) developed disease recurrence and died of their disease; one low-risk and two high-risk tumour patients. Survival without recurrence did not differ between the two risk groups. No consistent pattern existed in surgical staging between the two risk groups. A prospectively-assigned definition of risk would minimise variations in clinical practice by providing a basis for a more tailored approach to adjuvant treatments.

The role of investigations for term stillbirths.

The aim of the study was to document the role of laboratory investigations for unexpected stillbirths at term. It was a retrospective casenote review of 75 unexpected stillbirths at term from 1995 to 1999, at the National Maternity Hospital, Dublin, Republic of Ireland. Investigations performed included blood tests, chromosomal analysis, autopsy and placental histology. Perinatal autopsy was the most informative investigation with positive findings in 49% of cases. There were positive placental findings in 37% of cases. Six of the 26 cases showed abnormal karyotyping. Of the blood tests performed, the Kleihauer-Betke test was most informative, revealing a feto-maternal haemorrhage in 8% of cases and anticardiolipin antibodies were positive in 4% of cases. FBC, TORCH and glycosylated Hb were negative in all 75 patients. Despite thorough investigations 32 of cases (43%) remained unexplained.

Ischemic fasciitis: an unusual vulvovaginal spindle cell lesion.

Ischemic fasciitis is a benign reactive lesion that most commonly occurs in elderly, immobile patients in weight-bearing areas that are subject to intermittent ischemia with subsequent tissue breakdown and regenerative changes. The lesion can be clinically and pathologically mistaken for malignancy. Here we describe the first reported case of ischemic fasciitis of the vulvovaginal region in a 20-year-old paraplegic woman who presented with a clinically suspicious vulvar swelling. Histologic examination showed surface ulceration and underlying fibrinoid necrosis with surrounding reactive atypical fibroblast-like cells and small blood vessels, the characteristic histologic features of ischemic fasciitis.

The role of placental cytogenetic cultures in intrauterine and neonatal deaths.

Knowledge of chromosomal status is useful in helping to explain fetal and neonatal deaths, but maceration has been felt to compromise the success rate. As well as skin fibroblast culture, the placenta can be sampled and this may be useful where permission for autopsy is refused. We examined placental cytogenetic results from 250 cases over a 5-year period. There was a success rate of 84.4%, with neither weight (< or > 500 g) nor maceration status showing a significant impact. Placentas sampled within 1 day were significantly more likely to give a positive culture. The results suggested that placental cytogenetic cultures should be attempted in all perinatal deaths where such information is desired.

Menstrual cycle dependent fluctuations in NK and T-lymphocyte subsets from non-pregnant human endometrium.

PROBLEM: To establish the relative proportions of endometrial lymphocyte subpopulations during the menstrual cycle. METHOD OF STUDY: Lymphocytes were investigated by flow cytometry, during the early proliferative (EP), late proliferative (LP), early secretory (ES) and late secretory (LS) phases of the menstrual cycle. RESULTS: Between the LP and LS phases, there was an increase in NK cells from 26.4% to 83.2% (P = 0.0017) of the CD45+ population. T cells decreased from 55.06% to 6.7% (P = 0.0017). Within the T-cell population, CD8+ cells decreased significantly from 63.0% in the LP phase to 54.2% in the LS phase (P = 0.04). In contrast, potential regulatory populations such as double negative (DN) T cells CD3+CD4-CD8- and natural T cells (NT) CD3+CD56+, increased significantly in the LS phase (P = 0.05; P = 0.03). gammadelta T cells, predominantly of the DN and NT phenotypes remained consistently low at all stages of the cycle (2.34%). CONCLUSION: Endometrial lymphocyte fluctuations during the menstrual cycle may reflect hormonal regulation of maternal immunity, thereby promoting tolerance at the time of implantation.

Concentrations of antimony in infants dying from SIDS and infants dying from other causes.

OBJECTIVES: Raised concentrations of antimony have been found in infants dying of sudden infant death syndrome (SIDS). The presumed source of this antimony is toxic gases generated from fire retardants that are present in cot mattresses. The aim of this study was to determine the role of antimony in SIDS. DESIGN: Samples of liver, brain, serum, and urine were collected from all patients dying from SIDS and a group of aged matched control infants who had died of other causes. SETTING: Nationwide study in Ireland. SUBJECTS: 52 infants dying from SIDS and 19 control infants aged > 7 days and < 1 year. RESULTS: The median concentration of antimony in the liver and brain of infants dying of SIDS was < 1 ng/g, with no difference detected between the infants dying from SIDS and the control infants. The range of antimony in the serum of infants dying of SIDS was 0.09-0.71 microg/litre (median, 0.26). Although no difference was found between infants dying from SIDS and control infants, SIDS infants were found to have higher concentrations when compared with healthy infants in the 1st year of life, probably as a result of release of antimony into serum after death. Urine antimony concentrations in infants dying from SIDS were < 3.91 ng/mg (corrected for creatinine) and similar to values found both in control infants and healthy infants. CONCLUSION: There is no evidence to support a causal role for antimony in SIDS.

Uterine carcinosarcomas in patients receiving tamoxifen. A report of 19 cases.

Uterine carcinosarcomas are highly aggressive neoplasms with a tendency to early extrauterine spread, high stage at diagnosis, and a poor prognosis. Tamoxifen is widely used as adjuvant therapy in the treatment of breast cancer and is known to cause endometrial proliferative lesions, including adenocarcinoma. In recent years, there have been occasional reports of uterine carcinosarcoma in patients taking tamoxifen. The aim of this study is to describe the clinicopathologic profile in 19 women taking tamoxifen who subsequently developed uterine carcinosarcoma. Nineteen patients were identified from cases of uterine carcinosarcoma diagnosed at three institutions. The case notes were examined and the pathology reviewed. The age at diagnosis of carcinosarcoma ranged from 47 to 91 years (mean 71 years). All patients were postmenopausal and received 20 mg tamoxifen daily; the duration of treatment ranged from 1 to 15 years (mean 7.1 years). The cumulative dose of tamoxifen ranged from 7.3 to 109.5 g. Ten tumors were stage I, one stage II, seven stage III, and one stage IV. Ten of the carcinosarcomas were homologous, and nine contained heterologous elements in the form of rhabdomyoblasts (six cases) or malignant cartilage (three cases). The overall prognosis was extremely poor. Fifteen patients died within 35 months of the diagnosis of carcinosarcoma (mean 12 months). Two patients are alive with very short follow-up periods, and two were lost to follow-up. The reported data support an association between tamoxifen therapy and the development of uterine carcinosarcoma. The risk is likely to be highest in those patients who have been taking tamoxifen for a prolonged period. A majority of tumors in the present study were stage I, and this suggests that uterine carcinosarcomas in patients taking tamoxifen may be diagnosed at an earlier stage than those arising de novo. However, the prognosis was still poor even with low-stage disease. Properly controlled epidemiological studies are necessary to confirm an association between tamoxifen and uterine carcinosarcoma.

Optimisation of a technique for isolating lymphocyte subsets from human endometrium.

Human endometrium is a rich source of lymphocytes which may have unique immunoregulatory functions. The aim of this study was to compare current procedures for endometrial tissue disaggregation, and optimise a method for isolation of endometrial lymphocytes. Tissue was obtained from 41 women undergoing elective hysterectomy or dilation and curettage (D&C) for reasons of benign non-endometrial pathology. Specimens were exposed to reduction/chelation, mechanical or enzymatic disruption. Optimal single cell suspensions of high yields (mean 8.8 x 10(6) range 3.5-18 x 10(6)lymphs) and good viability (60%) were obtained, using a combination of collagenase IV (200 U/ml) and DNase I (35 U/ml). Suspensions were further purified by density gradient centrifugation. Multi-colour flow cytometry was used for analysis of endometrial lymphocyte subsets. Cell suspensions were stained with mAbs specific for CD3, CD4, CD8, CD56, CD45 and CD14, and it was clearly shown that the developed method had no effect on surface glycoprotein expression. Phenotypic analysis revealed consistent populations of endometrial large granular lymphocytes (CD56+CD3-) 54.16%, and T-cells (CD3+) 37.73%. This technique was applicable to the characterisation of T-cell populations, including CD8+ (56.6%), CD4+ (44.0%), and particularly smaller populations of CD4+CD8+(3.56%), CD4-CD8-(3.34%) and CD56+(6.3%) due to it's sensitivity. In conclusion, optimised enzymatic digestion, in combination with flow cytometry provides an effective method for phenotypic examination of small endometrial lymphocyte subpopulations.

Adenocarcinoma of the uterine cervix in Ireland and Sweden: human papillomavirus infection and biologic alterations.

Paraffin-embedded samples from cervical adenocarcinomas, 19 cases from Irish patients and 19 cases from Swedish patients, were analyzed by polymerase chain reaction for the presence of infection with human papillomavirus (HPV). The results were compared with DNA ploidy, proliferation activity, and p53 and p21/WAF1 expression. The studies were performed to discover whether high-risk HPV infection in adenocarcinomas of the uterine cervix is associated with an increased proliferative activity and genomic instability. The results show that the majority (84.6%) of patients 59 years of age or younger showed HPV infection. The overall prevalence of HPV DNA was 60.5%, with the high-risk types, 16 and 18, the most frequent. HPV-16 had a prevalence of 23.7% (9 of 38), and HPV-18 had a prevalence of 26.3% (10 of 38). The HPV-positive tumors predominantly showed a tetraploid DNA distribution pattern, whereas HPV-negative tumors more frequently showed highly scattered aneuploid DNA profiles. Both HPV-positive and HPV-negative cases displayed high proliferative activity, as indicated by high Ki-67 and cyclin A immunoreactivity. Tumor suppressor gene analysis detected low p53 expression and high p21/WAF1 expression in HPV-positive patients and high p53 expression without simultaneously increased p21/WAF1 (indicative of mutated p53) in HPV-negative cases in the groups of women older than 59 years of age.

Monoclonal antibodies raised to paraffin wax embedded archival tissue; feasibility study of their potential to detect novel antigenic markers.

A study to determine the feasibility of using archival paraffin wax embedded tissue to generate monoclonal antibodies is described. Specifically, monoclonal antibodies were raised to paraffin wax embedded normal human kidney tissue to test the possibility of producing antibodies to such tissue samples prior to attempting generation of antibodies to valuable archival tissue. Multiple sections (10 x 5 microm) were pooled and dewaxed as for immunohistochemical procedures and combined with Freund's adjuvant for immunization of BALB/c mice in vivo. Immunized spleen cells were fused with SP2 myeloma cells and subsequent clones screened on paraffin wax embedded normal human kidney sections, a range of cell lines and normal mouse tissue. Supernatants from 11 wells (from a total of 90 wells screened) showed different staining patterns on sections of paraffin wax embedded kidney. One clone, 1/11C, (isotype IgG1) which exhibited strong staining on all kidney tubules by immunohistochemical studies (glomeruli interstitium and vessels were unstained) and identified a band at 52 kDa on immunoblots of dewaxed kidney tissue (as used for immunogen) was chosen for further characterization. Immunoblotting of five mammalian cell lines showed differential expression of this 52 kDa band (distinct expression on 3/5, weak expression on 2/5 cell lines) whereas, all cell lines displayed a band at 44 kDa and a third band at 70 kDa was observed on 2/5 cell lines. In mouse tissue extracts, the 52 kDa band was identified in kidney tissue only (not in the lung, liver or spleen) with the 44 kDa and 70 kDa bands weakly expressed in all tissues. This preliminary investigation of a novel approach to identifying possible new antigenic markers or producing monoclonal antibodies which react better to known antigens on sections of paraffin wax embedded tissue showed that this method is feasible. The need to have a comprehensive screening system in place and the ability to identify potentially useful clones after the initial screening is paramount due to the relative scarcity of screening material (archival tissue sections) and the tedious nature of the screening method.

A new mdr-1 encoded P-170 specific monoclonal antibody: (6/1C) on paraffin wax embedded tissue without pretreatment of sections.

AIMS: The generation and characterisation of a monoclonal antibody that specifically recognises the mdr-1 encoded protein, P-glycoprotein (P-170), on routinely processed formalin fixed, paraffin wax embedded tissue sections. METHODS: The monoclonal antibody, designated 6/1C, was produced following a combination of in vivo and in vitro immunisation regimens in Balb/c mice with a synthetic 12 amino acid peptide that corresponds to amino acids 21-32 (believed to be intracellularly located) of P-170 and has insignificant homology with the mdr-3 encoded P-170. Antibody 6/1C was characterised by western blotting and immunocytochemistry on cytospins of paired multidrug resistant or sensitive cell lines, including mdr-1 and mdr-3 transfected cells, and by immunohistochemistry on normal and malignant formalin fixed paraffin wax embedded tissue sections. RESULTS: Antibody 6/1C showed a single band at 170 kDa on western blots of multidrug resistant cell lysates and mdr-1 transfected cell lysates that was absent on similar preparations of drug sensitive cells and mdr-3 transfected cells. Immunocytochemical studies on cytospins of multidrug resistant cells and mdr-1 transfected cells revealed strong inner plasma membrane/cytoplasmic staining. Staining was negligible on drug sensitive cells and cells transfected with the mdr-3 gene. Immunohistochemical studies on formalin fixed, paraffin wax embedded normal adult kidney, liver, and breast tissue and a range of fetal tissues exhibited staining patterns of a variety of secretory surfaces consistent with documented mdr-1 specific staining. Specific staining of malignant cells in similarly treated sections of breast tumours was seen also with antibody 6/1C. Staining on paraffin wax embedded tissue with this antibody did not require any pretreatment of tissue sections. CONCLUSIONS: This new monoclonal antibody, chosen for its specificity with the mdr-1 encoded P-170 and its reactivity on routinely fixed paraffin wax embedded tissue samples without pretreatment, appears to be useful for the investigation of P-170 in archival material. It is especially useful for retrospective studies on pretreatment and post-treatment tissue sections, and could help establish when and how rapidly mdr-1 associated drug resistance develops during chemotherapeutic regimens. Immunohistochemical assessment of P-170 expression in many cancers has potential for diagnostic purposes and may influence the choice of chemotherapeutic drugs used in the treatment of refractory tumours.

Genotypic mapping of HPV and assessment of EBV prevalence in endocervical lesions.

AIMS: To examine the prevalence of human papillomavirus (HPV) and Epstein-Barr virus (EBV) in low grade glandular intraepithelial lesions of the cervix, adenocarcinoma with high grade glandular intraepithelial lesions combined, and adenocarcinomas; and to perform a genotyping mapping analysis of endocervical carcinomas to determine the extent of HPV infections in such lesions. MATERIAL: Archival paraffin wax embeded material from the files of the departments of pathology, National Maternity Hospital, Dublin, and University College Cork, Ireland. METHODS: HPV prevalence was examined using type specific HPV PCR, general primer HPV PCR (pan HPV screen), nonisotopic in situ hybridisation (NISH), and PCR in situ hybridisation (PCR-ISH). In situ hybridisation was performed using fluorescein labelled oligonucleotide cocktail for eber transcripts of EBV. Genotypic analysis was performed, in all cases where possible, using a grid system. RESULTS: HPV 16 and 18 were predominantly identified in low grade glandular intraepithelial lesions, high grade glandular intraepithelial lesions, and adenocarcinomas, with HPV prevalence increasing with grade of dysplasia. EBV was only identified in subepithelial lymphocytes in a minority of cases. No link could be shown between HPV and EBV in endocervical lesions. HPV infection was not clonal in endocervical cancer and coexistent adjacent cervical intraepithelial neoplasia, where present, tended to show a similar HPV type. CONCLUSIONS: The restriction of HPV types 16 and 18 to endocervical lesions suggests that their effect is restricted and specific to endocervical mucosa, but the mechanism of interaction is currently unknown.

Multiple drug resistance-related messenger RNA expression in archival formalin-fixed paraffin-embedded human breast tumour tissue.

A method is described by which RNA, suitable for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, can be extracted from formalin-fixed paraffin-embedded (FFPE) tissues and subsequently used for detecting the expression of several genes. Using this technique, RNA can be extracted from specimens, quantified, reverse transcribed and regions of interest amplified and analysed within 36 h. The tissue specimens included in this study were from human breast carcinoma, investigating a range of genes associated with the development and/or maintenance of multiple drug resistance (MDR). This technique, applied to archival tissues, offers great potential for increasing our understanding of alterations in expression levels of genes associated with MDR. The method developed is also applicable to studies on expression of other genes in paraffin-embedded tissues.

The echogenic thalamus in hypoxic ischaemic encephalopathy.

This paper reports 16 term infants in whom an echogenic thalamus was identified on cranial ultrasonography. Fourteen of the patients suffered severe birth asphyxia. The prognostic significance of this finding and the underlying pathogenesis is assessed.