The role of ultrasound-guided cytology of groin lymph nodes in the management of squamous cell carcinoma of the vulva: 5-year experience in 44 patients.

AIM: To assess the accuracy of ultrasound combined with fine-needle aspiration cytology (FNAC) in the detection of lymph node metastasis in patients with squamous cell carcinoma of the vulva. MATERIALS AND METHODS: The groin nodes of 44 consecutive patients with primary squamous cell carcinoma of the vulva undergoing groin node dissection were assessed with ultrasound and FNAC. The results were compared with histology from subsequent inguinofemoral lymph node dissection. Twenty-nine patients underwent bilateral groin node dissections and 15 unilateral providing comparable data for 73 groins. RESULTS: Histology demonstrated metastatic disease in 28 groins and no evidence of metastatic disease in 45. Ultrasound agreed with the histology in 67 of the 73 groins (92%), with two false-positives, four false-negatives and two indeterminate appearances. Cytology agreed with the histology in 65 of 72 FNAC samples obtained (90%), with six false-negatives, and one indeterminate result. No false-positive cytology results were seen. Ultrasound and FNAC together failed to detect metastatic disease in four groins, one with an indeterminate ultrasound appearance, another with indeterminate cytology, the two others each having a single positive inguinal node despite a negative ultrasound and FNAC. CONCLUSION: The combination of ultrasound and FNAC provides a sensitive and specific tool for pre-operative assessment and may prevent unnecessary groin dissection and the attendant morbidity in selected patients with vulval cancer.

Squamous cell carcinoma of the breast: clinico-pathologic implications and outcome.

AIMS: Pure squamous cell carcinoma (SCC) of the breast is a rare tumour and little is known about long-term outcome. We report our experience of a consecutive series of patients. METHODS: All patients with SCC treated at our institution between 1970 and 2001 were included. The pathological features, outcome and prognosis were studied. RESULTS: Eleven patients were identified. The median age was 55 (38-90) years and median follow-up was 62 (3-332) months. Four tumours were T1, three were T2 and three were T3 (one tumour size was unknown). There were seven poorly differentiated and three moderately differentiated SCC. Tumour grade could not be assessed in one patient. Primary treatment was mastectomy in six patients, wide local excision in four patients and radiotherapy in one patient. There was lymph node (LN) involvement in two patients. Oestrogen receptor status was assessed in seven patients and only one tumour was positive. Adjuvant chemotherapy was given to three patients and five patients received adjuvant radiotherapy. Two patients developed local recurrence at 5 and 12 months and three patients developed distant metastasis at 2, 36 and 306 months. Three patients were treated with chemotherapy at recurrence. Three patients have died of the disease, two are alive with disease and six remain well. The 2- and 5-year overall survival was 80% (SE=13%) and 67% (SE=16%) respectively. Large tumour size and positive LN status were prognostic indicators of poor outcome. CONCLUSION: SCC of the breast adopts an aggressive course with outcome comparable to poorly differentiated breast adenocarcinoma.

A retrospective study comparing the individual modalities of triple assessment in the pre-operative diagnosis of invasive lobular breast carcinoma.

AIMS: Early invasive lobular breast carcinoma (ILC) is associated with few symptoms and signs. The individual sensitivity of clinical examination, mammography, ultrasonography, cytology and core biopsy have each been reported to be of limited value. The aim of this study was to evaluate the accuracy of triple assessment in the pre-operative detection of patients identified to have ILC from their surgical pathology. METHODS: Pure ILC was defined as tumours containing at least 90% lobular features. The triple assessment of 273 patients diagnosed primarily at our institution were reviewed. RESULTS: 87.5% of women were symptomatic and 12.5% were screen detected. The mean patient age was 59 (range 30-81) years and the median tumour size was 26 (range 5-110) mm. The main mammographic abnormalities were a spiculated lesion (33.3%), an ill-defined mass (33.3%) or architectural distortion (23.5%). The sensitivities for detecting ILC of each modality were: clinical examination (76.6%), mammography (79.8%), ultrasound examination (93.9%), fine-needle aspiration cytology (FNAC) (60.5%) and core biopsy (90.8%). Combining the three modalities of clinical examination, imaging and cyto/pathology increased the pre-operative detection rate of ILC. CONCLUSION: Triple assessment is useful in the diagnosis of ILC. As the features of ILC may be subtle, a high index of suspicion is required to facilitate early diagnosis.

Cellular characteristics of nipple aspiration fluid during the menstrual cycle in healthy premenopausal women.

Cellular characteristics of nipple aspiration fluid during the menstrual cycle in healthy premenopausal women Fifteen healthy premenopausal female volunteers underwent weekly nipple aspiration of ductal fluid from both breasts during two menstrual cycles to investigate the variability of the cellular profile of the ductal fluid. Ductal fluid was successfully obtained using breast massage and nipple-areolar suction from 247/280 (89%) breasts. 83% of samples available for cytological analysis were cellular and 30% of cellular aspirates contained ductal epithelial cells identified using standard morphological criteria. No significant variation in cell number or cell type was identified during the menstrual cycle. All samples tested had an 'H' score of zero for oestrogen receptor. Seven out of 14 women expressed the proliferation marker Mcm-2 in the cells of at least one of the specimens, with no evidence of a menstrual cycle influence on expression. In conclusion, the cellular profile of breast ductal fluid did not vary consistently during the menstrual cycle, permitting future breast cancer screening studies incorporating serial nipple aspirations to be performed independent of the phase of the cycle.

Investigating lymphadenopathy--report on the first 12 months of the lymph node diagnostic clinic at the Royal Marsden Hospital.

The lymph node diagnostic clinic was set up at the Royal Marsden Hospital to provide a direct access service for general practitioners. In the first year 82 patients were seen. The malignancy pick-up rate was 19.5% which compares very favourably to rates in breast and colorectal clinics. Patient and general practitioner satisfaction with the service was high.

The role of high resolution ultrasound with guided cytology of groin lymph nodes in the management of squamous cell carcinoma of the vulva: a pilot study.

The accuracy of high resolution ultrasound with guided fine needle aspiration cytology in detecting inguinal lymph node involvement was assessed in 24 women undergoing radical vulvectomy and groin node dissection for squamous cell vulval cancer. Of the 43 groins dissected, ultrasound correctly diagnosed the lymph node status in 36, with five false positive and two false negative results. Cytology in 40 groins showed no false positive and five false negative results. The sensitivity and specificity for the combined techniques were 83% and 82% respectively. Assessed together, the combined technique failed to detect metastatic disease in two groins; in both cases the extent of nodal metastatic involvement was a solitary focus < 3 mm in diameter. The ultrasound and fine needle aspiration procedure is safe and well tolerated and can be repeated as needed for surveillance. The authors suggest that this procedure should be evaluated further to determine whether a policy of individual selection for lymphadenectomy can be implemented based on this technique.

Quantitative changes in cytological molecular markers during primary medical treatment of breast cancer: a pilot study.

AIM: To quantify the changes in biological molecular markers during primary medical treatment in patients with operable breast cancer and to assess their possible relationship with response to treatment. METHODS: The treatment group consisted of 31 patients with operable breast carcinomas, median age 57 years (range 41-67), treated with four 3-weekly cycles of chemotherapy with Mitoxantrone, methotrexate (+/- mitomycin C), and tamoxifen before surgery. Fine needle aspiration (FNA) was used to obtain samples from patients prior to and at 10 or 21 days post-treatment. The following molecular markers were assessed: estrogen receptor (ER), progesterone receptor (PgR), p53, Bcl-2, and Ki67 measured by immunocytochemistry, and ploidy and S-phase fraction (SPF) by flow cytometry. To evaluate the reproducibility of the technique, repeat FNA was performed in a separate non-treatment control group of 20 patients and the same molecular markers assessed, two weeks after the first sample with no intervening treatment. RESULTS: The non-treatment control group showed a high reproducibility for the measurement of molecular markers from repeat FNA. In the treatment group there was a non-significant reduction in SPF and a significant reduction (p = 0.005) in Ki67. Patients who responded to neoadjuvant therapy were more likely to have a reduction in these two markers than those who failed to respond. Similarly, a reduction in ER scores was observed between the first and second samples (p = 0.04). For PgR, the change between the first and second samples was not significant although there was a significant difference between responders and non-responders (p = 0.03). All nine patients with an increase in PgR were responders. No significant changes in p53 or Bcl-2 were observed during treatment. CONCLUSION: Molecular markers can be adequately measured from FNA samples prior to and during neoadjuvant therapy. Changes in cellular proliferation and hormone receptors have been shown that may be related to tumour response. These relationships should be assessed in a larger cohort of patients.

Cytological evaluation of biological prognostic markers from primary breast carcinomas.

This study was undertaken to evaluate our ability to detect multiple molecular markers of prognosis and response to treatment in fine needle aspirates (FNA) from patients with primary breast carcinomas. 147 patients with operable primary breast carcinomas who had been recruited to a randomized trial of primary medical therapy (PMT) versus adjuvant chemoendocrine therapy were analysed. FNAs were taken prior to therapy and from this multiple slides were produced using cytospin cytocentrifugation and stored at -80 degrees C for subsequent immunocytochemical analysis (ICA). ICA was performed for oestrogen receptor (ER), progesterone receptor (PgR), p53, Ki67, and Bcl-2. Part of the aspirate was snap frozen and used for flow cytometric analysis of ploidy and S-phase fraction (SPF). In a subgroup of 50 patients who had surgery prior to systemic therapy, as well as FNAs, sections were also taken from paraffin-embedded blocks and stained by ICA for ER, PgR and p53 for validation. In these patients ER was additionally measured by enzyme immunoassay (EIA) from frozen tissue taken at surgery. ER, PgR, p53, Bcl-2, and Ki67 were successfully detected by ICA while ploidy and SPF were successfully measured by flow cytometry from FNA material. The percentage positive values obtained were reasonable and as follows: 74% for ER, 70% for PgR, 36% for p53, 80% for Bcl-2,68% of tumours were aneuploid and 32% diploid. Significant relationships between these measurements were observed in accordance with expectations. The concordance for ER, PgR, and p53 from FNA when compared to ICA of matching histological sections was 91.5%, 75.5%, and 75% respectively. For ER the concordance between measurement by ICA of cytological and histological samples and by EIA of frozen tissue was 82.5% and 84% respectively. These results indicate that multiple molecular markers can be adequately tested on cytological preparations from primary breast tumours. These markers can be used to determine prognosis and predict response to PMT.

Prediction of response to neoadjuvant chemoendocrine therapy in primary breast carcinomas.

Our aim was to determine whether biological molecular markers can predict response to neoadjuvant chemoendocrine therapy in patients with early breast cancer. Ninety patients (median age 56 years; range, 28-69 years) with primary operable breast carcinoma were studied. They were treated with four 3-weekly cycles of chemotherapy with mitozantrone, methotrexate (+/- mitomycin C), and tamoxifen prior to surgery. Fine-needle aspiration was used to obtain samples from patients prior to therapy, and the following parameters were assessed: estrogen receptor (ER), progesterone receptor (PgR), p53, Ki67, Bcl-2, and c-erbB-2 measured by immunocytochemistry, and ploidy and S-phase fraction (SPF) by flow cytometry. The tumors of 78% of the subjects responded (complete response, 9%; partial response, 69%) and 22% did not (no change, 20%; progressive disease, 2%). Response rates according to disease stage and patient age were as follows: T1, 74%; T2, 79%; T3/T4, 78%; age 50, 79% (P = not significant). Response rates for other parameters were as follows: ER-positive, 82%, and -negative, 70%; PgR-positive, 86%, and -negative, 71%; p53-positive, 74%, and -negative, 81%; Bcl-2-positive, 85%, and -negative 61%; c-erbB-2-positive, 57%, and -negative, 93%; Ki67 high, 77%, and low, 81%; SPF high, 77%, and low, 77%; aneuploid, 71%; and diploid, 85%. Only the difference for c-erbB-2 was statistically significant (P = 0.007). A trend for higher response rates to neoadjuvant chemoendocrine therapy for tumors that were positive for ER, PgR, and Bcl-2 was observed but did not reach statistical significance. Tumors negative for c-erbB-2 had a higher response rate, which was statistically significant. In contrast, Ki67, ploidy, SPF, and p53 failed to predict for response.

Measurement of S-phase fraction and ploidy in sequential fine-needle aspirates from primary human breast tumours treated with tamoxifen.

Sequential fine-needle aspirates (FNAs) for cytodiagnosis and flow cytometry were taken from 21 patients with primary breast carcinoma at intervals ranging from 1 to 3 months after the commencement of first-line tamoxifen therapy. Nine patients achieved a sustained complete or near complete response over a 3-9 month period. The tumour cells from seven out of nine of these patients were initially aneuploid, while the remaining two patients had diploid tumours. An analysis of sequential FNAs showed that, in three out of the seven aneuploid tumours, only benign epithelial cells could be detected by cytology in the post-tamoxifen sample. In the remaining six cases, including the two diploid tumours, there was no change in ploidy but a reduction in S-phase fraction (SPF) to approximately 50% of the pretreatment level. In all cases, these changes in ploidy or SPF were seen with a mean lead time of 4 months before the tumour had reached clinical complete remission. None of these patients have relapsed after a mean follow-up period of 18 months. The tumours of 12 patients achieved no more than a temporary partial response to primary tamoxifen therapy. In seven out of eight of these cases, which were all initially aneuploid, sequential FNAs during tamoxifen therapy revealed either an increase or no change in the SPF with the tumour remaining aneuploid. In the remaining four cases the tumours were all recorded as being diploid in the pretreatment sample. However, although three of these cases had a temporary partial response to tamoxifen, an aneuploid component was picked up in repeat sequential FNAs with a mean lead time of 5 months before clinical confirmation of eventual disease progression. We conclude that changes in ploidy and SPF detected by flow cytometry may predict initial response and the likelihood of relapse of breast tumours to tamoxifen before clinical changes become evident. These data justify a larger study.

Comparison of two methods of storing breast fine needle aspirates (FNAs) using oestrogen receptor immunocytochemical assay as a method of evaluating the storage methods.

Two methods of storing fine needle aspirates were compared in 14 patients with breast cancer. The methods of storage were: (1) as a Cytospin slide prepared immediately from the aspirated material and stored at -80 degrees C; (2) as a suspension of cells in tissue culture medium, stored at -80 degrees C. The effect of storage on the cells was assessed by means of an oestrogen receptor immunocytochemical assay (ER-ICA). An ER positivity of 100% was obtained by ER-ICA staining of cells after storage method 1, whilst all of the specimens stored by method 2 were ER-negative. The data demonstrate that cells stored in tissue culture medium at -80 degrees C are not suitable for ER measurement. The storage method of choice for specimens intended for ERICA is as a Cytospin slide. The ER status of cells deposited on Cytospin slides prepared immediately and stored at -80 degrees C for 2 years could be demonstrated despite the delay in processing the specimen.

Gynaecological cytology and pelvic ultrasonography in patients with breast cancer taking tamoxifen compared with controls.

The objective of this study was to look at the effect of tamoxifen on the endometrium by comparing gynaecological cervical and endometrial cytology in breast cancer patients on tamoxifen for 3 years compared with controls. In addition, pelvic ultrasonography was employed to detect ovarian abnormalities and to measure endometrial thickness. Patients followed-up after primary surgical therapy for breast cancer were invited for gynaecological assessment consisting of clinical examination pelvic ultrasonography and a cervical smear and endometrial sampling. The patients taking tamoxifen (n = 49) has been on adjuvant hormone therapy for a minimum of 3 years. Control patients (n = 45) were also being followed-up for breast cancer. On examination the tamoxifen patients had very similar clinical findings to the control patients with regard to the cervix (normal in 84% of tamoxifen takers compared to 87% of controls). The uterus was clinically enlarged in eight patients on tamoxifen and in none of the control patients (P = 0.006) and only one ovarian cyst was clinically detectable in a patient taking tamoxifen. Pelvic ultrasonography between the two groups of patients was not statistically different (chi 2 test) and ovarian cysts were noted in nine patients from each group (tamoxifen patients 18% vs control patients 20%, n.s.). There was a highly significant difference in endometrial thickness in premenopausal patients (9.2 mm) compared with postmenopausal patients (6.4 mm, P = 0.001). There was also a suggestion that endometrial thickness was greater in tamoxifen treated patients (P = 0.08). In general, a greater proportion of patients taking tamoxifen had cervical and endometrial cells exhibiting hyperplastic nuclei, and in endocervical smears, this difference achieved statistical significance (Mann-Whitney test, P = 0.046). These findings show that a significantly increased proportion of patients taking tamoxifen had endocervical nuclear hyperplasia, and a trend towards increased endometrial thickness. These findings confirm that tamoxifen has mild oestrogenic activity. However, the lack of any difference in the incidence of dysplasia suggests that the carcinogenic potential of tamoxifen on the uterus is very low and the beneficial effects of tamoxifen as an adjuvant therapy for breast cancer outweighs its theoretical risks.

Immunocytochemical staining for the c-erbB-2 gene product in breast aspirates: a preliminary report.

The results of the determination of c-erbB-2 gene expression by immunocytochemical staining of cytological aspirates, prepared by cytocentrifugation, have been compared with paraffin-embedded tissue sections from the same tumour. Our results show equivalent staining in 20/22 cases, with six cases being both scored positive and fourteen cases being both negative. Two samples gave conflicting results. One case was scored as being positive on the cytological aspirate, whereas in the tissue sections taken from the same tumour positive staining was only seen in areas of non-invasive intraduct carcinoma. This sample was scored as being negative. In another case, cytoplasmic staining with less than 50% of the cells showing any positivity was observed in the cytospin sample, with negative staining in the corresponding tissue section. We conclude that expression of c-erbB-2 immunostaining is detectable on cytological preparations prepared by cytocentrifugation but must be interpreted with caution in tumours which may have a large intraduct component or which give predominant cytoplasmic staining.

The role of routine urine and parametrial aspirate cytology in staging carcinoma of the cervix.

The role of routine urine and parametrial aspirate cytology as a means of refining the staging of carcinoma of the cervix was assessed in a series of 45 consecutive patients undergoing examination under anaesthetic and cytoscopy. Only 2/45 (4%) of urine samples were positive and 3/37 (8%) of parametrial aspirates were abnormal. In none of these patients was additional information obtained over that already available from cytoscopy, digital examination and routine radiology. These procedures have no role in the routine clinical staging of cervical carcinoma.