BRAF p.Val600Glu (V600E) mutation detection in thyroid fine needle aspiration cell block samples: a feasibility study.

Assessing BRAF mutation status in thyroid fine needle aspiration (FNA) cytology samples by both immunohistochemistry (IHC) and molecular methods has been documented in recent literature. We aim to highlight issues relating to quality and quantity of cellular material and DNA extracted from cell block samples.BRAF mutation status was assessed by both molecular and IHC methods in cell block material from thyroid FNA samples over a range of diagnostic categories, and correlated with available follow-up resection specimens.Of 39 samples there were 14 cases with 'inconclusive' cytology (Bethesda diagnostic categories 3, 4 or 5) and 25 cases with malignant cytology. Follow-up information was available in 38 of 39 cases and resection material for comparison in 18 of 39 case. Detection of BRAF mutation in cell block samples by combined molecular and IHC methods showed 100% specificity and 71.4% sensitivity compared to subsequent histologically confirmed BRAF mutated papillary thyroid carcinoma. IHC detected BRAF mutation in two (8.2%) cases which were negative by molecular methods and confirmed mutation positive by IHC and molecular methods on subsequent histology. Low extracted DNA concentration did not appear to preclude detection of BRAF mutation, although cell blocks with lower tumour cell content were over-represented in cases that were wild-type on FNA material and BRAF mutant on subsequent histology.BRAF mutation detection in cell block material is feasible and highly specific for papillary thyroid carcinoma. Best results are obtained by a combination of molecular and IHC methods.

Practical issues concerning the implementation of Ki-67 proliferative index measurement in breast cancer reporting.

Commercial molecular tests which rely heavily on proliferation markers to stratify breast cancer are in increasing demand, but are expensive and not widely available. There is heightened interest in the use of Ki-67 immunohistochemistry as a marker of proliferation. This study sought to examine practical issues in the incorporation of Ki-67 measurement into breast cancer reporting.We conducted a prospective study of Ki-67 proliferative activity in 85 breast carcinomas in 70 patients. We considered whether dual staining with cytokeratin and Ki-67 was necessary to exclude background cells in automated digital image analysis (DIA) and how well a semi-quantitative assessment (SQA) method of Ki-67 proliferation and formal manual counting by two pathologists correlated with DIA.Our study showed good correlation between single and dual stained specimens by DIA (Spearman correlation coefficient 0.8), with a kappa statistic of 0.51 (moderate agreement) but with significantly fewer positive cells identified in dual stained sections. There was fair correlation between SQA and DIA by two pathologists (Spearman correlation coefficient 0.7 and 0.7). Using a ≥10% cut-off to define cases with a 'low' and 'high' proliferative index gave a kappa statistic of 0.25 and 0.32 (fair agreement). There was fair correlation between formal manual counts between two pathologists (Spearman correlation coefficient 0.7; kappa 0.32). Repeat DIA on all cases showed excellent correlation (Spearman coefficient 0.98; kappa 1.0).Automated digital analysis of Ki-67 PI is likely to be more accurate and consistent than semi-quantitative assessment and more practicable than formal manual counting. There remain challenges in standardisation of technique within and across laboratories, interpretation of results and in evaluating clinical relevance.

A diagnosis of malignant pleural mesothelioma can be made by effusion cytology: results of a 20 year audit.

AIMS: Cytological diagnosis of malignant pleural mesothelioma (MPM) is controversial, but has been used in our institution for over 30 years. To assess the role of effusion cytology in mesothelioma diagnosis we conducted an audit of pleural fluid cytology results over a 20 year period (1988-2007). METHODS: Pleural samples were received from 6285 patients; data linkage with Western Australian Cancer and Mesothelioma Registries demonstrated that 815 of these patients had a diagnosis of MPM. Cytological examination of a pleural effusion specimen had been performed in 517 (63%) of these 815 patients. RESULTS: Definitive cytological diagnosis of MPM was made in 377/517 cases, resulting in an 'absolute' sensitivity of 73%. An additional 66 patients were diagnosed as atypical/suspicious, resulting in a 'complete' sensitivity of 86%. If only biopsy/necropsy proven cases are considered, the absolute sensitivity is 68% and the complete sensitivity is 82%. There were no false positive diagnoses of malignancy; two patients with metastatic adenocarcinoma were initially diagnosed as MPM, prior to the availability of specific mesothelial markers, resulting in a positive predictive value of 99%. CONCLUSIONS: Effusion cytology is an inexpensive, minimally invasive procedure which should be included in the diagnostic work-up of cases of suspected MPM.

Experience with standardized thyroid fine-needle aspiration reporting categories: follow-up data from 529 cases with "indeterminate" or "atypical" reports.

BACKGROUND.: A significant number of thyroid fine-needle aspiration cytology (FNAC) cases yield inconclusive results. The recent National Cancer Institute guidelines and those published by other societies are important contributions to standardizing the diagnostic approach. Nevertheless, there are significant issues in the application of guidelines and the evaluation of their clinical efficacy. Data from individual departments can be useful in demonstrating the role of standardized reporting. METHODS.: The authors followed 529 consecutive cases with inconclusive thyroid FNA results that were analyzed in a single laboratory in Western Australia. In that laboratory, standardized reporting in categories has been in place for a decade, and inconclusive cases have been subdivided into indeterminate and atypical groups. Follow-up data was obtained for 341 indeterminate cases (17.2% of total thyroid FNA accessions) and for 188 atypical cases (9.5% of accessions). RESULTS.: In total, 127 nodules with atypical results (67.6%) underwent surgical excision compared with 131 nodules with indeterminate results (38.4%; P < .0001). In 96 excised nodules with atypical results (75.6%), the excised specimens were identified as neoplastic compared with 61 excised nodules with indeterminate results (46.6%; P < .0001). In addition, 31 excised nodules with atypical results (24.4%) had a malignancy proven compared with 17 excised nodules with indeterminate results (13%; P < .05). In addition, 51 of 82 repeat FNAs (62.2%) among patients who had indeterminate results yielded a more specific diagnosis compared with 2 of 9 repeat FNAs (22.2%) among patients who had atypical results (P < .05). CONCLUSIONS.: The routine subcategorization of patients who had inconclusive thyroid FNA reports into indeterminate and atypical groups resulted in statistically significant differences in the likelihood of neoplasia and malignancy. Patients who had indeterminate results were more likely to benefit from repeat FNAC than patients who had atypical results. The current results indicated that patients who fall into these 2 categories are likely to benefit from different clinical management protocols. Cancer Cancer Cytopathol 2010. © 2010 American Cancer Society.

Can HPV DNA testing on FNA material determine anogenital origin in metastatic squamous cell carcinoma?

AIMS: Currently there are no diagnostic techniques that can precisely determine the primary site of a metastatic squamous cell carcinoma (SCC). Anogenital SCC has a high prevalence of high-risk (HR) human papillomavirus (HPV) DNA, particularly in the cervix where the value approaches 100%, whereas non-anogenital SCC generally has a low prevalence. The aim of this study was to examine whether the finding of HR HPV DNA in a fine needle aspiration (FNA) of metastatic SCC could be used to determine a likely anogenital origin. METHODS: Polymerase chain reaction (PCR) and viral sequencing were used to identify HR HPV DNA in cell block and needle rinse material derived from FNA samples in a series of metastatic SCC. RESULTS: HPV DNA was detected in nine of 12 (75%) metastatic anogenital SCC, and type 16 was sequenced in seven. HPV DNA was detected in only one of 18 (5.6%) metastatic SCC from other sites such as lung, oral cavity and skin. The positive control case was an oral cavity tumour and type 33 was sequenced. CONCLUSIONS: Although reservations remain, particularly in relation to the prevalence of HPV DNA in non-anogenital sites, HPV DNA testing by PCR: (1) can be successfully performed on FNA material, and (2) in the correct clinical context, can guide clinicians in assigning a site of origin. HPV DNA detection was the major indicator to the primary site in one occult tumour and several where the previous diagnosis of anogenital cancer was not known at presentation.

Human papillomavirus genotypes and their association with cervical neoplasia in a cohort of Western Australian women.

Human papillomavirus (HPV) is known to be the cause of almost all cervical cancers. The genotypes have been classified into high and low risk types according to their oncogenic potential. However, data for many of the genotypes are limited and some (HPV-26, 53, and 66) have no agreed status. A study was undertaken to determine the HPV genotype distribution in women of Western Australia and the association with cervical neoplasia. Liquid based cervical samples from a cohort of 282 Western Australian women were tested for HPV DNA by PCR followed by DNA sequencing to determine HPV genotypes. HPV-53 and HPV-16 were the most common genotypes found in this population. In addition 86 archived liquid based cervical samples from women with cervical intraepithelial neoplasia grades 1-3 (CIN 1-3) were tested for HPV DNA. Also 32 archived paraffin biopsy samples from women with squamous cell carcinoma were also tested. HPV-16 was the most common genotype found in these samples. Of the cohort of Western Australian women tested, 27% were found to contain HPV and approximately half of these contained known high-risk HPV genotypes, but only 30% of these were types 16 or 18. The data from this study indicate that HPV-53 is not oncogenic based on an R value and odds ratio (OR) of zero. The data also suggest that HPV-73 may be oncogenic, while HPV-66 is unlikely to be. Two high-risk HPV genotypes that are associated with the Asian region (HPV-52 and HPV-58) were found in Western Australian women suggesting a possible epidemiological link between women in these countries.

Predictive value of diagnoses of endocervical glandular abnormalities in cervical smears.

AIMS: To determine the positive predictive value (PPV) of cervical smear diagnoses of 'definite' and 'possible' endocervical adenocarcinoma in situ or invasive adenocarcinoma, and whether diagnostic accuracy can be improved. METHODS: The study examined cervical smears reported as definite or possible high-grade glandular abnormality between 1992 and 1998. PPV was calculated by comparing smear diagnoses with the subsequent histopathology report. All available smears were reviewed without knowledge of follow-up results, and were reclassified by consensus. RESULTS: Thirty-two smears were diagnosed as high-grade glandular lesions, with adequate biopsy follow-up in 31 cases (96.9%). A high-grade epithelial abnormality (HGEA) was detected in 29 cases (PPV, 93.5%), with a high-grade glandular lesion in 24 (PPV, 77.4%). Very few smears were reclassified on review. Seventy-three smears were initially diagnosed in the 'inconclusive' glandular or indeterminate cell-type category. There was adequate biopsy follow up for 54 cases (74.0%). On follow-up, 31 cases had a HGEA (PPV, 57.4%), with 14 cases having a high-grade glandular abnormality (PPV, 25.9%). In the review of 'inconclusive' smears, 12 were reclassified as squamous abnormalities and none of these had a glandular lesion on biopsy. Eight were reclassified as negative; seven contained endometrial stroma and the glandular cells in question were considered to be of lower uterine segment (LUS) origin. No significant lesion was present on follow-up of these cases. CONCLUSIONS: For clinicians using our laboratory, large loop excision of the transformation zone (LLETZ) or cone biopsy should follow a 'definite' cytological diagnosis of a high-grade endocervical glandular lesion. However, cone biopsy may not be the appropriate initial management in the 'possible' high-grade glandular group because of a significantly lower predictive value of the diagnosis. The slide review highlighted the importance of (1) caution in classifying sheets of abnormal cells as glandular, and (2) endometrial stroma as a marker of LUS material.

Adenocarcinoma in situ of the cervix.

BACKGROUND: The current study examines 1) the sensitivity of detection and 2) sampling and screening/diagnostic error in the cytologic diagnosis of adenocarcinoma in situ (AIS) of the cervix. The data were taken from public and private sector screening laboratories reporting 25,000 and 80,000 smears, respectively, each year. METHODS: The study group was comprised of women with a biopsy diagnosis of AIS or AIS combined with a high-grade squamous intraepithelial lesion (HSIL) who were accessioned by the Western Australian Cervical Cytology Registry (WACCR) between 1993-1998. Cervical smears reported by the Western Australia Centre for Pathology and Medical Research (PathCentre) or Western Diagnostic Pathology (WDP) in the 36 months before the index biopsy was obtained were retrieved. A true measure of the sensitivity of detection could not be determined because to the authors' knowledge the exact prevalence of disease is unknown at present. For the current study, sensitivity was defined as the percentage of smears reported as demonstrating a possible or definite high-grade epithelial abnormality (HGEA), either glandular or squamous. Sampling error was defined as the percentage of smears found to have no HGEA on review. Screening/diagnostic error was defined as the percentage of smears in which HGEA was not diagnosed initially but review demonstrated possible or definite HGEA. Sensitivity also was calculated for a randomly selected control group of biopsy proven cases of Grade 3 cervical intraepithelial neoplasia (CIN 3) accessioned at the WACCR in 1999. RESULTS: For biopsy findings of AIS alone, the diagnostic "sensitivity" of a single smear was 47.6% for the PathCentre and 54.3% for WDP. Nearly all the abnormalities were reported as glandular. The sampling and screening/diagnostic errors were 47.6% and 4.8%, respectively, for the PathCentre and 33.3% and 12.3%, respectively, for WDP. The results from the PathCentre were better for AIS plus HSIL than for AIS alone, but the results from WDP were similar for both groups. For the CIN 3 control cases, the "sensitivity" of a single smear was 42.5%. CONCLUSIONS: To the authors' knowledge epidemiologic studies published to date have not demonstrated a benefit from screening for precursors of cervical adenocarcinoma. However, in the study laboratories as in many others, reasonable expertise in diagnosing AIS has been acquired only within the last 10-15 years, which may be too short a period in which to demonstrate a significant effect. The results of the current study provide some encouraging baseline data regarding the sensitivity of the Papanicolaou smear in detecting AIS. Further improvements in sampling and cytodiagnosis may be possible.

Atypical ductal hyperplasia and atypia of uncertain significance in core biopsies from mammographically detected lesions: correlation with excision diagnosis.

AIMS: To assess: (1) the prevalence of reporting of atypical ductal hyperplasia (ADH) and intraductal atypia of uncertain significance (AUS) in a series of core biopsies from mammographically detected lesions, (2) the proportion of cases where excision revealed breast carcinoma, and (3) whether any diagnoses should be revised on review. METHODS: Breast core biopsy reports from the Sir Charles Gairdner Hospital Breast Assessment Centre for the years 1999-2000 were retrieved. Slides from cases reported as ADH or AUS were reviewed as well as slides from the excision biopsies. RESULTS: There were 1048 core biopsies from 911 women. Breast carcinoma was diagnosed in 197 samples (18.8%) including 88 with invasive carcinoma (8.4%), 109 with ductal carcinoma in situ (DCIS) (10.4%). Three biopsies (0.3%) 'suspicious' of invasive carcinoma proved to be so. Of 52 samples (5.0%) with a diagnosis of ADH or AUS, 46 were excised, showing seven invasive carcinomas, 15 DCIS, 11 ADH, two lobular carcinoma in situ (LCIS), nine fibrocystic change (FCC), one mucocoele-like lesion and one fibroadenoma. The 22 malignancies represented 47.8% of the excised lesions. On review, seven of the 52 original core diagnoses were downgraded to benign hyperplasia. Five underwent excision, revealing two FCC, one complex sclerosing lesion, and two incidental lesions unrelated to the mammographic abnormality, including a microscopic tubular carcinoma and a focus of LCIS. In one case reviewed as unsatisfactory, excision showed invasive carcinoma. Lesions of particular interest included a case of high-grade DCIS with local regression in the core biopsy (so-called 'bumt out DCIS'), and one case diagnosed on excision as micropapillary ADH, where the review diagnosis was micropapillary DCIS. CONCLUSIONS: ADH and AUS were reported in 5.0% of biopsies. There was a high rate of carcinoma (47.8%) in subsequent excisions. Very few diagnoses were revised on review. Current protocols for excision of lesions with a 14-gauge core biopsy diagnosis of ADH/AUS appear justified. Literature review suggests that vacuum-assisted core sampling with 11-gauge needles will not remove the need for excision. Further study of local regression of DCIS and micropapillary lesions will be worthwhile.