Benjamin Dessauvagie

Validation of mitosis counting by automated phosphohistone H3 (PHH3) digital image analysis in a breast carcinoma tissue microarray

Summary Mitosis counting in H&E stained sections is the most informative constituent of the Nottingham histological grade in breast carcinoma prognosis. Phosphohistone H3 (PHH3) immunohistochemistry (IHC) is a highly specific marker of mitoses, with practical application in identifying mitoses in poorly fixed or distorted tissue and is of prognostic significance in breast carcinoma. Our aim was to assess methods of PHH3 IHC mitosis counting in a tissue microarray (TMA) of 2 mm cores from 36 resected breast carcinomas. Mitoses in H&E and PHH3 stained slides were manually scored by pathologist consensus and expressed as counts/2 mm2. PHH3 stained cores were also evaluated by automated digital image analysis (DIA). Results were compared using Spearman correlation. A strong and significant correlation was observed between manual PHH3 and manual H&E mitotic counts (correlation = 0.81; p < 0.0001) and between automated PHH3 DIA and manual H&E mitotic counts (correlation = 0.79; p < 0.0001). More mitoses were identified with PHH3 IHC than with H&E. Manual and DIA PHH3 counts were strongly and significantly correlated (correlation = 0.83; p < 0.0001) and of similar absolute values. PHH3 DIA is a valid alternative to manual counting with potential application in breast cancer reporting and prognostication.

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

Summary 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.

Squamous metaplasia of lactiferous ducts (SMOLD)

The aim of this review is to illustrate the mammographic and sonographic appearances of squamous metaplasia of the lactiferous ducts (SMOLD) and to discuss the disease processes of this uncommon breast disease, which shows a strong correlation with smoking. The most common mammographic appearance is of a retro-areolar asymmetrical density. Ultrasonography of the symptomatic breast typically shows a retro-areolar, predominately medial, ill-defined, hypoechoic lesion with either abscess or sinus/fistula formation. Duct dilatation and continuity with lactiferous ducts is commonly seen. Increased vascularity is occasionally seen on colour Doppler ultrasound. Pathology tissue confirmation is always required and this can be by histology of a core biopsy or excision specimen, or fine-needle aspiration (FNA) cytology. Occasionally smears of an associated abundant nipple or sinus discharge may be of value.

Handling of radioactive seed localisation breast specimens in the histopathology laboratory: the Western Australian experience

Summary Radio-guided occult lesion localisation using iodine-125 seeds (ROLLIS) is a novel method of localisation for impalpable in situ and invasive carcinomas that has been the subject of a recent pilot study and pilot study extension in Western Australia. Robust protocols for radiation safety, specimen labelling, specimen tracking, seed retrieval and seed disposal were developed at two Western Australian laboratories to minimise the risk of seed loss. The processes are safe and effective with no significant radiation exposure to pathologists and with acquisition of all seeds intact and undamaged. The success can be attributed to developing specific seed retrieval techniques, suited to local preferences at each institution, with input from surgeons, radiologists and medical physics personnel. These techniques are now routine and will continue in the randomised control phase of the ROLLIS study.

CD117 and CD43 are useful adjuncts in the distinction of adenoid cystic carcinoma from adenoid basal cell carcinoma

Summary Distinction of cutaneous adenoid cystic carcinoma (ACC) from adenoid basal cell carcinoma (BCC) is an occasional diagnostic dilemma in dermatopathology. We examined the immunohistochemical staining patterns with CD117 and CD43 in ACCs and BCCs, including BCCs with an adenoid growth pattern, to determine whether a combination of these markers can assist in the differential diagnosis. Fifteen cases each of ACC and BCC, including seven BCCs with a partial or entirely adenoid growth pattern were immunohistochemically stained for CD117 and CD43. The stains were interpreted semi-quantitatively. Staining for CD43 and CD117 was significantly more common in ACC than in BCC. Forty percent of ACCs showed staining for CD43, while no cases of BCC were positive. CD117 was positive in all cases of ACC, with 93% showing moderate or strong staining. BCC were less frequently positive, with only 20% of cases showing labelling of weak or moderate intensity. Immunohistochemical positivity for CD117 and CD43 are likely to be helpful adjuncts in the separation of cutaneous ACC from adenoid BCC.

Evaluation of the use of microbubbles in the ultrasound assessment of the axilla in breast cancer patients

Contrast-enhanced ultrasound of the axilla can be used to identify the axillary sentinel lymph node. We introduced this into our practice in 2013. During the study period there was an upgrade of our US equipment. The purpose of our audit was to see the negative predictive value of CEUS biopsy of the SLN.

Genetic characterisation of molecular targets in carcinoma of unknown primary

BackgroundCarcinoma of unknown primary (CUP) is a metastatic epithelial malignancy in the absence of an identifiable primary tumour. Prognosis for patients with CUP is poor because treatment options are generally limited to broad spectrum chemotherapy. A shift towards personalised cancer management based on mutation profiling offers the possibility of new treatment paradigms. This study has explored whether actionable, oncogenic driver mutations are present in CUP that have potential to better inform treatment decisions.MethodsCarcinoma of unknown primary cases (n = 21) were selected and DNA was isolated from formalin-fixed paraffin embedded sections prior to amplification and sequencing. Two distinct yet complementary targeted gene panels were used to assess variants in up to 76 known cancer-related genes for the identification of biologically relevant and actionable mutations.ResultsVariants were detected in 17/21 cases (81%) of which 11 (52%) were potentially actionable with drugs currently approved for use in known primary cancer types or undergoing clinical trials. The most common variants detected were in TP53 (47%), KRAS (12%), MET (12%) and MYC (12%). Differences at the molecular level were seen between common CUP histological subtypes. CUP adenocarcinomas and poorly differentiated carcinomas harboured the highest frequency of variants in genes involved in signal transduction pathways (e.g. MET, EGFR, HRAS, KRAS, and BRAF). In contrast, squamous cell carcinoma exhibited a higher frequency of variants in cell cycle control and DNA repair genes (e.g. TP53, CDKN2A and MLH1).ConclusionTaken together, mutations in biologically relevant genes were detected in the vast majority of CUP tumours, of which half provided a potentially novel treatment option not generally considered in CUP.

Interobserver variation in the diagnosis of fibroepithelial lesions of the breast: a multicentre audit by digital pathology

Aim Fibroepithelial lesions (FELs) of the breast span a morphological continuum including lesions where distinction between cellular fibroadenoma (FA) and benign phyllodes tumour (PT) is difficult. The distinction is clinically important with FAs managed conservatively while equivocal lesions and PTs are managed with surgery. We sought to audit core biopsy diagnoses of equivocal FELs by digital pathology and to investigate whether digital point counting is useful in clarifying FEL diagnoses. Method Scanned slide images from cores and subsequent excisions of 69 equivocal FELs were examined in a multicentre audit by eight pathologists to determine the agreement and accuracy of core needle biopsy (CNB) diagnoses and by digital point counting of stromal cellularity and expansion to determine if classification could be improved. Results Interobserver variation was high on CNB with a unanimous diagnosis from all pathologists in only eight cases of FA, diagnoses of both FA and PT on the same CNB in 15 and a ‘weak’ mean kappa agreement between pathologists (k=0.36). ‘Moderate’ agreement was observed on CNBs among breast specialists (k=0.44) and on excision samples (k=0.49). Up to 23% of lesions confidently diagnosed as FA on CNB were PT on excision and up to 30% of lesions confidently diagnosed as PT on CNB were FA on excision. Digital point counting did not aid in the classification of FELs. Conclusion Accurate and reproducible diagnosis of equivocal FELs is difficult, particularly on CNB, resulting in poor interobserver agreement and suboptimal accuracy. Given the diagnostic difficulty, and surgical implications, equivocal FELs should be reported in consultation with experienced breast pathologists as a small number of benign FAs can be selected out from equivocal lesions.

Optical palpation for the visualization of tumor in human breast tissue

Accurate and effective removal of tumor in one operation is an important goal of breast-conserving surgery. However, it is not always achieved. Surgeons often utilize manual palpation to assess the surgical margin and/or the breast cavity. Manual palpation, however, is subjective and has relatively low resolution. Here, we investigate a tactile imaging technique, optical palpation, for the visualization of tumor. Optical palpation generates maps of the stress at the surface of tissue under static preload compression. Stress is evaluated by measuring the deformation of a contacting thin compliant layer with known mechanical properties using optical coherence tomography. In this study, optical palpation is performed on 34 freshly excised human breast specimens. Wide field-of-view (up to ~46 × 46 mm) stress images, optical palpograms, are presented from four representative specimens, demonstrating the capability of optical palpation to visualize tumor. Median stress reported for adipose tissue, 4 kPa, and benign dense tissue, 8 kPa, is significantly lower than for invasive tumor, 60 kPa. In addition, we demonstrate that optical palpation provides contrast consistent with a related optical technique, quantitative micro-elastography. This study demonstrates that optical palpation holds promise for visualization of tumor in breast-conserving surgery.

Expression of proliferation genes in formalin-fixed paraffin-embedded (FFPE) tissue from breast carcinomas. Feasibility and relevance for a routine histopathology laboratory.

Aim Breast carcinoma proliferative activity, histological grade and commercial molecular tests are all important in prognostication and treatment. There is a particular need for improved, standardised techniques for subclassification of grade 2 breast cancers into low-risk and high-risk prognostic groups. In this study we investigated whether gene expression profiling of five proliferation genes was feasible using breast cancer tissue in a clinical setting and whether these profiles could enhance pathological assessment. Methods Expression of five proliferation gene mRNAs; Ki-67, STK 15, CCNB1, CCND1 and MYBL2, was quantified in 27 breast carcinomas and compared with Ki-67 proliferation index (PI) and Nottingham mitotic score. Results Expression of Ki-67, STK15 and MYBL2 mRNA showed moderate Spearmans correlation with Ki-67 PI (p<0.01), but CCND1 and CCNB1 showed weak, non-significant correlation. Individual gene expression did not associate with mitotic score but combined mRNA expression correlated with both Ki-67 PI (p=0.018) and mitotic score (p=0.03; 0.007). Conclusions This study confirms mRNA analysis in breast carcinoma formalin-fixed, paraffin-embedded samples is feasible and suggests gene expression profiling, using a small set of five proliferation genes, has potential in aiding histological grading or assessment of proliferative activity of breast cancers. To fully evaluate the clinical applicability of this approach, a larger cohort study with long-term follow-up data is required.