Pathologist Computer-Aided Diagnostic Scoring of Tumor Cell Fraction: A Swiss National Study.

Tumor cell fraction (TCF) estimation is a common clinical task with well-established large interobserver variability. It thus provides an ideal test bed to evaluate potential impacts of employing a tumor cell fraction computer-aided diagnostic (TCFCAD) tool to support pathologists' evaluation. During a National Slide Seminar event, pathologists (n = 69) were asked to visually estimate TCF in 10 regions of interest (ROIs) from hematoxylin and eosin colorectal cancer images intentionally curated for diverse tissue compositions, cellularity, and stain intensities. Next, they re-evaluated the same ROIs while being provided a TCFCAD-created overlay highlighting predicted tumor vs nontumor cells, together with the corresponding TCF percentage. Participants also reported confidence levels in their assessments using a 5-tier scale, indicating no confidence to high confidence, respectively. The TCF ground truth (GT) was defined by manual cell-counting by experts. When assisted, interobserver variability significantly decreased, showing estimates converging to the GT. This improvement remained even when TCFCAD predictions deviated slightly from the GT. The standard deviation (SD) of the estimated TCF to the GT across ROIs was 9.9% vs 5.8% with TCFCAD (P < .0001). The intraclass correlation coefficient increased from 0.8 to 0.93 (95% CI, 0.65-0.93 vs 0.86-0.98), and pathologists stated feeling more confident when aided (3.67 ± 0.81 vs 4.17 ± 0.82 with the computer-aided diagnostic [CAD] tool). TCFCAD estimation support demonstrated improved scoring accuracy, interpathologist agreement, and scoring confidence. Interestingly, pathologists also expressed more willingness to use such a CAD tool at the end of the survey, highlighting the importance of training/education to increase adoption of CAD systems.

Evaluation of type-specific antibodies to high risk-human papillomavirus (HPV) proteins in patients with oropharyngeal cancer.

OBJECTIVES: High risk human papillomavirus (HR-HPV) infection leads to a subgroup of oropharyngeal cancer (OPSCC) characterized by improved treatment response. However an universally accepted definition of an HR-HPV-attributable cancer is lacking. METHODS: Detailed, type-specific HPV antibody responses were analyzed by multiplex serology in HR-HPV-attributable OPSCC patients, defined by p16INK4A overexpression and HR-HPV DNA detection by PCR amplification and sequencing. RESULTS: Fifty patients were prospectively enrolled. 26/50 (52%) tumor samples were positive for both p16INK4A expression and HR-HPV DNA (22 HPV16, 4 HPV33). Seropositivity was present in 26/26 HPV-attributable OPSCC and one p16INK4A-positive/HPV DNA-negative case. The sensitivity and specificity to diagnose an HR-HPV-attributable tumor was 100% and 96%, respectively for anti-E6 reactivity, 82% and 100%, respectively for anti-E2 reactivity, and clearly lower for anti-E7, anti-E1, anti-E4 and anti-L1-reactivity. 3yr-overall (OS) and disease specific survival (DSS) was higher in patients with HR-HPV-attributable tumors (OS 88% vs 64%, p=0.02; DSS 90% vs 80%, p=0.07) and seropositive patients (OS 88% vs 62%, p=0.01; DSS 92% vs 78%, p=0.05) than HR-HPV-negative or seronegative patients. CONCLUSIONS: Detection of HR-HPV type-specific antibodies highly correlated with HPV-attributable OPSCC and was associated with better survival. HR-HPV antibodies are promising diagnostic, prognostic and potentially screening markers in HR-HPV-attributable OPSCC.

Assessment of mean EGFR gene copy number is a highly reproducible method for evaluating FISH in histological and cytological cancer specimens.

The aims of this study were first, to systematically assess the inter-observer reproducibility of mean Epidermal Growth Factor Receptor (EGFR) gene copy number (MCN) in histological and cytological specimens from lung and non-lung cancers, second to compare the performance of this quantitative approach to the current Colorado criteria for the assessment of fluorescence in situ hybridization (FISH) positivity and third to develop a model to convert cytology into histology MCN. EGFR FISH analysis was performed on 170 histological and 153 cytological specimens. The MCN and Colorado criteria were assessed by two independent observers and agreement evaluated using Bland-Altman plots and kappa values. Conversion of cytology into histology MCN was tested on two randomized subgroups of specimens. Applying the Colorado criteria, agreement between observers was moderate with histology (k=0.5) and excellent with cytology (k=0.94). Positivity in histology versus cytology led to fair agreement (k=0.33). MCN was significantly greater in cytology compared to histology (p<0.001) with excellent inter-observer agreement in both sample types (r=0.99 and 0.89, respectively). The average difference in MCN between observers was -0.003 (95%CI: -0.05 to 0.05) and 0.008 (95%CI: -0.09 to 0.11) for cytology and histology, respectively. A reliable conversion model with an R(2)-value of 0.91 in the validation subgroup (p<0.001) was obtained. The MCN is a reproducible scoring method for evaluating EGFR FISH in samples from lung and non-lung cancers. This quantitative scoring system may constitute an alternative to current methods of gene copy number assessment and will further help to optimize patient selection for targeted therapies.

The prognostic value of cytology and fluorescence in situ hybridization in the follow-up of nonmuscle-invasive bladder cancer after intravesical Bacillus Calmette-Guérin therapy.

Molecular markers reliably predicting failure or success of Bacillus Calmette-Guérin (BCG) in the treatment of nonmuscle-invasive urothelial bladder cancer (NMIBC) are lacking. The aim of our study was to evaluate the value of cytology and chromosomal aberrations detected by fluorescence in situ hybridization (FISH) in predicting failure to BCG therapy. Sixty-eight patients with NMIBC were prospectively recruited. Bladder washings collected before and after BCG instillation were analyzed by conventional cytology and by multitarget FISH assay (UroVysion, Abbott/Vysis, Des Plaines, IL) for aberrations of chromosomes 3, 7, 17 and 9p21. Persistent and recurrent bladder cancers were defined as positive events during follow-up. Twenty-six of 68 (38%) NMIBC failed to BCG. Both positive post-BCG cytology and positive post-BCG FISH were significantly associated with failure of BCG (hazard ratio (HR)= 5.1 and HR= 5.6, respectively; p < 0.001 each) when compared to those with negative results. In the subgroup of nondefinitive cytology (all except those with unequivocally positive cytology), FISH was superior to cytology as a marker of relapse (HR= 6.2 and 1.4, respectively). Cytology and FISH in post-BCG bladder washings are highly interrelated and a positive result predicts failure to BCG therapy in patients with NMIBC equally well. FISH is most useful in the diagnostically less certain cytology categories but does not provide additional information in clearly malignant cytology.

Multitarget fluorescence in situ hybridization elucidates equivocal lung cytology.

The category of equivocal respiratory cytology is a common diagnostic dilemma to both cytopathologists and clinicians. Chromosomal alterations are a hallmark of cancer but are rare or absent in benign conditions. The goal of this study was to test the ability of multitarget fluorescence in situ hybridization (FISH) for dissecting equivocal respiratory cytology into reactive and malignant categories. A consecutive series of 54 Papanicolaou-stained cytologic specimens of the lung was analyzed. The Papanicolaou-stained atypical cell groups were photographed, and the exact locations on the specimens were saved using automated stage and relocation software. The specimens were hybridized with a multitarget FISH probe that contains a mixture of fluorescent probes to the centromeric region of chromosome 6 and to the 5p15, 8q24 (site of the MYC gene) and 7p12 (site of the EGFR gene) loci. The hybridized atypical cells were selectively scored after relocation. A final diagnosis was available in 45 patients, revealing lung carcinoma in 55.5% (n = 25), no evidence of malignancy in 37.8% (n = 17), and pulmonary metastasis of another primary carcinoma in 6.7% (n = 3). FISH results were negative in all 17 patients with benign pulmonary disease and positive in 20 of the 25 patients (80%) with lung carcinoma (p < 0.0001). The sensitivity, specificity, and positive and negative predictive values for detection of malignancy were 79%, 100%, 100%, and 74%, respectively. These data suggest that multitarget FISH in conjunction with automated relocation is a powerful approach for the elucidation of equivocal lung cytology.