The diagnosis of squamous intraepithelial lesions in cervical tissue specimens is subject to substantial variability. Adjunctive immunohistochemical (IHC) staining for p16 has been shown to add objective biomarker information to morphologic interpretation of hematoxylin and eosin (H&E)-stained tissues. In the CERvical Tissue AdjunctIve aNalysis (CERTAIN) study, we systematically analyzed the impact of adjunctive p16 IHC on the accuracy (agreement with reference pathology results) of diagnosing cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) in the United States. Eleven hundred cervical biopsies were divided into 4 sets of 275 cases by stratified randomization. All H&E slides from each set were interpreted by 17 to 18 individual surgical pathologists, for a total of 19,250 reads by 70 surgical pathologists. After a wash-out period and blinding to original results, cases were re-read by the same pathologists using H&E+p16-stained slides. Using expert consensus diagnoses on H&E+p16 as reference, adjunctive p16 IHC use significantly improved diagnostic agreement of surgical pathologists by 4.7% (95% confidence interval [CI], 3.9, 5.4; P<0.0001). This improvement was driven by an increase of 11.5% (95% CI, 9.3, 13.5; P<0.0001) in sensitivity and an increase of 3.0% (95% CI, 2.2, 3.7; P<0.0001) in specificity. Diagnostic performance was significantly increased as well when expert consensus diagnoses established on H&E only was used as reference. Furthermore, interobserver reliability improved significantly from moderate (H&E: κ=0.58) to substantial (H&E+p16: κ=0.73; P<0.0001). Adjunctive use of p16 IHC provides more accurate and reproducible diagnostic results in the interpretation of cervical biopsies, ensuring that more patients are treated correctly without treating more patients.

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Pathologists have had increasing responsibility for quantitating immunohistochemistry (IHC) biomarkers with the expectation of high between-reader reproducibility due to clinical decision-making especially for patient therapy. Digital imaging-based quantitation of IHC clinical slides offers a potential aid for improvement; however, its clinical adoption is limited potentially due to a conventional field-of-view annotation approach. In this study, we implemented a novel solely morphology-based whole tumor section annotation strategy to maximize image analysis quantitation results between readers. We first compare the field-of-view image analysis annotation approach to digital and manual-based modalities across multiple clinical studies (~120 cases per study) and biomarkers (ER, PR, HER2, Ki-67, and p53 IHC) and then compare a subset of the same cases (~40 cases each from the ER, PR, HER2, and Ki-67 studies) using whole tumor section annotation approach to understand incremental value of all modalities. Between-reader results for each biomarker in relation to conventional scoring modalities showed similar concordance as manual read: ER field-of-view image analysis: 95.3% (95% CI 92.0-98.2%) vs digital read: 92.0% (87.8-95.8%) vs manual read: 94.9% (91.4-97.8%); PR field-of-view image analysis: 94.1% (90.3-97.2%) vs digital read: 94.0% (90.2-97.1%) vs manual read: 94.4% (90.9-97.2%); Ki-67 field-of-view image analysis: 86.8% (82.1-91.4%) vs digital read: 76.6% (70.9-82.2%) vs manual read: 85.6% (80.4-90.4%); p53 field-of-view image analysis: 81.7% (76.4-86.8%) vs digital read: 80.6% (75.0-86.0%) vs manual read: 78.8% (72.2-83.3%); and HER2 field-of-view image analysis: 93.8% (90.0-97.2%) vs digital read: 91.0 (86.6-94.9%) vs manual read: 87.2% (82.1-91.9%). Subset implementation and analysis on the same cases using whole tumor section image analysis approach showed significant improvement between pathologists over field-of-view image analysis and manual read (HER2 100% (97-100%), P=0.013 field-of-view image analysis and 0.013 manual read; Ki-67 100% (96.9-100%), P=0.040 and 0.012; ER 98.3% (94.1-99.5%), p=0.232 and 0.181; and PR 96.6% (91.5-98.7%), p=0.012 and 0.257). Overall, whole tumor section image analysis significantly improves between-pathologist's reproducibility and is the optimal approach for clinical-based image analysis algorithms.

Conventional histological stains, such as hematoxylin plus eosin (H&E), and immunohistochemistry (IHC) are mainstays of histology that provide complementary diagnostic information. H&E and IHC currently require separate slides, because the stains would otherwise obscure one another. This consumes small specimen, limiting the total amount of testing. Additionally, performing H&E and IHC on different slides does not permit comparison of staining at the single cell level, since the same cells are not present on each slide, and alignment of tissue features can be problematic due to changes in tissue landscape with sectioning. We have solved these problems by performing conventional staining and IHC on the same slide using invisible IHC chromogens, such that the chromogens are not visible when viewing the conventional stain and the conventional stain is excluded from images of the IHC. Covalently deposited chromogens provided a convenient route to invisible chromogen design and are stable to reagents used in conventional staining. A dual-camera brightfield microscope system was developed that permits simultaneous viewing of both visible conventional stains and invisible IHC chromogens. Simultaneous staining was demonstrated on several formalin-fixed paraffin-embedded tissue specimens using single and duplex IHC, with chromogens that absorb ultraviolet and near infrared light, followed by H&E staining. The concept was extended to other conventional stains, including mucicarmine special stain and Papanicoulou stain, and further extended to cytology specimens. In addition to interactive video review, images were recorded using multispectral imaging and image processing to provide flexible production of color composite images and enable quantitative analysis.