Representative CIN 1 staining patterns.

<p>Columns a-d: Four H&E stained CIN 1 specimens (a, b, d, HPV 16 positive; c, HPV 35 positive); row e-h: HPV <i>E6/E7</i> RNA CISH staining patterns; row i-l: <i>p16</i> mRNA CISH staining patterns; row m-p: p16^INK4a IHC staining patterns. The first specimen (a) may represent an early lesion displaying low-level HPV staining (e) mostly in the lower half of the lesion with occasional superficial cells showing diffusely stained nuclei (black arrows, e-h). <i>p16</i> mRNA signals were undetected. p16^INK4a staining was scored as negative. The second specimen (b) shows a productive phase HPV staining pattern as indicated by the abundant diffusely stained nuclei throughout the epithelial thickness (f). <i>p16</i> mRNA signals were barely detectable (j). p16^INK4a staining shows patchy positivity (n). Specimen c also shows a productive phase pattern of HPV expression (g). <i>p16</i> mRNA signals were detectable in the lower third of the epithelium (k) matching p16^INK4a IHC staining (o). Most CIN 1 lesions showed the staining patterns shown for specimens b, f, j, n and c, g, k, o. An exception to this staining trend was found in specimen d, which showed strong staining for HPV in the lower part of the lesion and occasional diffuse staining nuclei (h). <i>p16</i> mRNA signals were detectable in the lower epithelial layers and strong p16^INK4a staining was detected throughout the lesion; possibly, this specimen represents a CIN lesion entering into a transformative phase directly from CIN 1 morphology. All images were originally taken using a 20X objective lens. Scale bar: 50 µm.</p

Correspondence of HPV <i>E6/E7</i> RNA staining patterns with HPV infective biology.

<p><b>A.</b> HPV infection of basal cells occurring via micro-abrasions to the cervical epithelium may give rise to latent and subsequently productive (permissive) infections. The transforming phase is associated with upregulated <i>E6</i> and <i>E7</i> expression Blue arrow). <b>B.</b> The data from the present study suggests HPV <i>E6</i> and <i>E7</i> RNA expression detected by chromogenic <i>in situ</i> hybridization corresponds with HPV infective mode which in turn correlates with CIN grade. [Image A. (references 15 and 16) is reproduced with kind permission courtesy of Professor Magnus von Knebel Deobervitz, University of Heidelberg, Germany.].</p

Summary of major findings.

<p>Summary of major findings.</p

Representative CIN 3 staining patterns.

<p>Columns a-d: Four H&E stained CIN 3 specimens (all HPV 16 positive); e-h: HPV <i>E6/E7</i> RNA CISH staining patterns; i-l: <i>p16</i> mRNA CISH staining patterns; m-p: p16^INK4a IHC staining patterns. All CIN 3 lesions showed staining patterns consistent with the transformative phase of HPV infection. HPV staining was detected throughout the thickness of the epithelium as strongly staining nuclear and cytoplasmic dots. Occasional diffusely staining nuclei were detected in some lesions in the outermost superficial layer; focal ‘CIN 2 patterns’ were noted along with some CIN 3 (e). <i>p16</i> mRNA staining was notable qualitatively stronger and detectable in all layers (I, j, k, l). All images were originally taken using a 20X objective lens. Scale bar: 50 µm.</p

HPV genotype distribution amongst the CIN 1, CIN 2 and CIN 3 study specimens.

<p>HPV genotype distribution amongst the CIN 1, CIN 2 and CIN 3 study specimens.</p

Summary of CIN staining patterns of high-risk HPV RNA and <i>p16</i> mRNA by RNAscope CISH, p16^INK4a IHC and HPV DNA CISH.

<p>≤BME: staining confined to basal through to mid-epithelial layers; BME+: staining extending up to the surface epithelium; SupD: superficial layer diffusely stained nuclei detected.</p><p>*Chi-Square Test for Trend.</p

HPV <i>E6/E7</i> RNA <i>In Situ</i> Hybridization Signal Patterns as Biomarkers of Three-Tier Cervical Intraepithelial Neoplasia Grade

<div><p>Cervical lesion grading is critical for effective patient management. A three-tier classification (cervical intraepithelial neoplasia [CIN] grade 1, 2 or 3) based on H&E slide review is widely used. However, for reasons of considerable inter-observer variation in CIN grade assignment and for want of a biomarker validating a three-fold stratification, <i>CAP-ASCCP LAST</i> consensus guidelines recommend a two-tier system: low- or high-grade squamous intraepithelial lesions (LSIL or HSIL). In this study, high-risk HPV <i>E6/E7</i> and <i>p16</i> mRNA expression patterns in eighty-six CIN lesions were investigated by RNAscope chromogenic <i>in situ</i> hybridization (CISH). Specimens were also screened by immunohistochemistry for p16^INK4a (clone E6H4), and by tyramide-based CISH for HPV DNA. HPV genotyping was performed by GP5+/6+ PCR combined with cycle-sequencing. Abundant high-risk HPV RNA CISH signals were detected in 26/32 (81.3%) CIN 1, 22/22 (100%) CIN 2 and in 32/32 (100%) CIN 3 lesions. CIN 1 staining patterns were typified (67.7% specimens) by abundant diffusely staining nuclei in the upper epithelial layers; CIN 2 lesions mostly (66.7%) showed a combination of superficial diffuse-stained nuclei and multiple dot-like nuclear and cytoplasmic signals throughout the epithelium; CIN 3 lesions were characterized (87.5%) by multiple dot-like nuclear and cytoplasmic signals throughout the epithelial thickness and absence/scarcity of diffusely staining nuclei (trend across CIN grades: P<0.0001). These data are consistent with productive phase HPV infections exemplifying CIN 1, transformative phase infections CIN 3, whereas CIN 2 shows both productive and transformative phase elements. Three-tier data correlation was not found for the other assays examined. The dual discernment of diffuse and/or dot-like signals together with the assay’s high sensitivity for HPV support the use of HPV <i>E6/E7</i> RNA CISH as an adjunct test for deciding lesion grade when CIN 2 grading may be beneficial (e.g. among young women) or when ‘LSIL vs. HSIL’ assignment is equivocal.</p></div

<i>ESR1</i> predicts response to tamoxifen on YTMA 130 Subset.

<p>Kaplan-Meier curves all show 10-year recurrence-free survival. (A) Cases from the YTMA 130 Subset were split by the median <i>ESR1</i> AQUA score and shows no prognostic value. (B) <i>ESR1</i> high (AQUA >4.13) cases from the YTMA 130 Subset were split by tamoxifen treatment status. <i>ESR1</i> high patients who received tamoxifen had a statistically significant reduced risk of recurrence. (C) <i>ESR1</i> low (AQUA <4.13) cases from the YTMA 130 Subset were split by tamoxifen treatment status and show no statistically significant trend. (D) ER positivity on YTMA 130 determined by QIF with SP1 is prognostic. (E) ER positive cases from YTMA 130 were split by tamoxifen treatment status. Patients who received tamoxifen had a reduced risk of recurrence trending towards significance. All p values were calculated using the log-rank test.</p

Comparison between <i>ESR1</i> and ER protein on Yale breast cancer cohorts.

<p>The natural log of the nuclear ER AQUA score determined by QIF using SP1 is shown on the y-axis and the AQUA score for <i>ESR1</i> determined by qISH is on x-axis for YTMA 128, n = 167 with scores for both SP1 and <i>ESR1</i> (A) and YTMA 130 1993–2005 Subset, n = 195 with scores for both SP1 and <i>ESR1</i> (B). The dotted line represents the threshold for ER protein positivity. (C) Representative images from 4 cases on YTMA 128 for cytokeratin (green), DAPI (blue), and <i>ESR1</i> or ER SP1 (red).</p

Multivariate Analysis for YTMA 130 Subset.

<p>Abbreviations: ER, estrogen receptor; IHC, immunohistochemistry; PgR, progesterone receptor.</p