Probes for INS

Increased testing for human papillomavirus (HPV) in oropharyngeal carcinomas has broadened the range of HPV-associated malignancies identified at this site. While HPV-related oropharyngeal non-keratinizing squamous cell carcinomas (SCC) are known to have a better prognosis than their non-HPV counterparts, HPV positivity may not alter the aggressive nature of HPV-associated small cell neuroendocrine carcinomas (SCNEC). We report a unique case of a mixed non-keratinizing type HPV-associated tonsillar SCC with SCNEC differentiation, and provide a comparison with the rare reported cases of such mixed carcinomas in the literature. Our patient is only the second such case positive for HPV genotype 18 and the only case in which this HPV-related mixed tonsillar tumor occurred in a patient with small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL). The case discussion supports the concept that HPV positivity does not confer a better prognosis in such mixed non-keratinizing type SCC with SCNEC. Our report also alerts pathologists to the need to evaluate for the possibility of a coexisting neuroendocrine component when oropharyngeal squamous cell carcinoma (OPSCC) is diagnosed, as its presence will affect the patients’ clinical management and prognosis

Dysregulated expression of oncogenic types of E6 and E7 is necessary for human papillomavirus (HPV)-driven carcinogenesis. An HPV E6/E7 mRNA in situ hybridization (ISH) assay covering 18 common high-risk types ("HR-RISH," aka HR-HPV RNA18 ISH) has not been extensively studied in the anogenital tract or validated on automated technology. We herein compare HR-RISH to DNA polymerase chain reaction (PCR), p16 immunohistochemistry, and a previously available HPV DNA ISH assay in HPV-related anogenital and head and neck (H&N) neoplasia. A total of 102 squamous intraepithelial lesions (16 CIN1, 25 CIN3, 3 AIN1, 12 AIN3, 9 VIN3)/invasive squamous cell carcinomas (17 cervical, 2 anal, 18 H&N) as well as 10 normal and 15 reactive cervix samples were collected. HR-RISH, DNA ISH, and p16 immunohistochemistry were performed on whole formalin-fixed, paraffin-embedded sections. RNA ISH for 6 low-risk HPV types (LR-RISH) was also performed. RNA and DNA ISH assays used automated systems. HR-HPV PCR was performed on morphology-directed formalin-fixed, paraffin-embedded punches. HR-RISH was ≥97% sensitive for PCR+ and p16+ neoplasia, as well as morphologically defined anogenital high grade squamous intraepithelial lesion/invasive squamous cell carcinoma. HR-RISH was also positive in 78% of anogenital low grade squamous intraepithelial lesion, including 81% of CIN1. Furthermore, a subset of PCR-negative/invalid and p16-negative lesions was positive for HR-RISH. Only 1 problematic reactive cervix sample and no normal cervix samples stained. These results demonstrate that HR-RISH is a robust method for the detection of HR-HPV-related neoplasia and provides insight into HPV pathobiology. Performance meets or exceeds that of existing assays in anogenital and H&N lesions and may play a role in resolving diagnostically challenging CIN1 versus reactive cases.

Cervical low-grade squamous intraepithelial lesions (LSIL) (aka cervical intraepithelial neoplasia, grade 1 [CIN1]) can present considerable diagnostic challenges and are associated with poor interobserver reproducibility and overdiagnosis. Furthermore, ancillary studies such as p16 immunohistochemistry have shown little utility in resolving the LSIL versus negative/reactive differential. Human papillomavirus (HPV) RNA in situ hybridization (ISH) has shown promise as a diagnostic aid in this setting, but has not been studied in a large case series. We herein investigate high-risk and low-risk HPV RNA ISH in 126 cervical biopsies originally diagnosed as LSIL/CIN1 and compare HPV RNA ISH results to expert-adjudicated morphologic diagnosis to assess whether this assay can help routine cases attain the existing "gold standard" of morphologic consensus diagnosis. We also assess whether this criterion standard can be further improved by integration of HPV RNA ISH results. A consensus diagnosis of intraepithelial lesion (CIN1) was confirmed in 61% of cases, whereas 57% were HPV RNA. HPV-RNA positivity was 84% sensitive and 86% specific for an expert-adjudicated diagnosis of CIN1. Conversely, consensus diagnosis was 90% sensitive and 78% specific for the presence of HPV RNA. Integrating RNA ISH into morphologic review led to further reclassification of 10% of cases, resulting in 95% sensitivity and 98% specificity of HPV RNA ISH for a CIN1 diagnosis and 98% sensitivity and 92% specificity of CIN1 for the presence of HPV RNA. These findings suggest that judicious use of HPV RNA ISH can improve the accuracy of LSIL/CIN1 diagnosis for morphologically ambiguous cases.

Abstract

High-risk human papillomavirus (HPV)-related oropharyngeal squamous cell carcinomas have a more favorable prognosis than HPV-negative ones. p16 immunohistochemistry has been recommended as a prognostic test in clinical practice. Several p16 antibodies are available, and their performance has not been directly compared. We evaluated three commercially available p16 antibody clones (E6H4, JC8 and G175-405) utilizing 199 cases of oropharyngeal squamous cell carcinoma from a tissue microarray, read by three pathologists with three different cutoffs for positivity: any staining, >50% and >75%. Positive predictive values for high-risk HPV status by RNA in situ hybridization for the E6H4, JC8 and G175-405 clones were 98%, 100% and 99% at the 75% cutoff, but negative predictive values were much more variable at 86%, 69% and 56%, respectively. These improved using the 50% cutoff, becoming similar for all three antibodies. Intensity varied substantially, with 85% of E6H4, 72% of JC8 and 67% of G175-405 showing strong (3+) intensity. With Kaplan-Meier survival plots at the 75% cutoff, the E6H4 clone showed the largest differential in disease specific and overall survival between p16-positive and -negative results. Decreasing the cutoff to 50% increased correlation with HPV in situ hybridization and improved the survival differential for the JC8 and G175-405 clones without worsening of performance for the E6H4 clone. Interobserver agreement was also assessed by kappa scores and was highest for the E6H4 clone. Overall, these study results show modest but important performance differences between the three different p16 antibody clones, suggesting that the E6H4 clone performs best because of strongest staining intensity, greatest differential in outcomes between positive and negative results, lowest interobserver variability, and lowest background, nonspecific staining. The results also suggest that a 75% cutoff is very functional but that, in this patient population with high HPV incidence, 50% and any staining cutoffs may be more effective, particularly for the non-E6H4 clones.

Abstract

BACKGROUND:

The aim of this study was to identify the presence and frequency of human papillomavirus (HPV) nucleic acid in p16-positive oral squamous cell carcinomas (OSCCs), to assess whether the virus was transcriptionally active and to assess the utility of p16 overexpression as a surrogate marker for HPV in OSCC.

METHODS:

Forty-six OSCC patients treated between 2007 and 2011 with available formalin-fixed paraffin-embedded (FFPE) specimens were included. Twenty-three patients were positive for p16 by immunohistochemistry (IHC) and these were matched with 23 patients with p16-negative tumours. Laser capture microdissection of the FFPE OSCC tissues was undertaken to isolate invasive tumour tissue. DNA was extracted and tested for high-risk HPV types using a PCR-ELISA method based on the L1 SPF10 consensus primers, and a real-time PCR method targeting HPV-16 and HPV-18 E6 region. Genotyping of HPV-positive cases was performed using a reverse line blot hybridization assay (Inno-LiPA). RNAScope^® (a chromogenic RNA in situ hybridization assay) was utilized to detect E6/E7 mRNA of known high-risk HPV types for detection of transcriptionally active virus.

RESULTS:

HPV DNA was found in 3 OSCC cases, all of which were p16 IHC-positive. Two cases were genotyped as HPV-16 and one as HPV-33. Only one of the HPV-16 cases was confirmed to harbour transcriptionally active virus via HPV RNA ISH.

CONCLUSION:

We have shown that the presence of transcriptionally active HPV rarely occurs in OSCC and that p16 is not an appropriate surrogate marker for HPV in OSCC cases. We propose that non-viral mechanisms are responsible for the majority of IHC p16 overexpression in OSCC.

Intake of hemoglobin by the hemoglobin-haptoglobin receptor CD163 leads to a distinct alternative non-foam cell antiinflammatory macrophage phenotype that was previously considered atheroprotective. Here, we reveal an unexpected but important pathogenic role for these macrophages in atherosclerosis. Using human atherosclerotic samples, cultured cells, and a mouse model of advanced atherosclerosis, we investigated the role of intraplaque hemorrhage on macrophage function with respect to angiogenesis, vascular permeability, inflammation, and plaque progression. In human atherosclerotic lesions, CD163+ macrophages were associated with plaque progression, microvascularity, and a high level of HIF1α and VEGF-A expression. We observed irregular vascular endothelial cadherin in intraplaque microvessels surrounded by CD163+ macrophages. Within these cells, activation of HIF1α via inhibition of prolyl hydroxylases promoted VEGF-mediated increases in intraplaque angiogenesis, vascular permeability, and inflammatory cell recruitment. CD163+ macrophages increased intraplaque endothelial VCAM expression and plaque inflammation. Subjects with homozygous minor alleles of the SNP rs7136716 had elevated microvessel density, increased expression of CD163 in ruptured coronary plaques, and a higher risk of myocardial infarction and coronary heart disease in population cohorts. Thus, our findings highlight a nonlipid-driven mechanism by which alternative macrophages promote plaque angiogenesis, leakiness, inflammation, and progression via the CD163/HIF1α/VEGF-A pathway.