[Study of the Human Papillomavirus (HPV) prevalence in head and neck carcinomas in a French monocentric cohort of 372 patients]. / Étude de la prévalence du papillomavirus (HPV) dans les cancers des voies aéro-digestives sur une cohorte unicentrique française de 372 patients.

INTRODUCTION: French data about HPV role in head and neck carcinomas are sparse, although French patients are mostly heavy smokers. In this series of oropharyngeal et non-oropharyngeal tumors, we aimed to determine what were the clinicopathological features associated with HPV and evaluate survival of patients according to HPV status. METHODS: Three hundred and seventy-two cases of head and neck squamous cell carcinomas were reviewed and clinicopathological data were detailed. For each case, we performed a HPV PCR and an immunostaining against p16 protein (paraffin embedded tissues). RESULTS: The series contained 90% of heavy smokers and 36% of tumors were located in oropharynx. HPV DNA was detected in 46% of oropharyngeal carcinomas and 16% of non-oropharyngeal carcinomas. Genotype 16 was the most frequently detected (84%). Clinicopathological features significantly associated with HPV DNA were: oropharyngeal location; absence of tobacco smoking; nodal involvement; poorly-differentiated non-keratinizing histology; positive p16 immunostaining. HPV infection was significantly associated with a longer survival for oropharyngeal carcinomas. It was not the case for non-oropharyngeal carcinomas. CONCLUSION: In this French series with lot of heavy smokers, under half of carcinomas are HPV induced. Clinicopathological features and survival data associated with HPV infection are the same as those classically described in literature.

HPV RNA CISH score identifies two prognostic groups in a p16 positive oropharyngeal squamous cell carcinoma population.

HPV-related and HPV-unrelated oropharyngeal squamous cell carcinomas are two distinct entities according to the Union for International Cancer Control, with a better prognosis conferred to HPV-related oropharyngeal squamous cell carcinomas. However, variable clinical outcomes are observed among patients with p16 positive oropharyngeal squamous cell carcinoma, which is a surrogate marker of HPV infection. We aimed to investigate the prognostic value of RNA CISH against E6 and E7 transcripts (HPV RNA CISH) to predict such variability. We retrospectively included 50 histologically confirmed p16 positive oropharyngeal squamous cell carcinomas (p16 positive immunostaining was defined by a strong staining in 70% or more of tumor cells). HPV RNA CISH staining was assessed semi-quantitatively to define two scores: RNA CISH "low" and RNA CISH "high". Negative HPV RNA CISH cases were scored as RNA CISH "low". This series contained 29 RNA CISH low cases (58%) and 21 RNA CISH high cases (42%). Clinical and pathologic baseline characteristics were similar between the two groups. RNA CISH high staining was associated with a better overall survival in both univariate and multivariate analyses (p = 0.033 and p = 0.042, respectively). Other recorded parameters had no prognostic value. In conclusion, HPV RNA CISH might be an independent prognostic marker in p16 positive oropharyngeal squamous cell carcinomas and might help guide therapeutics.

[Current events in immunotherapy for upper aerodigestive tract cancer]. / Actualités sur l'immunothérapie en pathologie des voies aérodigestives supérieures.

Head and neck (HN) carcinomas (mostly represented by squamous cell carcinomas [SCC]) still have a poor prognosis, which could be dramatically improved with immunotherapy. Tumor's microenvironment changes, caused by many endogenous or exogenous events, can correlate with prognosis and therapeutic response. Here, we review recent data regarding HNSCC, nasopharyngeal carcinomas (NPC) and salivary gland malignant tumors, all three being potential target of immunotherapies. About half of HNSCC exhibit PD-L1 expression, this expression being upregulated in HPV-positive tumors. In recent clinical trials, a better therapeutic response to anti-PD-1 has been obtained in patients with higher PD-L1 expression. Food and Drug Administration (FDA) approved the use of these therapeutics without the screening of patients regarding PD-L1 status. Activation status, density and localisation of TIL as well as PD-L2, γ-interferon, inflammatory cytokines, epithelial-mesenchymal transition phenotype and mutational burden may all be potential therapeutic response markers. In Epstein-Barr Virus (EBV)-induced nasopharyngeal non-keratinizing cancer, PD-L1 is over-expressed compared to EBV negative-tumors. A 22 % response rate has been observed under anti-PD-1 treatment, among PD-L1-positive HNSCC patients. There is little data regarding microenvironment of salivary gland cancer. PD-L1 shows great heterogeneity in localisation, when expressed. A 11 % response rate has been obtained under anti-PD-1 treatment among PD-L1-positive NPC patients. A better understanding of immune checkpoint regulation processes needs to be achieved to allow patients with HN carcinomas to benefit from these promising immunotherapies.

Evaluating the Functional Impairment of Assault Victims in a Judicial Context: A Multicentered Retrospective Study in Seven French Forensics Units.

Functional impairment is of major importance in the evaluation of assault victims. French law assesses the seriousness of the violence in terms of the functional impairment experienced by the victims, quantified by the duration of the victims' inability to fulfill their usual daily activities, measured in days of "total incapacity to work" (TIW). Significant variations in the duration of TIW have been reported depending on the examination centers or physician. To date, few studies, all monocentric, have focused on the determinants of this variability. We aimed to study the functional impairment of assault victims by searching for determinants of TIW. We conducted a retrospective multicenter study, including assault victims from seven forensic medicine units. The data were automatically extracted from the medical certificates drawn up and gathered within the ORFéAD database. Thirty-eight variables were studied, relating to the victim's characteristics, the circumstances of the examination and the assault, the physical and psychological symptoms, and the duration of TIW. A total of 5,663 victims were included, including 2,438 (43%) women. The median age was 32 years (min; max [10; 98]). The median duration of TIW was 2 days (min; max [0; 182]). Male gender, age, time to examination, examination center, traumatic injuries (ecchymosis, hematoma, wound, bone fracture), use of a weapon, and functional limitation were associated with the duration of TIW (p < .05). The associations formerly identified in a monocentric setting were confirmed and new determinants were identified. This study has allowed a better understanding of the factors influencing the evaluation of functional impairment and determination of the TIW of assault victims. This first study using ORféAD is intended to be supplemented by the participation of other forensic units, and the inclusion of other variables, such as violence type, victim categories, or the examining physician.

Histological Severity Risk Factors Identification in Juvenile-Onset Recurrent Respiratory Papillomatosis: How Immunohistochemistry and AI Algorithms Can Help?

Juvenile-onset recurrent respiratory papillomatosis (JoRRP) is a condition characterized by the repeated growth of benign exophytic papilloma in the respiratory tract. The course of the disease remains unpredictable: some children experience minor symptoms, while others require multiple interventions due to florid growth. Our study aimed to identify histologic severity risk factors in patients with JoRRP. Forty-eight children from two French pediatric centers were included retrospectively. Criteria for a severe disease were: annual rate of surgical endoscopy ≥ 5, spread to the lung, carcinomatous transformation or death. We conducted a multi-stage study with image analysis. First, with Hematoxylin and eosin (HE) digital slides of papilloma, we searched for morphological patterns associated with a severe JoRRP using a deep-learning algorithm. Then, immunohistochemistry with antibody against p53 and p63 was performed on sections of FFPE samples of laryngeal papilloma obtained between 2008 and 2018. Immunostainings were quantified according to the staining intensity through two automated workflows: one using machine learning, the other using deep learning. Twenty-four patients had severe disease. For the HE analysis, no significative results were obtained with cross-validation. For immunostaining with anti-p63 antibody, we found similar results between the two image analysis methods. Using machine learning, we found 23.98% of stained nuclei for medium intensity for mild JoRRP vs. 36.1% for severe JoRRP (p = 0.041); and for medium and strong intensity together, 24.14% for mild JoRRP vs. 36.9% for severe JoRRP (p = 0.048). Using deep learning, we found 58.32% for mild JoRRP vs. 67.45% for severe JoRRP (p = 0.045) for medium and strong intensity together. Regarding p53, we did not find any significant difference in the number of nuclei stained between the two groups of patients. In conclusion, we highlighted that immunochemistry with the anti-p63 antibody is a potential biomarker to predict the severity of the JoRRP.

The Microenvironment of Head and Neck Cancers: Papillomavirus Involvement and Potential Impact of Immunomodulatory Treatments.

Cancer progression can be understood as the result of deregulation of tumors' immune microenvironments. Recent studies of the alterations of microenvironments highlight their significant influence on the prognosis of patients with head and neck squamous cell carcinoma (HNSCC). It is necessary to better characterize tumor-infiltrating lymphocytes by focusing, in particular, on the tumor escape mechanisms from immune surveillance. One of the best described tumor immune system evasion mechanisms is the expression of co-stimulation molecules that constitute so-called "immune checkpoints". These molecules regulate the immune response by either activating or inhibiting its effects. The programmed cell death 1 (PD-1) surface protein is an inhibitory co-stimulation molecule that induces exhaustion of activated T-lymphocytes (TLs, T cells) through binding with its ligands, PD-L1 and PD-L2. Half of HNSCCs exhibit PD-L1 expression with higher expression identified in human papillomavirus (HPV) positive tumors. Numerous studies have shown differences between the microenvironments of HPV+ and HPV- cancers. Notably, infiltrations of exhausted CD4+ PD1+ and CD8+ PD1+ T cells are far higher in the microenvironment of HPV+ tumors. The FDA has approved the use of molecules that target PD-1 for the treatment of HNSCC. The first results of clinical trials with anti-PD-1 blockers in HNSCC show improved patient survival, particularly long-term survival without recurrence. However, discordant results were sometimes observed, and improvements in defining cellular predictive markers are necessary. With the development of immunotherapies, pathologists play a role in the selection of patients who are eligible for specific treatments and assessment of their prognosis in greater detail. An automated, quantitative in situ imaging system that integrates both multispectral imaging and automated slide scanning could be developed in pathology laboratories. The evaluation of PD-L1 expression has only been used to stratify the administration of first-line immunotherapy. The validation of these tests and their routine interpretation is essential. No specific recommendation is adopted for HPV+ HNSCC.

Juvenile-Onset Recurrent Respiratory Papillomatosis Aggressiveness: In Situ Study of the Level of Transcription of HPV E6 and E7.

Juvenile-onset recurrent respiratory papillomatosis (JoRRP) is a condition related to HPV 6 and 11 infection which is characterized by the repeated growth of benign exophytic papilloma in the respiratory tract. Disease progression is unpredictable: some children experience minor symptoms, while others require multiple interventions due to florid growth. The aim of this study was to explore the biomarkers of JoRRP severity on a bicentric cohort of forty-eight children. We performed a CISH on the most recent sample of papilloma with a probe targeting the mRNA of the E6 and E7 genes of HPV 6 and 11 and an immunostaining with p16INK4a antibody. For each patient HPV RNA CISH staining was assessed semi-quantitatively to define two scores: 1+, defined as a low staining extent, and 2+, defined as a high staining extent. This series contained 19 patients with a score of 1+ and 29 with a score of 2+. Patients with a score of 2+ had a median of surgical excision (SE) per year that was twice that of patients with a score of 1+ (respectively 6.1 versus 2.8, p = 0.036). We found similar results with the median number of SE the first year. Regarding p16INK4a, all patients were negative. To conclude, HPV RNA CISH might be a biomarker which is predictive of disease aggressiveness in JoRRP, and might help in patient care management.

Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis.

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated with a better outcome than alcohol- and tobacco-related OPSCC. Chromogenic in situ hybridization (CISH) of HPV viral RNA could allow the semiquantitative evaluation of viral transcripts of the oncogenic proteins E6 and E7 and an in situ visualization with a good spatial resolution. This technique allows the diagnosis of an active infection with the visualization of HPV transcription in the tumoral HPV-infected cells. An advantage of this technique is the avoidance of contamination from nonneoplastic HPV-infected cells adjacent to the tumor. Overall, its good diagnosis performances have it considered to be the gold standard for active HPV infection identification. Since E6 and E7 viral protein interaction with cell proteins pRb and p53 is mandatory for cell transformation, HPV RNA CISH is functionally relevant and acutely reflects active oncogenic HPV infection. This technique is clinically relevant as well since "low" or "high" HPV transcription levels helped the identification of two prognosis groups among HPV-related p16-positive head and neck cancer patients. Here we present the protocol for manual HPV RNA CISH performed on formalin-fixed paraffin-embedded (FFPE) slides with a kit obtained from the manufacturer. Instead of chromogenic revelation, RNA in situ hybridization may also be performed with fluorescent revelation (RNA FISH). It may also be combined with conventional immunostaining.

The health of female arrestees in police cells: A descriptive study.

INTRODUCTION: Little information is available regarding the medical status and health care needs of female arrestees. Our objective was to evaluate the perceived health and somatic or psychiatric disorders reported by female arrestees in police cells. MATERIAL AND METHODS: We conducted an observational study in a regional reference department of forensic medicine in France. We studied female arrestees examined in police cells (01/01/2013-06/30/2013). Data were collected regarding individuals' medical characteristics, addictive behaviours, and perceived health status, as well as reported assaults or recent traumatic injuries. We recorded medical decisions regarding fitness for detention in police cells. RESULTS: A total of 438 women (median age, 29; range, 13-67) accounted for 5% of the 7408 examined arrestees. Females considered their overall health as good or very good in 314/395 cases (70%). Women reported chronic somatic or psychiatric disorders more frequently than men (89/379, 23% vs. 757/6,135, 12%, p < 0.001 and 59/379, 15% vs. 392/6319, 6%, p < 0.001, respectively). Daily tobacco consumption and cannabis use were reported by 255/403 (63%) and 98/438 female arrestees (22%), respectively. Physical assaults were reported in 113/415 cases (27%). Female arrestees were considered fit for detention in 92% of cases. Among 24 pregnant arrestees, 6 (25%) were unfit for detention, 2 (8%) were fit for custody during daytime only and 16 (67%) were fit for detention if certain conditions were met. CONCLUSIONS: Detention in police custody involves a minority of females. Females are older and report somatic or psychiatric disorders more frequently than males.

Immunotherapy in head and neck cancers: A new challenge for immunologists, pathologists and clinicians.

Cancer occurrence can be understood as the result of dysfunctions in immune tumoral microenvironment. Here we review the recent understandings of those microenvironment changes, regarding their causes and prognostic significance in head and neck (HN) carcinoma. We will focus on HN squamous cell cancer (SCC) and nasopharyngeal carcinomas (NPC). Their overall poor prognosis may be improved with immunotherapy in a subset of patients, as supported by current clinical trials. However, finding reliable markers of therapeutic response is crucial for patient selection, due to potential severe adverse reactions and high costs. Half of HNSCC exhibit PD-L1 expression, this expression being higher in HPV-positive tumors. In recent clinical trials, a better therapeutic response to anti-PD-1 was obtained in patients with higher PD-L1 expression. The Food and Drug Administration (FDA) approved the use of these therapeutics without stating a need for patient selection regarding PD-L1 status. Activation status, density and localization of TIL as well as PD-L2, γ-interferon, inflammatory cytokines, epithelial-mesenchymal transition phenotype and mutational burden may all be potential therapeutic response markers. In Epstein-Barr Virus (EBV)-induced nasopharyngeal non-keratinizing cancer, PD-L1 is over-expressed compared to EBV-negative tumors. A 22% response rate has been observed under anti-PD-1 treatment among PD-L1-positive NPC patients. A better understanding of immune checkpoint regulation processes may allow patients to benefit from these promising immunotherapies.

Evaluation of the efficacy of the 4 tests (p16 immunochemistry, polymerase chain reaction, DNA, and RNA in situ hybridization) to evaluate a human papillomavirus infection in head and neck cancers: a cohort of 348 French squamous cell carcinomas.

It is now established that human papillomavirus (HPV) plays a role in the development of a subset of head and neck squamous cell carcinomas (SCCs), notably oropharyngeal (OP) SCCs. However, it is not clear which test one should use to detect HPV in OP and non-OP SCCs. In this study, using 348 head and neck SCCs (126 OP SCCs and 222 non-OP SCCs), we evaluated diagnostic performances of different HPV tests in OP and non-OP SCCs: polymerase chain reaction, p16 immunostaining, in situ hybridization targeting DNA (DNA-CISH) and RNA (RNA-CISH), combined p16 + DNA-CISH, and combined p16 + RNA-CISH. HPV DNA (polymerase chain reaction) was detected in 26% of all tumors (44% of OP SCCs and 17% of non-OP SCCs). For OP SCCs, RNA-CISH was the most sensitive stand-alone test (88%), but p16 + RNA-CISH was even more sensitive (95%). Specificities were the same for RNA-CISH and DNA-CISH (97%), but it was better for p16 + RNA-CISH (100%). For non-OP SCCs, all tests had sensitivities less than 50%, and RNA-CISH, DNA-CISH, and p16 + DNA-CISH had 100%, 97%, and 99% specificities, respectively. As a stand-alone test, RNA-CISH is the most performant assay to detect HPV in OP SCCs, and combined p16 + RNA-CISH test slightly improves its performances. However, RNA-CISH has the advantage of being one single test. Like p16 and DNA-CISH, RNA-CISH performances are poor in non-OP SCCs to detect HPV, and combining tests does not improve performances.