Preneoplastic Lesions in Surgical Specimens Do Not Worsen the Prognosis of Patients Who Underwent Surgery for Pancreatic Adenocarcinoma: Post-Hoc Analysis of the PRODIGE 24-CCTG PA 6 Trial.

OBJECTIVE: The prognosis of pancreatic cancer after curative surgery is burdened by frequent recurrence. The aim of this study was to evaluate the impact of dysplasia in the surgical specimen on disease-free survival (DFS). METHODS: A post-hoc analysis of the phase III PRODIGE 24-CCTG PA 6 trial was performed. From April 2012 to October 2016, 493 patients were included in the primary study. Assessment for dysplasia in the surgical specimens was secondarily performed. Dysplasia was defined based on presence and grade of three most common pre-malignant lesions (intraductal papillary mucinous neoplasm (IPMN), mucinous cystic neoplasm (MCN) and pancreatic intraepithelial neoplasia (PanIN). The primary endpoint was DFS validated through multivariate analysis. RESULTS: Two hundred twenty-six patients (45.9%) had a preneoplastic lesion. PanIN lesions were found in 193 patients (39.2%), including 100 high-grade lesions (20.6%); 43 patients had IPMN lesions (8.7%), including high-grade lesions in 32 (6.5%). Three MCN were described (0.6%). In bivariate analysis, the presence of dysplasia was not associated with poorer DFS (HR = 0.82, 95% CI [0.66; 1.03]). In multivariate analysis, risk factors for poorer DFS were poorly differentiated/undifferentiated tumor, N1 status, R1 surgical margins and perineural invasion. CONCLUSIONS: The presence of dysplasia in the surgical specimen after pancreatic cancer surgery does not worsen DFS.

Antitumor Effect and Induced Immune Response Following Exposure of Hexaminolevulinate and Blue Light in Combination with Checkpoint Inhibitor in an Orthotopic Model of Rat Bladder Cancer.

Previous studies have found that use of hexaminolevulinate (HAL) and blue light cystoscopy (BLC) during treatment of bladder cancer had a positive impact on overall survival after later cystectomy, indicating a potential treatment effect beyond improved diagnostic accuracy. The aim of our study was to determine whether HAL and BL mimicking clinically relevant doses in an orthotopic rat model could have therapeutic effect by inducing modulation of a tumor-specific immune response. We also assessed whether administration with a checkpoint inhibitor could potentiate any effects observed. Rats were subjected to HAL BL alone and in combination with anti-PD-L1 and assessed for anti-tumor effects and effects on immune markers. Positive anti-tumor effect was observed in 63% and 31% of rats after, respectively, 12 and 30 days after the procedure, together with a localization effect of CD3+ and CD8+ cells after 30 days. Anti-tumor effect at 30 days increases from 31% up to 38% when combined with intravesical anti-PD-L1. In conclusion, our study demonstrated treatment effects with indications of systemic immune activation at diagnostic doses of HAL and blue light. The observed treatment effect seemed to be enhanced when used in combination with intravesically administrated immune checkpoint inhibitor.

A NGS-based Blood Test For the Diagnosis of Invasive HPV-associated Carcinomas with Extensive Viral Genomic Characterization.

PURPOSE: Use of circulating tumor DNA (ctDNA) for diagnosis is limited regarding the low number of target molecules in early-stage tumors. Human papillomavirus (HPV)-associated carcinomas represent a privileged model using circulating viral DNA (ctHPV DNA) as a tumor marker. However, the plurality of HPV genotypes represents a challenge. The next-generation sequencing (NGS)-based CaptHPV approach is able to characterize any HPV DNA sequence. To assess the ability of this method to establish the diagnosis of HPV-associated cancer via a blood sample, we analyzed ctHPV DNA in HPV-positive or HPV-negative carcinomas. EXPERIMENTAL DESIGN: Patients (135) from France and Senegal with carcinoma developed in the uterine cervix (74), oropharynx (25), oral cavity (19), anus (12), and vulva (5) were prospectively registered. Matched tumor tissue and blood samples (10 mL) were taken before treatment and independently analyzed using the CaptHPV method. RESULTS: HPV prevalence in tumors was 60.0% (81/135; 15 different genotypes). Viral analysis of plasmas compared with tumors was available for 134 patients. In the group of 80 patients with HPV-positive tumors, 77 were also positive in plasma (sensitivity 95.0%); in the group of 54 patients with HPV-negative tumors, one was positive in plasma (specificity 98.1%). In most cases, the complete HPV pattern observed in tumors could be established from the analysis of ctHPV DNA. CONCLUSIONS: In patients with carcinoma associated with any HPV genotype, a complete viral genome characterization can be obtained via the analysis of a standard blood sample. This should favor the development of noninvasive diagnostic tests providing the identification of personalized tumor markers. See related commentary by Rostami et al., p. 5158.

Differences in clinical and imaging characteristics between p16-positive non-smokers and p16-positive smokers or p16-negative patients in oropharyngeal carcinoma.

The eighth edition of the TNM classifies oropharyngeal squamous cell carcinomas (OSCCs) depending on p16 status. Some imaging features are reportedly associated more frequently with p16-positive (P16+) OSCC than p16-negative (p16-) OSCC. However, classical risk factors such as tobacco use were not specifically considered when assessing these imaging features. We aimed to evaluate whether P16+ OSCCs have different epidemiological, clinical, prognostic and imaging features depending on smoking status, and to compare P16+ and p16- groups. A retrospective study of data from 85 patients with P16+ OSCC (41 non-smokers, 44 smokers) and 36 with p16- OSCC from 2011 to 2020 was carried out, assessing epidemiological data, clinical aspects of the tumour and presence of adenopathy. Staging was assessed according to the seventh and eighth editions of the TNM. Compared with P16+ OSCC non-smokers, P16+ OSCC smokers had tumours that were less well-defined (36.6% vs 77.8%, p < 0.001), more ulcerated (85.4% vs 44.4%, p < 0.001) and more necrotic (53.7% vs 25%, p = 0.012). There was also less downstaging from N2 or N3 of the seventh edition of the TNM to N1 of the eighth edition for smokers than non-smokers (22.7% vs 43.9%, p = 0.042). Compared with p16- tumours, more P16+ tumours had well-defined contours (55.8% vs 22.2%, p = 0.001), were exophytic (89.6% vs 72.2%, p = 0.023), less necrotic (40.3% vs 80.6%, p < 0.001), less ulcerated (97.2% vs 66.2%, p = 0.006) and involved less muscle tissue (26.0% vs 47.2%, p = 0.027).P16+ OSCCs of smokers show clinical, imaging and prognostic differences with P16+ OSCCs of non-smokers.

Expression of immune response biomarkers (PD­L1, p16, CD3+ and CD8+ TILs) in recurrent head and neck squamous cell carcinoma within previously irradiated areas.

The immune landscape of head and neck squamous cell carcinoma in pretreated areas remains poorly documented. We aimed to assess the tumor microenvironment for biomarkers of antitumor immune responses in tumors in previously irradiated areas compared with de novo tumors. This retrospective monocentric study analyzed 100 paraffin­embedded surgical samples of invasive head and neck squamous cell carcinoma (oral cavity, oropharynx, larynx, hypopharynx) from patients who underwent surgery between January 2010 and November 2017. We compared the immune microenvironment in 50 de novo tumors and 50 tumors recurring within irradiated areas. We used immunohistochemistry to assess p16 status, CD3+/CD8+ tumor­infiltrating lymphocytes (TILs), and programmed death­ligand 1 (PD­L1) expression on tumor and immune cells in stromal and intratumoral components. CD3+ TIL counts were significantly lower in intratumoral and stromal components (P=0.003 and P=0.020, respectively) in the irradiated area cohort; there was no significant difference between CD8+ TIL counts in the two cohorts. The percentage of tumors with PD­L1+ tumor cells (tumor proportion score ≥1%) was significantly lower within the irradiated area cohort than the de novo cohort (56.0% vs. 86.0%, P<0.001). There were also significantly fewer tumors with PD­L1+ immune cells in the irradiated area cohort. Predominantly, tumors from the irradiated area cohort had microenvironments classified as 'adaptive immune resistance'. There was persistence of cytotoxic cells in tumors in the irradiated areas but lower PD­L1 expression and CD3+ TIL counts than in the de novo tumors. This offers an initial hypothesis to explain why these lesions are less responsive to immunotherapy, even though they may still have antitumor capacities. Assessment of immune response biomarkers in patients treated with immunotherapy in randomized trials is required.

Identification of Specific Tumor Markers in Vulvar Carcinoma Through Extensive Human Papillomavirus DNA Characterization Using Next Generation Sequencing Method.

OBJECTIVES: A subset of vulvar carcinomas (VC) are associated with human papillomavirus (HPV) DNA. This trait can be used to identify tumor markers for patient's follow-up. A large diversity of HPV prevalence in VC has been reported, but no data are available concerning the insertional HPV status in this tumor type. Therefore, we have used an innovative next generation sequencing (NGS)-based CaptHPV method able to provide an extensive characterization of HPV DNA in tumors. MATERIAL AND METHODS: Tumor tissue specimens from 55 patients with VC were analyzed using p16 immunohistochemistry, in situ hybridization, polymerase chain reaction, and CaptHPV-NGS assays. RESULTS: Our analyses showed that 8 (14.5%) of 55 cases were associated with HPV 16 DNA. No other HPV genotypes were identified. The HPV genome was in a free episomal state only in one case and both episomal and integrated into the tumor cell genome in 7. There was a single insertion in 5 cases and multiple sites, scattered at different chromosomal loci in two. ISH data suggest that some of these might reflect tumor heterogeneity. Viral integration targeted cellular genes among which were TP63, CCDC148, LOC100133091, PKP1, and POLA2. Viral integration at the PKP1 locus was associated with partial gene deletion, and no PKP1 protein was detected in tumor tissue. CONCLUSIONS: Using the NGS-based innovative capture-HPV approach, we established a cartography of HPV 16 DNA in 8 VC cases and identified novel genes targeted by integration that may be used as specific tumor markers. In addition, we established a rationale strategy for optimal characterization of HPV status in VC.

HPV insertional pattern as a personalized tumor marker for the optimized tumor diagnosis and follow-up of patients with HPV-associated carcinomas: a case report.

BACKGROUND: In clinical oncology, only a few applications have been developed using HPV as a personalized tumor marker, a lack most probably related to the limited information obtained by the classical Polymerase Chain Reaction (PCR) approach. To overcome this limitation, we have recently developed the capture-based Next-Generation Sequencing (NGS) "CaptHPV" assay, designed to provide an extensive and comprehensive molecular characterization of HPV DNA sequences associated with neoplasias, ie the sequence of the viral genome (245 genotypes), its physical state, viral load, integration site and genomic alterations at integration locus. These data correspond to highly specific tumor markers that can be used to improve diagnosis and patient's follow-up. CASE PRESENTATION: We report here a case that is a straightforward and practical illustration of the power of the CaptHPV method. A patient developed successively a carcinoma of the anal canal and of the tongue. The two tumors were squamous cell carcinoma, found associated with HPV16 using PCR. In order to document a possible metastasis to the tongue from the anal cancer, we performed CaptHPV analysis on the two tumors. The analysis of the anal carcinoma found 55 viral/human hybrid reads allowing the identification of the HPV16 DNA integration in the 4q25 chromosomal band locus with a 178,808 bp deletion in the cell genome. Molecular analysis of the tongue tumor disclosed 6110 reads of HPV16, with a viral pattern strictly identical to that of the anal tumor. A total of 131 hybrid reads between HPV16 and the cell genome were found, corresponding exactly to the same locus of integration of viral DNA at the 4q25 site. The 178,808 bp genomic deletion was also found in the lingual tumor. The exact identity of HPV insertional signatures in the two tumors, demonstrates unambiguously that the tongue tumor derived from the anal cancer whereas neither histological immunophenotyping nor classical viral analysis using PCR could allow a definitive diagnosis. CONCLUSION: Our observation indicates that the establishment of a detailed cartography of HPV DNA sequences in a tumor specimen provides crucial information for the design of specific biomarkers that can be used for diagnostic, prognostic or predictive purposes.

Evaluation and validation of HPV real-time PCR assay for the detection of HPV DNA in oral cytobrush and FFPE samples.

Specific HPV genotypes have been recognized as risk factors inducing head and neck cancers (HNC). The aim of this study was to validate a real-time PCR assay to detect accurately High Risk HPV DNA in Formalin Fixed Paraffin Embedded (FFPE) and oral cytobrush samples and compare the results with conventional PCR. Repeatability, reproducibility and limit of detection of Cobas assay were estimated for oral cytobrush and FFPE samples of patients with HNC. 53 samples of patients with a HNC were then used for assay comparison with conventional PCR. Finally, 26 samples of patients with anogenital neoplasia cancer were analyzed as control and assays comparison. Among the 53 samples of patients with HNC, 12 (26.7%) were HPV positive, 33 (73.3%) were HPV negative and 8 (15.1%) were non contributive with the Cobas assay. Among the 26 samples of patients with anogenital neoplasia, 15 (57.7%) were HPV positive and 11 were HPV negative (42.3%). One sample was found with an HPV 16 and HPV 18 co-infection. Only 3 samples were found with discrepant results. Cobas assay was found suitable for routine HPV detection with a very good repeatability and reproducibility for all HPV genotypes (CV < 0.6% and <0.4% respectively). Sensitivity and specificity for Cobas assay were 91.7% [61.5%;99.8%] and 96.9% [83.8%;99.9%] respectively. Ten nanograms of DNA were sufficient for the detection of HPV 16, HPV 18 and HPV in FFPE and oral cytobrush samples. Cobas assay was found comparable to conventional PCR and can detect accurately and rapidly HPV DNA in FFPE and oral cytobrush samples for the management of HNC and other types of HPV-associated neoplasia.