Mouth Neoplasms

Cases were all patients residing in the designated "départements" newly diagnosed with a primary, malignant tumor of the oral cavity, pharynx, sinonasal cavities, larynx, bronchi and lung (International Classification of Diseases, 10th Revision, codes C00-C14; C30-C34), during the study period which varied according to study center (see Table 2). Only histologically confirmed cases aged 75 or less at diagnosis were eligible. All histological types were included.
Poor survival for some of the cancers included in this study did not allow to rely on routine inclusion of cases in the registries for their identification. A procedure was set up to expedite case identification, in order to reduce the delay between diagnosis and interview of cases. Cases were identified from pathology laboratories, which are the first to know of a case's diagnosis, and from hospital departments. A list of laboratories and hospital departments that had reported lung and head and neck cancer cases in the two previous years was established by each registry. Interviewers contacted these laboratories and hospitals regularly to collect names and addresses of eligible patients and their physicians.
The interviewers then contacted the physician and requested permission to include their patients in the study. A letter was then sent to the patients' home, or handed to them at the hospital if they were hospitalized, informing them about the study and requesting their participation. If the patients agreed, direct contact was then made with them to arrange an interview and to collect written consent.

Our goal was to identify a prognostic signature for recurrence in OSCC, based on the hypothesis that gene expression deregulation occurring in OSCC would be an early indicator of recurrence if gene expression changes are present in a subset of histologically normal surgical resection margins. We performed gene expression profiling of both resection margins and tumors with the purpose of (1) identifying over-expressed genes in tumors as potential markers of recurrence in histologically normal margins, and (2) finding a subset of those genes predictive of recurrence. In order to generate a very high-confidence gene set, we augmented the analysis of our data with a meta-analysis of five published microarray studies [24 (link)-28 (link)] to reliably identify a set of genes significantly deregulated in OSCC compared to normal oral tissues. These five public data sets were selected based on the availability of raw microarray data, as well as for the inclusion of both oral cavity tumors and either adjacent normal tissues or oral tissues from healthy individuals. Although data from Pyeon et al. [28 (link)] included HPV positive and HPV negative head and neck carcinomas from different anatomic sites, we selected oral carcinomas only, which are mainly negative for HPV infection, as shown in a recent study performed by our group [29 (link)]. This meta-analysis sample set was composed of a total of 199 samples (141 OSCCs, 38 adjacent normal tissues and 20 healthy normal tissues) from 141 oral cancer patients and 20 healthy individuals (without cancer) (Table 3). We pre-processed data from the different array platforms with updated chip definition files, as described above, to correct outdated probe mapping information from older platforms. We used a Rank Product analysis for the public studies, which considered only the ranking of genes by differential expression between pairs of samples within studies [20 (link),30 (link)], avoiding batch and platform-related effects which would occur from directly combining expression values from the different studies. Genes were selected with evidence of up-regulation in tumors with a False Discovery Rate (FDR) of 0.01 and fold-change ≥ 2. We chose to focus on over-expressed genes only, since histologically normal margins may contain only a fraction of genetically altered cells, and the presence of genetically normal cells would likely make down-regulated genes unreliable markers. By using the intersection of genes identified both by meta-analysis and the in-house array training set, we retained only genes that were reproducibly over-expressed compared to normal oral tissues from healthy patients and histologically normal margins. These strict selection criteria for gene signature candidates, based on prior hypothesis, helped to reduce the risk of over-fitting during Cox regression analysis.

Eligibility criteria are described in Table 1. The ORGAVADS study focuses on patients with surgically resectable HNSCC who undergo surgery at François Baclesse Center and Caen University Hospital Center. After patient screening according to criteria, and the patient’s non-opposition, the patient will be enrolled in the study. An identification number will be thus assigned to each patient to be used throughout the study.Table 1

ORGAVADS study inclusion and exclusion criteria

Inclusion criteria	Non-inclusion criteria
Patient  ≥ 18 years	Pregnant women
Histologically confirmed squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx or larynx	Patient deprived of liberty or placed under the authority of a tutor
Patients for whom oncologic surgery is planned or who have recently undergone surgery of the tumor of oral cavity, oropharynx, hypopharynx or larynx
Subject affiliated to a social security regimen
No opposition to participate to the study

The entire data set consists of expression data for single cells obtained from 18 patients (5 patients with matching primary and metastatic samples) in two locations: a primary tumor site (oral cavity) with 1,426 cancer cells and 2,817 non-cancer cells, and a metastatic site (lymph node) with 788 cancer cells and 546 non-cancer cells. The non-cancer (normal) cells include fibroblasts, endothelial cells, and B and T cells, amongst others; however, we only consider fibroblasts and endothelial cells. In our analysis, there are four cellular categories: metastatic in lymph node, normal in lymph node, primary tumor in oral cavity, and normal in oral cavity.

Client owned dogs with spontaneously occurring oral and perioral neoplasia were prospectively enrolled from May 2019-May 2022. Inclusion criteria included dogs with any oral pathology that had confirmed or highly suspicious cervical metastasis (cN+ neck) based on palpation, diagnostic imaging, or cytology. Dogs with the cN0 neck were recruited if the primary oral pathology was associated with a ≥20% risk of locoregional spread. Specifically, based on current literature, this included T1-T3 OMM [3 (link),4 (link),8 (link),19 (link),40 (link)–42 (link)], T2-T3 OSCC [2 (link),19 (link),43 (link),44 (link)], and T1-T3 oral/perioral MCT [1 (link),9 (link)]. Furthermore, dogs with a primary oral tumor and distant metastasis were deemed at high risk for LN metastasis and were also eligible for enrollment. Both grossly visible masses and excisional biopsy scars were permitted if they met the other inclusion criteria. Dogs were excluded if prior cervical dissection or locoregional radiation therapy had occurred, or if there was a contra-indication to intravenous contrast administration.
Tumor size was categorized based on the World Health Organization grading scheme [45 ]. Prior chemotherapy, radiation therapy or immunotherapy were not permitted. The study was approved by the Institutional Animal Care and Use Committee (IACUC #2201-39790A). Written client consent was obtained prior to enrollment. Clinical data, including signalment, weight, tumor histology, tumor longest diameter, and tumor location were recorded. In dogs presenting with microscopic disease, the original documented tumor diameter was recorded. Tumor location was categorized by laterality and as rostral maxilla, caudal maxilla, rostral mandible, caudal mandible, mucosal (not directly overlying bone), or tongue. Caudal was defined as distal to the second premolar [46 (link)].