Faculty Bibliography

AIM/UNASSIGNED:Oral cancer patients suffer pain at the site of the cancer, which degrades quality of life (QoL). The University of California San Francisco Oral Cancer Pain Questionnaire (UCSFOCPQ), the only validated instrument specifically designed for measuring oral cancer pain, measures the intensity and nature of pain and the level of functional restriction due to pain. PURPOSE/UNASSIGNED:The aim of this study was to compare pain reported by untreated oral cancer patients on the UCSFOCPQ with pain they reported on the Brief Pain Inventory (BPI), an instrument widely used to evaluate cancer and non-cancer pain. PATIENTS AND METHODS/UNASSIGNED:The correlation between pain measured by the two instruments and clinical characteristics were analyzed. Thirty newly diagnosed oral cancer patients completed the UCSFOCPQ and the BPI. RESULTS/UNASSIGNED:Pain severity measurements made by the UCSFOCPQ and BPI were concordant; however, the widely used BPI average pain over 24 hours score appeared less sensitive to detect association of oral cancer pain with clinical characteristics of patients prior to treatment (nodal status, depth of invasion, DOI). A BPI average score that includes responses to questions that measure both pain severity and interference with function performs similarly to the UCSFOCPQ in detection of associations with nodal status, pathologic T stage (pT stage), stage and depth of invasion (DOI). CONCLUSION/UNASSIGNED:Pain assessment instruments that measure sensory and interference dimensions of oral cancer pain correlate with biologic features and clinical behavior.

Oral cancer patients suffer pain at the site of the cancer. Calcitonin gene related polypeptide (CGRP), a neuropeptide expressed by a subset of primary afferent neurons, promotes oral cancer growth. CGRP also mediates trigeminal pain (migraine) and neurogenic inflammation. The contribution of CGRP to oral cancer pain is investigated in the present study. The findings demonstrate that CGRP-immunoreactive (-ir) neurons and neurites innervate orthotopic oral cancer xenograft tumors in mice. Cancer increases anterograde transport of CGRP in axons innervating the tumor, supporting neurogenic secretion as the source of CGRP in the oral cancer microenvironment. CGRP antagonism reverses oral cancer nociception in preclinical oral cancer pain models. Single-cell RNA-sequencing is used to identify cell types in the cancer microenvironment expressing the CGRP receptor components, receptor activity modifying protein 1 Ramp1 and calcitonin receptor like receptor (CLR, encoded by Calcrl). Ramp1 and Calcrl transcripts are detected in cells expressing marker genes for Schwann cells, endothelial cells, fibroblasts and immune cells. Ramp1 and Calcrl transcripts are more frequently detected in cells expressing fibroblast and immune cell markers. This work identifies CGRP as mediator of oral cancer pain and suggests the antagonism of CGRP to alleviate oral cancer pain.

INTRODUCTION/BACKGROUND:Oral cancer patients suffer severe chronic and mechanically-induced pain at the site of the cancer. Our clinical experience is that oral cancer patients report new sensitivity to spicy foods. We hypothesized that in cancer patients, mechanical and chemical sensitivity would be greater when measured at the cancer site compared to a contralateral matched normal site. METHODS:We determined mechanical pain thresholds (MPT) on the right and left sides of the tongue of 11 healthy subjects, and at the cancer and contralateral matched normal site in 11 oral cancer patients in response to von Frey filaments in the range of 0.008 to 300 g (normally not reported as painful). We evaluated chemical sensitivity in 13 healthy subjects and seven cancer patients, who rated spiciness/pain on a visual analog scale in response to exposure to six paper strips impregnated with capsaicin (0-10 mM). RESULTS:Mechanical detection thresholds (MDT) were recorded for healthy subjects, but not MPTs. By contrast, MPTs were measured at the site of the cancer in oral cancer patients (7/11 patients). No MPTs were measured at the cancer patients' contralateral matched normal sites. Measured MPTs were correlated with patients' responses to the University of California Oral Cancer Pain Questionnaire. Capsaicin sensitivity at the site of the cancer was evident in cancer patients by a leftward shift of the cancer site capsaicin dose-response curve compared to that of the patient's contralateral matched normal site. We detected no difference in capsaicin sensitivity on the right and left sides of tongues of healthy subjects. CONCLUSIONS:Mechanical and chemical sensitivity testing was well tolerated by the majority of oral cancer patients. Sensitivity is greater at the site of the cancer than at a contralateral matched normal site.

Oral cancer pain is attributed to the release from cancers of mediators that sensitize and activate sensory neurons. Intraplantar injection of conditioned media (CM) from human tongue cancer cell line HSC-3 or OSC-20 evokes nociceptive behavior. By contrast, CM from noncancer cell lines, DOK, and HaCaT are non-nociceptive. Pain mediators are carried by extracellular vesicles (EVs) released from cancer cells. Depletion of EVs from cancer cell line CM reverses mechanical allodynia and thermal hyperalgesia. CM from non-nociceptive cell lines become nociceptive when reconstituted with HSC-3 EVs. Two miRNAs (hsa-miR-21-5p and hsa-miR-221-3p) are identified that are present in increased abundance in EVs from HSC-3 and OSC-20 CM compared to HaCaT CM. The miRNA target genes suggest potential involvement in oral cancer pain of the toll like receptor 7 (TLR7) and 8 (TLR8) pathways, as well as signaling through interleukin 6 cytokine family signal transducer receptor (gp130, encoded by IL6ST) and colony stimulating factor receptor (G-CSFR, encoded by CSF3R), Janus kinase and signal transducer and activator of transcription 3 (JAK/STAT3). These studies confirm the recent discovery of the role of cancer EVs in pain and add to the repertoire of algesic and analgesic cancer pain mediators and pathways that contribute to oral cancer pain.

Cancer invading into nerves, termed perineural invasion (PNI), is associated with pain. Here we show that oral cancer patients with PNI report greater spontaneous pain and mechanical allodynia compared with patients without PNI, suggesting unique mechanisms drive PNI-induced pain. We studied the impact of PNI on peripheral nerve physiology and anatomy using a murine sciatic nerve PNI model. Mice with PNI exhibited spontaneous nociception and mechanical allodynia. PNI induced afterdischarge in A high threshold mechanoreceptors (AHTMRs), mechanical sensitization (i.e., decreased mechanical thresholds) in both A and C HTMRs, and mechanical desensitization in low threshold mechanoreceptors (LTMRs). PNI resulted in nerve damage, including axon loss, myelin damage, and axon degeneration. Electrophysiological evidence of nerve injury included decreased conduction velocity, and increased percentage of both mechanically-insensitive and electrically-unexcitable neurons. We conclude that PNI-induced pain is driven by nerve injury and peripheral sensitization in HTMRs.

Oral cancer patients often have severe, chronic, and mechanically induced pain at the site of the primary cancer. Oral cancer pain is initiated and maintained in the cancer microenvironment and attributed to release of mediators that sensitize primary sensory nerves. This study was designed to investigate the histopathology associated with painful oral cancers in a preclinical model. The relationship of pain scores with pathologic variables was also investigated in a cohort of 72 oral cancer patients. Wild-type mice were exposed to the carcinogen, 4-nitroquinoline 1-oxide (4NQO). Nociceptive (pain) behavior was measured with the dolognawmeter, an operant device and assay for measuring functional and mechanical allodynia. Lesions developed on the tongues and esophagi of the 4NQO-treated animals and included hyperkeratoses, papillomas, dysplasias, and cancers. Papillomas included lesions with benign and dysplastic pathological features. Two histologic subtypes of squamous cell carcinomas (SCCs) were identified-SCCs with exophytic and invasive components associated with papillary lesions (pSCCs) and invasive SCCs without exophytic histology (iSCCs). Only the pSCC subtype of tongue cancer was associated with nociceptive behavior. Increased tumor size was associated with greater nociceptive behavior in the mouse model and more pain experienced by oral cancer patients. In addition, depth of invasion was associated with patient-reported pain. The pSCC histology identifies 4NQO-induced tongue cancers that are expected to be enriched for expression and release of nociceptive mediators.

Oral cancer patients experience pain at the site of the primary cancer. Patients with metastatic oral cancers report greater pain. Lack of pain identifies patients at low risk of metastasis with sensitivity = 0.94 and negative predictive value = 0.89. In the same cohort, sensitivity and negative predictive value of depth of invasion, currently the best predictor, were 0.95 and 0.92, respectively. Cancer pain is attributed to cancer-derived mediators that sensitize neurons and is associated with increased neuronal density. We hypothesized that pain mediators would be overexpressed in metastatic cancers from patients reporting high pain. We identified 40 genes overexpressed in metastatic cancers from patients reporting high pain (n = 5) compared to N0 cancers (n = 10) and normal tissue (n = 5). The genes are enriched for functions in extracellular matrix organization and angiogenesis. They have oncogenic and neuronal functions and are reported in exosomes. Hierarchical clustering according to expression of neurotrophic and axon guidance genes also separated cancers according to pain and nodal status. Depletion of exosomes from cancer cell line supernatant reduced nociceptive behavior in a paw withdrawal assay, supporting a role for exosomes in cancer pain. The identified genes and exosomes are potential therapeutic targets for stopping cancer and attenuating pain.

Pain is rated by oral cancer patients as the worst symptom and significantly impairs a patient's ability to eat, talk, and drink. Mediators, secreted from oral cancer microenvironment, excite primary afferent neurons, which in turn generate pain. Oral cancer cells release TNFalpha which induces acute inflammation and nociception in mice. We hypothesize that TNFalpha activates Schwann cells to amplify pain signals. First, we confirmed the involvement of TNFalpha in oral cancer pain in patients and animal models. We found that oral cancer tissues collected from patients have higher TNFalpha concentration compared to anatomically matched normal tissues. Differences in TNFalpha concentration between the tumor and anatomically matched normal tissues correlate positively with total pain scores. In a Nitroquinoline 1-oxide (4NQO) mouse oral cancer model we demonstrated reduced mechanical hypersensitivity (P<0.05, N=8) with the dolognawmeter gnawing assay when TNFalpha was neutralized with C-87. Using a non-contact co-culture model, we found that HSC-3 cells induced a more activated human primary Schwann cells phenotype with increased proliferation (P<0.05) and migration (P<0.05); introduction of C-87 in the co-culture reduced Schwann cell proliferation (P<0.05) and migration (P<0.05) induced by HSC-3 cells. After removal of the co-cultured cancer cells, cancer-activated Schwann cells secrete greater amounts of TNFalpha and nerve growth factor (NGF), another known nociceptive mediator in the oral cancer microenvironment, compared to Schwann cells initially co-cultured with DOK (P<0.05) or naive Schwann cells (P<0.05). To determine whether activated Schwann cells mediate oral cancer pain, we cultured Schwann cells in hypoxic conditions - a known cancer stimulus that induces robust Schwann cell activation. Schwann cell supernatant was then collected and injected into the mouse cheek. Supernatant from hypoxia-activated Schwann cells induced greater facial allodynia (measured with von Frey filaments) in mice (P<0.05, N=7), compared to supernatant from Schwann cells cultured in normoxic conditions (N=5). C-87 significantly reduced facial allodynia caused by hypoxiaactivated Schwann cells (P<0.05, N=5). We infer from our results that TNFalpha plays a role in the activation of Schwann cells and that cancer-activated Schwann cells are a source of nociceptive mediators in the cancer microenvironment. Inhibition of Schwann cell activation might be clinically useful for alleviating oral cancer pain

The incidence of oral cancer in the United States is increasing, especially in young people and women. Patients with oral cancer report severe functional pain. Using a patient cohort accrued through the New York University Oral Cancer Center and immune-competent mouse models, we identify a sex difference in the prevalence and severity of oral cancer pain. A neutrophil-mediated endogenous analgesic mechanism is present in male mice with oral cancer. Local naloxone treatment potentiates cancer mediator-induced orofacial nociceptive behavior in male mice only. Tongues from male mice with oral cancer have significantly more infiltrating neutrophils compared to female mice with oral cancer. Neutrophils isolated from the cancer-induced inflammatory microenvironment express beta-endorphin and met-enkephalin. Furthermore, neutrophil depletion results in nociceptive behavior in male mice. These data suggest a role for sex-specific, immune cell-mediated endogenous analgesia in the treatment of oral cancer pain.