Assessing Prognostic Value of Quantitative Neck Dissection Quality Measures in Patients With Clinically Node-Negative Oral Cavity Squamous Cell Carcinoma.

Importance: In clinically localized (T1-2) oral cavity squamous cell carcinoma (OCSCC), regional lymph node metastasis is associated with a poor prognosis. Given the high propensity of subclinical nodal disease in these patients, upfront elective neck dissections (END) for patients with clinically node-negative disease are common and associated with better outcomes. Unfortunately, even with this risk-adverse treatment paradigm, disease recurrence still occurs, and our understanding of the factors that modulate this risk and alter survival have yet to be fully elucidated. Objective: To investigate the prognostic value of lymph node yield (LNY), lymph node ratio (LNR), and weighted LNR (wLNR) in patients with clinically node-negative T1-2 OCSCC. Design, Setting, and Participants: In this cohort study, data were collected retrospectively from 7 tertiary care academic medical centers. Overall, 523 patients with cT1-2N0 OCSCC who underwent elective neck dissections after primary surgical extirpation were identified. Exposures: Lymph node yield was defined as the number of lymph nodes recovered from elective neck dissection. Lymph node ratio was defined as the ratio of positive nodes against total LNY. Weighted LNR incorporated information from both LNY and LNR into a single continuous metric. Main Outcomes and Measures: Locoregional control (LRC) and disease-free survival (DFS) were both evaluated using nonparametric Kaplan-Meier estimators and semiparametric Cox regression. Results: On multivariable analysis, LNY less than or equal to 18 lymph nodes was found to be significantly associated with decreased LRC (aHR, 1.53; 95% CI, 1.04-2.24) and DFS (aHR, 1.46; 95% CI, 1.12-1.92) in patients with pN0 disease, but not those with pN-positive disease. Importantly, patients with pN0 disease with LNY less than or equal to 18 and those with pN1 diseasehad nearly identical 5-year LRC (69.7% vs 71.4%) and DFS (58.2% vs 55.7%). For patients with pN-positive disease, LNR greater than 0.06 was significantly associated with decreased LRC (aHR, 2.66; 95% CI, 1.28-5.55) and DFS (aHR, 1.65; 95% CI, 1.07-2.53). Overall, wLNR was a robust prognostic variable across all patients with cN0 disease, regardless of pathologic nodal status. Risk stratification via wLNR thresholds demonstrated greater optimism-corrected concordance compared with American Joint Committee on Cancer (AJCC) 8th edition nodal staging for both LRC (0.61 vs 0.57) and DFS (0.61 vs 0.58). Conclusions and Relevance: Movement toward more robust metrics that incorporate quantitative measures of neck dissection quality and regional disease burden, such as wLNR, could greatly augment prognostication in cT1-2N0 OCSCC by providing more reliable and accurate risk estimations.

Development and Validation of Machine Learning Models for Predicting Occult Nodal Metastasis in Early-Stage Oral Cavity Squamous Cell Carcinoma.

Importance: Given that early-stage oral cavity squamous cell carcinoma (OCSCC) has a high propensity for subclinical nodal metastasis, elective neck dissection has become standard practice for many patients with clinically negative nodes. Unfortunately, for most patients without regional metastasis, this risk-averse treatment paradigm results in unnecessary morbidity. Objectives: To develop and validate predictive models of occult nodal metastasis from clinicopathological variables that were available after surgical extirpation of the primary tumor and to compare predictive performance against depth of invasion (DOI), the currently accepted standard. Design, Setting, and Participants: This diagnostic modeling study collected clinicopathological variables retrospectively from 7 tertiary care academic medical centers across the US. Participants included adult patients with early-stage OCSCC without nodal involvement who underwent primary surgical extirpation with or without upfront elective neck dissection. These patients were initially evaluated between January 1, 2000, and December 31, 2019. Exposures: Largest tumor dimension, tumor thickness, DOI, margin status, lymphovascular invasion, perineural invasion, muscle invasion, submucosal invasion, dysplasia, histological grade, anatomical subsite, age, sex, smoking history, race and ethnicity, and body mass index (calculated as weight in kilograms divided by height in meters squared). Main Outcomes and Measures: Occult nodal metastasis identified either at the time of elective neck dissection or regional recurrence within 2 years of initial surgery. Results: Of the 634 included patients (mean [SD] age, 61.2 [13.6] years; 344 men [54.3%]), 114 (18.0%) had occult nodal metastasis. Patients with occult nodal metastasis had a higher frequency of lymphovascular invasion (26.3% vs 8.1%; P < .001), perineural invasion (40.4% vs 18.5%; P < .001), and margin involvement by invasive tumor (12.3% vs 6.3%; P = .046) compared with those without pathological lymph node metastasis. In addition, patients with vs those without occult nodal metastasis had a higher frequency of poorly differentiated primary tumor (20.2% vs 6.2%; P < .001) and greater DOI (7.0 vs 5.4 mm; P < .001). A predictive model that was built with XGBoost architecture outperformed the commonly used DOI threshold of 4 mm, achieving an area under the curve of 0.84 (95% CI, 0.80-0.88) vs 0.62 (95% CI, 0.57-0.67) with DOI. This model had a sensitivity of 91.7%, specificity of 72.6%, positive predictive value of 39.3%, and negative predictive value of 97.8%. Conclusions and Relevance: Results of this study showed that machine learning models that were developed from multi-institutional clinicopathological data have the potential to not only reduce the number of pathologically node-negative neck dissections but also accurately identify patients with early OCSCC who are at highest risk for nodal metastases.

Prognostic Impact of Metastatic Site and Pattern in Patients with Metastatic Head and Neck Cancer.

OBJECTIVES/HYPOTHESIS: Investigate the relationship between site and pattern of distant metastasis (DM) and overall survival (OS) in a multi-institutional cohort of patients with DM head and neck cancer (HNC). STUDY DESIGN: Retrospective review. METHODS: 283 patients treated at 4 academic centers in the Midwest HNC Consortium between 2000 and 2015 were retrospectively reviewed. Disease patterns were divided between solitary metastatic versus polymetastatic (≥2 sites) disease. Survival functions for clinically relevant variables were estimated using Kaplan-Meier and Cox proportional hazards models. RESULTS: Median OS for all patients was 9.0 months (95% confidence interval [CI]: 7.4-10.6). Lung (n = 220, 77.7%) was the most common site of DM, followed by bone (n = 90, 31.8%), mediastinal lymph nodes (n = 55, 19.4%), liver (n = 41, 14.5%), and brain (n = 17, 6.0%). Bone metastases were independently associated with the worst prognosis (hazard ratio [HR] = 1.6, 95% CI: 1.3-2.1). On univariate analysis, brain metastases were associated with improved prognosis (HR = 0.5, 95% CI: 0.3-0.9), although this was not statistically significant on the multivariate analysis. Polymetastatic disease was present in the majority of patients (n = 230, 81.3%) and was associated with a worse prognosis compared to solitary metastatic disease (HR = 1.4, 95% CI: 1.0-2.0). CONCLUSION: Our large, multi-institutional review indicates that both the metastatic pattern and site of DM impact OS. Polymetastatic disease and bone metastasis are associated with worse prognosis, independent of treatment received. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:E1838-E1846, 2021.

Macrophage inflammatory factors promote epithelial-mesenchymal transition in breast cancer.

The majority of breast cancers (90-95%) arise due to mediators distinct from inherited genetic mutations. One major mediator of breast cancer involves chronic inflammation. M1 macrophages are an integral component of chronic inflammation and the breast cancer tumor microenvironment (TME). Previous studies have demonstrated that up to 50% of the breast tumor comprise of tumor-associated macrophages (TAMs) and increased TAM infiltration has been associated with poor patient prognosis. Furthermore, breast cancer associated deaths are predominantly attributed to invasive cancers and metastasis with epithelial-mesenchymal transition (EMT) being implicated. In this study, we investigated the effects of cellular crosstalk between TAMs and breast cancer using an in vitro model system. M1 polarized THP-1 macrophage conditioned media (CM) was generated and used to evaluate cellular and functional changes of breast cancer lines T47D and MCF-7. We observed that T47D and MCF-7 exhibited a partial EMT phenotype in the presence of activated THP-1 CM. Additionally, MCF-7 displayed a significant increase in migratory and invasive properties. We conclude that M1 secretory factors can promote a partial EMT of epithelial-like breast cancer cells. The targeting of M1 macrophages or their secretory components may inhibit EMT and limit the invasive potential of breast cancer.

Hyperactive ERK and persistent mTOR signaling characterize vemurafenib resistance in papillary thyroid cancer cells.

Clinical studies evaluating targeted BRAFV600E inhibitors in advanced thyroid cancer patients are currently underway. Vemurafenib (BRAFV600E inhibitor) monotherapy has shown promising results thus far, although development of resistance is a clinical challenge. The objective of this study was to characterize development of resistance to BRAFV600E inhibition and to identify targets for effective combination therapy. We created a line of BCPAP papillary thyroid cancer cells resistant to vemurafenib by treating with increasing concentrations of the drug. The resistant BCPAP line was characterized and compared to its sensitive counterpart with respect to signaling molecules thought to be directly related to resistance. Expression and phosphorylation of several critical proteins were analyzed by Western blotting and dimerization was evaluated by immunoprecipitation. Resistance to vemurafenib in BCPAP appeared to be mediated by constitutive overexpression of phospho-ERK and by resistance to inhibition of both phospho-mTOR and phospho-S6 ribosomal protein after vemurafenib treatment. Expression of potential alternative signaling molecule, CRAF, was not increased in the resistant line, although formation of CRAF dimers appeared increased. Expression of membrane receptors HER2 and HER3 was greatly amplified in the resistant cancer cells. Papillary thyroid cancer cells were capable of overcoming targeted BRAFV600E inhibition by rewiring of cell signal pathways in response to prolonged vemurafenib therapy. Our study suggests that in vitro culture of cancer cells may be useful in assessing molecular resistance pathways. Potential therapies in advanced thyroid cancer patients may combine vemurafenib with inhibitors of CRAF, HER2/HER3, ERK, and/or mTOR to delay or abort development of resistance.

mTOR inhibitors sensitize thyroid cancer cells to cytotoxic effect of vemurafenib.

Treatment options for advanced metastatic thyroid cancer patients are limited. Vemurafenib, a BRAFV600E inhibitor, has shown promise in clinical trials although cellular resistance occurs. Combination therapy that includes BRAFV600E inhibition and avoids resistance is a clinical need. We used an in vitro model to examine combination treatment with vemurafenib and mammalian target of rapamycin (mTOR) inhibitors, metformin and rapamycin. Cellular viability and apoptosis were analyzed in thyroid cell lines by trypan blue exclusion and TUNEL assays. Combination of vemurafenib and metformin decreased cell viability and increased apoptosis in both BCPAP papillary thyroid cancer cells and 8505c anaplastic thyroid cancer cells. This combination was also found to be active in vemurafenib-resistant BCPAP cells. Changes in expression of signaling molecules such as decreased mTOR expression in BCPAP and enhanced inhibition of phospho-MAPK in resistant BCPAP and 8505c were observed. The second combination of vemurafenib and rapamycin amplified cell death in BCPAP cells. We conclude that combination of BRAFV600E and mTOR inhibition forms the basis of a treatment regimen that should be further investigated in in vivo model systems. Metformin or rapamycin adjuvant treatment may provide clinical benefits with minimal side effects to BRAFV600E-positive advanced thyroid cancer patients treated with vemurafenib.

Disruption of mutated BRAF signaling modulates thyroid cancer phenotype.

BACKGROUND: Thyroid cancer is the most common endocrine-related cancer in the United States and its incidence is rising rapidly. Since among various genetic lesions identified in thyroid cancer, the BRAFV600E mutation is found in 50% of papillary thyroid cancers and 25% of anaplastic thyroid cancers, this mutation provides an opportunity for targeted drug therapy. Our laboratory evaluated cellular phenotypic effects in response to treatment with PLX4032, a BRAFV600E-specific inhibitor, in normal BRAF-wild-type thyroid cells and in BRAFV600E-positive papillary thyroid cancer cells. METHODS: Normal BRAF-wild-type thyroid cells and BRAFV600E-mutated papillary thyroid cancer cells were subjected to proliferation assays and analyzed for cell death by immunofluorescence. Cell cycle status was determined using an EdU uptake assay followed by laser scanning cytometry. In addition, expression of proteins within the MAPK signal transduction pathway was analyzed by Western blot. RESULTS: PLX4032 has potent anti-proliferative effects selectively in BRAF-mutated thyroid cancer cells. These effects appear to be mediated by the drug's activity of inhibiting phosphorylation of signaling molecules downstream of BRAF within the pro-survival MAPK pathway. Interestingly, PLX4032 promotes the phosphorylation of these signaling molecules in BRAF-wild-type thyroid cells. CONCLUSIONS: These findings support further evaluation of combinational therapy that includes BRAFV600E inhibitors in thyroid cancer patients harboring the BRAFV600E mutation.

Epithelial expression of the cytosolic retinoid chaperone cellular retinol binding protein II is essential for in vivo imprinting of local gut dendritic cells by lumenal retinoids.

Dendritic cells (DCs) use all-trans retinoic acid (ATRA) to promote characteristic intestinal responses, including Foxp3(+) Treg conversion, lymphocyte gut homing molecule expression, and IgA production. How this ability to generate ATRA is conferred to DCs in vivo remains largely unstudied. Here, we observed that among DCs, retinaldehyde dehydrogenase (ALDH1), which catalyzes the conversion of retinal to ATRA, was preferentially expressed by small intestine CD103(+) lamina propria (LP) DCs. Retinoids induced LP CD103(+) DCs to generate ATRA via ALDH1 activity. Either biliary or dietary retinoids were required to confer ALDH activity to LP DCs in vivo. Cellular retinol-binding protein II (CRBPII), a cytosolic retinoid chaperone that directs enterocyte retinol and retinal metabolism but is redundant to maintain serum retinol, was required to confer ALDH activity to CD103(+) LP DCs. CRBPII expression was restricted to small intestine epithelial cells, and ALDH activity in CRBPII(-/-) DCs was restored by transfer to a wild-type recipient. CD103(+) LP DCs from CRBPII(-/-) mice had a decreased capacity to promote IgA production. Moreover, CD103(+) DCs preferentially associated with the small intestine epithelium and LP CD103(+) DC ALDH activity, and the ability to promote IgA production was reduced in mice with impaired DC-epithelia associations. These findings demonstrate in vivo roles for the expression of epithelial CRBPII and lumenal retinoids to imprint local gut DCs with an intestinal phenotype.

Effect of α4ß7 blockade on intestinal lymphocyte subsets and lymphoid tissue development.

BACKGROUND: Blockade of the integrin α4ß7 has promise as a therapy for inflammatory bowel disease. α4ß7 plays diverse roles in the intestinal immune system, including lymphocyte homing and lymphoid tissue formation; however, the effects of α4ß7 blockade on these processes during inflammation and their relationship to the efficacy of α4ß7 blockade and its potential untoward effects are largely unknown. METHODS: α4ß7 function was inhibited by genetic manipulation or antibody blockade. The effects of these manipulations on lymphoid tissues and the presence of lymphocyte subpopulations in the murine small intestine and colon were evaluated in the unchallenged state, during the acute injury dextran sodium sulfate model, and during the splenocyte transfer chronic inflammation model. RESULTS: α4ß7 inhibition resulted in a decrease in the B-lymphocyte population in the diffuse lamina propria and a decrease in the number of lymphoid aggregates in the uninflamed intestine and in the acute injury model. α4ß7 blockade did not reduce the Foxp3- T-lymphocyte population but did decrease the Foxp3+ T-lymphocyte population located selectively within the lymphoid aggregates in the uninflamed intestine and in the acute injury model. In contrast, α4ß7 blockade reduced the intestinal T-lymphocyte population and decreased the production of inflammatory cytokines in the T-lymphocyte mediated chronic inflammation model. CONCLUSIONS: These findings demonstrate differential use of α4ß7 by B-lymphocytes, Foxp3- T-lymphocytes, and Foxp3+ T-lymphocytes to home to the gut, and suggest that α4ß7 blockade may serve as a targeted therapy that selectively inhibits the accumulation of pathogenic T-lymphocyte populations in the chronically inflamed intestine.