Primary Lymph Node Metastases Research Articles

Abstract B35: Characterization of exosomes extracted from primary tumor samples of HPV+/- oropharyngeal cancer

Abstract Objective: To determine if distinctions in exosomal cargo contribute to differences in EMT between HPV-related and HPV-unrelated oropharyngeal disease and can provide a signature for prediction of tumor aggressiveness and metastatic potential. Subjects and Methods: Specimen from patients with HPV+/- OPSCC were collected according to an IRB-approved protocol including primary tumor, lymph node metastasis, distant metastasis, and serum when available. Exosomes isolated from HPV+ and HPV- tumors were analyzed with miARma-seq and proteomics. These same exosomes were cocultured with well-characterized HPV+ and HPV- cell lines as well as fibroblasts and normal keratinocytes. Tumor aggressiveness, EMT, and potential for metastasis were measured utilizing wound healing scratch assays and transwell invasion assay techniques based on the Boyden Chamber migration assay. Confirmation of epithelial to mesenchymal transition was evaluated by Western blot and qPCR using a panel of well-established EMT markers (e-cadherin, n-cadherin, vimentin, snail, slug, twist, ZEB1/2). Results: Tumors from HPV+ patients demonstrate exosomal content enriched in EMT markers compared to HPV-negative patient cultures. Exosomes extracted from primary tumor and lymph node metastasis of the same patient demonstrate differential expression of markers of EMT. Exosomes extracted from lymph node and cocultured with fibroblasts demonstrate increased invasion and migration. Conclusions: Exosomes can be extracted from primary patient tumor cultures of both primary tumor and lymph node metastasis. These exosomes demonstrate differential expression of markers of EMT. Exosomal contents can be exploited for biomarker discovery to aide in prognostication. Citation Format: Vivian F. Wu, Xiang Guo Dai, Michael Chopp. Characterization of exosomes extracted from primary tumor samples of HPV+/- oropharyngeal cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B35.

Prognostic impact of Claudin 18.2 in gastric and esophageal adenocarcinomas

IntroductionThe tight junction molecule Claudin 18.2 is selectively expressed in healthy and malignant gastric epithelial tissue and is a promising therapy target for high Claudin 18.2 expressing adenocarcinomas of the esophagogastric junction and stomach (AEG/S).MethodsThis study analyzed the prevalence, characteristics and prognostic impact of Claudin 18.2 expression in primary tumor, lymph node and distant metastasis in a large Caucasian AGE/S cohort with 414 patients.ResultsClaudin 18.2 was highly expressed in 17.1% of primary tumors, 26.7% of lymph node metastasis and 16.7% of distant metastasis. High Claudin 18.2 expression in lymph node metastasis and primary tumors correlated significantly (p < 0.001). High expression of Claudin 18.2 was neither associated with histomorphogical subtype, or tumor state, nor with overall survival.ConclusionIn Caucasian AEG/S patients, 17.1% appeared to be eligible for an anti-Claudin 18.2 therapy. Claudin 18.2 expression itself has no impact on prognosis and is not related to any tumor subtype.

Predicting Therapeutic Antibody Delivery into Human Head and Neck Cancers.

The efficacy of antibody-based therapeutics depends on successful drug delivery into solid tumors; therefore, there is a clinical need to measure intratumoral antibody distribution. This study aims to develop and validate an imaging and computation platform to directly quantify and predict antibody delivery into human head and neck cancers in a clinical study. Twenty-four patients received systemic infusion of a near-infrared fluorescence-labeled therapeutic antibody followed by surgical tumor resection. A computational platform was developed to quantify the extent of heterogeneity of intratumoral antibody distribution. Both univariate and multivariate regression analyses were used to select the most predictive tumor biological factors for antibody delivery. Quantitative image features from the pretreatment MRI were extracted and correlated with fluorescence imaging of antibody delivery. This study not only confirmed heterogeneous intratumoral antibody distribution in-line with many preclinical reports, but also quantified the extent of interpatient, intertumor, and intratumor heterogeneity of antibody delivery. This study demonstrated the strong predictive value of tumor size for intratumoral antibody accumulation and its significant impact on antibody distribution in both primary tumor and lymph node metastasis. Furthermore, this study established the feasibility of using contrast-enhanced MRI to predict antibody delivery. This study provides a clinically translatable platform to measure antibody delivery into solid tumors and yields valuable insight into clinically relevant antibody tumor penetration, with implications in the selection of patients amenable to antibody therapy and the design of more effective dosing strategies.

68Ga-PSMA PET/CT for Primary Lymph Node and Distant Metastasis NM Staging of High-Risk Prostate Cancer.

With the largest high-risk prostate cancer (PCa) cohort to date undergoing 68Ga-prostate-specific membrane antigen (PSMA) PET/CT primary staging, we aimed to, first, characterize the metastatic spread of PCa in relation to tumor 68Ga-PSMA uptake and the D'Amico classification and, second, compare 68Ga-PSMA PET/CT findings with radical prostatectomy and pelvic lymph node dissection (PLND) histopathology findings. Methods: The study included 691 consecutive newly diagnosed, biopsy-proven, treatment-naïve, D'Amico high-risk PCa patients primary-staged by 68Ga-PSMA PET/CT. PSMA SUVmax and metastatic findings were compared with prostate-specific antigen level, International Society of Urological Pathology (ISUP) grade, and clinical stage as traditional risk stratification parameters. Moreover, 68Ga-PSMA PET/CT findings were compared with histology findings in radical prostatectomy patients undergoing PLND. Undetected lymph node metastases (LNMs) underwent immunohistochemical PSMA staining. Results: Advanced disease (N1/M1) was observed in 35.3% of patients (244/691) and was associated with increasing prostate-specific antigen level, ISUP grade, and clinical stage. LNMs (N1/M1a) were detected in 31.4% (217/691) and bone metastases (M1b) in 16.8% (116/691). Advanced disease frequencies in patients with ISUP grades 2 and 3 were 10.8% (11/102) and 37.1% (33/89), respectively. Risk of advanced disease for cT2a, cT2b, and cT2c tumors was almost equal (24.2%, 27.9%, and 22.4%, respectively). We observed a weak correlation between SUVmax and biopsy ISUP grade (ρ = 0.21; P < 0.001) and a modest correlation between SUVmax and postprostatectomy ISUP grade (ρ = 0.38; P < 0.001). Sensitivity, specificity, positive and negative predictive value, and accuracy for LNM detection on 68Ga-PSMA PET/CT in the PLND cohort were 30.6%, 96.5%, 68.8%, 84.5%, and 83.1%, respectively. Undetected LNMs either were micrometastases located in the lymph node border or were without PSMA expression. Conclusion: In this high-risk PCa cohort, we identified advanced disease in about one third at diagnosis. ISUP grade was the superior predictor for advanced disease at diagnosis. We found a significant difference in frequency of advanced disease between ISUP grades 2 and 3, as supports the Gleason score 7 subdivision. Interestingly, we observed no significant differences in risk of advanced disease when comparing the different cT2 stages. The undetected LNMs were either PSMA-negative or micrometastases.

Total tumour volume as a prognostic factor in patients with resectable colorectal cancer liver metastases.

BackgroundAlthough total tumour volume (TTV) may have prognostic value for hepatic resection in certain solid cancers, its importance in colorectal liver metastases (CRLM) remains unexplored. This study investigated its prognostic value in patients with resectable CRLM.MethodThis was a retrospective review of patients who underwent hepatic resection for CRLM between 2008 and 2017 in a single institution. TTV was measured from CT images using three‐dimensional construction software; cut‐off values were determined using receiver operating characteristic (ROC) curve analyses. Potential prognostic factors, overall survival (OS) and recurrence‐free survival (RFS) were determined using multivariable and Kaplan–Meier analyses.ResultsSome 94 patients were included. TTV cut‐off values for OS and RFS were 100 and 10 ml respectively. Right colonic primary tumours, primary lymph node metastasis and bilobar liver metastasis were included in the multivariable analysis of OS; a TTV of 100 ml or above was independently associated with poorer OS (hazard ratio (HR) 6·34, 95 per cent c.i. 2·08 to 17·90; P = 0·002). Right colonic primary tumours and primary lymph node metastasis were included in the RFS analysis; a TTV of 10 ml or more independently predicted poorer RFS (HR 1·90, 1·12 to 3·57; P = 0·017). The 5‐year OS rate for a TTV of 100 ml or more was 41 per cent, compared with 67 per cent for a TTV below 100 ml (P = 0·006). Corresponding RFS rates with TTV of 10 ml or more, or less than 10 ml, were 14 and 58 per cent respectively (P = 0·009). A TTV of at least 100 ml conferred a higher rate of unresectable initial recurrences (12 of 15, 80 per cent) after initial hepatic resection.ConclusionTTV was associated with RFS and OS after initial hepatic resection for CRLM; TTV of 100 ml or above was associated with a higher rate of unresectable recurrence.

The daily practice reality of PD-L1 (CD274) evaluation in non-small cell lung cancer: A retrospective study.

Treatment with pembrolizumab, an anti-programmed cell death-1 (PDCD-1) monoclonal antibody for the treatment of non-small cell lung cancers (NSCLCs) requires prior immunohistochemical (IHC) analysis of the expression of the programmed death-ligand 1 (PD-L1) (also known as CD274 molecule) which is a heterogeneous and complex marker. The present study aimed to investigate how pathological and technical factors (such as tumor location and sampling type, respectively) may affect the PD-L1 evaluation in patients with NSCLC in the daily practice of pathology laboratories. The current study was retrospective, and included 454 patients with NSCLC, for whom PD-L1 expression analysis by IHC was prospectively performed between November 2016 and January 2018. The association between PD-L1 expression and the clinicopathological characteristics of patients was statistically investigated using either the χ2 and Fisher exact tests or the Mann-Whitney and Kruskal-Wallis tests, depending on whether PD-L1 expression was assessed in three large categories (<1, 1–49, ≥50%) or in more precise percentages. Furthermore, the same statistical methodology was used to analyze the heterogeneity of PD-L1 expression according to its sampling type (cytology, biopsy or surgical specimen) and its location (primary tumor, lymph node or distant metastasis). Intra- and inter-observer discrepancies were also studied using double-blind evaluation and concordance analyses based on the weighted κ coefficient. The results demonstrated a significant association between PD-L1 expression and sample location (P=0.005), histological type (P=0.026), total number of mutations (P=0.004) and KRAS proto-oncogene, GTPase mutations (P=0.024). In addition, sampling type did not influence PD-L1 expression. The inter- and intra-observer discrepancies were 15% and between 16 and 17.5%, respectively. The present study confirmed that evaluation of PD-L1 expression by IHC can be performed on all types of samples. In addition, the results from the current study highlighted the heterogeneity of PD-L1 expression among the different types of sample location. In complex cases, a second evaluation of PD-L1 expression by IHC would be performed due to intra- and inter-observer discrepancies.

Over expression of CDK4 and MDM2 in a patient with recurrent ALK-negative mediastinal inflammatory myofibroblastic tumor

Rationale:The diagnosis of anaplastic lymphoma kinase (ALK)-negative inflammatory myofibroblastic tumors (IMT) remains challenging because of their morphological resemblance with spindle cell sarcoma with myofibroblastic characteristics.Patient concerns:A 69-year-old female patient presented with loco-regional recurrent IMT several times within 8 years after primary treatment and neck lymph node metastasis 3.5 years after last recurrence.Diagnosis:The primary, recurrence, and lymph node metastasis lesions were diagnosed as ALK-negative IMTs based on the histopathological features.Interventions:Biopsy samples were obtained during repeated surgeries and evaluated for genomic alterations during first and recurrent presentations. The evaluation was done using pathway-driven massive parallel sequencing, and genomic alterations between primary and recurrent tumors were compared.Outcomes:Copy number gains and overexpression of mouse double minute 2 homolog (MDM2) and cyclin dependent kinase 4 (CDK4) were observed in the primary lesion, and additional gene amplification of Discoidin Domain Receptor Tyrosine Kinase 2 (DDR2), Succinate Dehydrogenase Complex II subunit C (SDHC), and thyroid stimulating hormone receptor (TSHR) Q720H were found in the recurrent tumors. Metastases to the neck lymph node were observed 3.5 years after recurrence.Lessons:Our results indicated genetic evolution in a microscopically benign condition and highlighted the importance of molecular characterization of fibro-inflammatory lesions of uncertain malignant potential.

A panel of protein kinase high expression is associated with postoperative recurrence in cholangiocarcinoma

BackgroundCancer recurrence is one of the most concerning clinical problems of cholangiocarcinoma (CCA) patients after treatment. However, an identification of predictive factor on Opisthorchis viverrini (OV)-associated CCA recurrence is not well elucidated. In the present study, we aimed to investigate the correlation of twelve targeted protein kinases with CCA recurrence.MethodsTwelve protein kinases, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2, 3, 4 (HER2, HER3, HER4), vascular endothelial growth factor receptor 3 (VEGFR3), vascular endothelial growth factor-C (VEGF-C), erythropoietin-producing hepatocellular carcinoma receptor type-A3 (EphA3), EphrinA1, phosphor-serine/threonine kinase 1 (p-Akt1), serine/threonine kinase 1 (Akt1), beta-catenin and protein Wnt5a (Wnt5a) were examined using immunohistochemistry. Pre-operative serum tumor markers, CA19–9 and CEA were also investigated.ResultsAmong twelve protein kinases, EGFR, HER4, and EphA3 were associated with tumor recurrence status, recurrence-free survival (RFS) and overall survival (OS). Multivariate cox regression demonstrated that EGFR, HER4, EphA3 or the panel of high expression of these proteins was an independent prognostic factor for tumor recurrence. The combination of high expression of these proteins with a high level of CA19–9 could improve the predictive ability on tumor recurrence. Moreover, the patients were stratified more accurately when analyzed using the combination of high expression of these proteins with primary tumor (T) or lymph node metastasis (N) status.ConclusionEGFR, HER4, EphA3 or the panel of high expression of these proteins is an independent prognostic factor for post-operative CCA recurrence.

Results of concurrent HER2 (CERB-B2) staining in the primary tumor and lymph node metastasis in advanced stage gastric carcinoma

Aim: We hypothesized that tumor cells with metastatic capacity and nodal metastasis would exhibit prominent HER2 overexpression in gastric cancer for which intratumoral heterogeneity is highly variable. This study purposed to investigate the mismatch for HER2 immunohistochemical staining between the primary tumor cells and the metastatic cells in patients with advanced-stage gastric cancer.Material and Methods: A hundred and forty-four patients with advanced staged gastric cancer, and lymph node metastasis who underwent surgical resection were enrolled in this retrospective study. Primary tumor and lymph node metastasis underwent concurrent immunohistochemical staining for addressing the HER2 positivity. The concordance of HER2 positivity between the primary tumor samples and the lymph node metastases was investigated.Results: There was a significant difference in HER2 positivity rate among the well, moderate, and poorly differentiated carcinomas, which was primarily driven by the high HER2 overexpression in well-differentiated subgroup. HER2 positivity was highly frequent in stage 3 tumors, whereas HER2 was negative in the majority of the stage 4 tumors. Tumor size was also significantly larger in subjects without HER2 overexpression compared to those with HER overexpression [6 (IQR=4.88) cm vs. 5.25 (IQR=3.5) cm, p = 0.037]. Concordance of primary tumors and the metastatic lymph nodes regarding HER2 positive IHC staining were 93.7%. Conclusion: HER2 might be positive in lymph node metastasis samples even if the primary tumor is negative for HER2. We suggest that HER2 IHC staining of the lymph node metastasis should be considered if the primary tumor is signet ring cell carcinoma, moderately or poorly differentiated, and negative for HER2 in subjects with gastric carcinoma and lymph node metastasis.

Ubiquitin specific peptidase 5 enhances STAT3 signaling and promotes migration and invasion in Pancreatic Cancer.

Purpose: Ubiquitin specific peptidase 5 (USP5) has been reported to promote the progression of several malignant tumors. It may affect cancer development via modulating cell cycle and colony formation. In pancreatic cancer, the biological function of USP5, especially in migration and invasion remains unclear.Methods: USP5 protein expression levels in primary pancreatic cancer and lymph node metastasis tissues were detected using immunohistochemistry (IHC). χ2 test, Kaplan-Meier analysis, univariate and multivariate analyses were used to evaluate the relationship between USP5 expression and clinicopathological feature. RT-qPCR were carried out to quantitate the mRNA expression levels of USP5 in pancreatic cancer cell lines. CCK8 and Colony formation assay were performed to prove how USP5 works in proliferation. Evaluation of tumor metastasis was made by Transwell and wound healing assay. EMT and STAT3 signaling related markers were detected by western blot.Results: (1) USP5 protein expression levels were related to tumor differentiation, CEA and CA19-9 level. (2) Univariate and multivariate analyses showed that high USP5 expression is an unfavorable prognostic factor for pancreatic cancer. Kaplan-Meier analysis directly indicated that patients with high USP5 expression had shorter overall survival. (3) Increased USP5 expression is related to pancreatic cancer in both proliferation and metastasis. (4) USP5 was proved to mediate STAT3 signaling in pancreatic cancer cells.Conclusions: The results suggest that USP5 is highly expressed and might have clinical significance for pancreatic cancer patients. High USP5 expression promotes both progression and metastasis by activating STAT3 signaling. Thus, USP5 might be a potential target in pancreatic cancer treatment.

CD274 (PD-L1), CDKN2A (p16), TP53, and EGFR immunohistochemical profile in primary, recurrent and metastatic vulvar cancer.

Vulvar squamous cell carcinoma can be divided by human papillomaviruses (HPV) status into two distinct clinicopathological and molecular entities. New agents targeting the tumor surface expression of programmed cell death-1/programmed cell death-ligand-1 are becoming a therapeutic option in an increasing number of carcinomas. We evaluate CD274 (PD-L1), CDKN2A (p16), tumor protein p53 (TP53), and epidermal growth factor receptor (EGFR) immunoexpression in primary tumors, recurrences and lymph node metastases and its correlations with prognosis and HPV status. We report 93 cases of vulvar squamous cell carcinoma diagnosed between 2002 and 2016 with the description of their clinicopathological features and prognosis data. Immunohistochemistry for CD274, CDKN2A, TP53, and EGFR was performed on tissue microarrays collecting from primary tumor, recurrences and lymph node metastasis. Kaplan-Meier estimator and multivariable Cox regression analysis controlling for FIGO stage and age were used. Patients who underwent surgery had a superior overall survival (HR = 0.51, 95% CI = 0.26-0.99 p = 0.04). Lymph node metastasis size ≥5 mm was associated with an inferior overall survival (HR = 1.88, 95% CI = 1.22-2.92 p = 0.004). CDKN2A expression was correlated with an inferior rate of recurrent disease (p = 0.02). In high-risk HPV DNA+ vulvar squamous cell carcinomas patients with CDKN2A- carcinomas showed a significantly worse overall survival than women with CDKN2A+ tumors (56% vs.100%, p = 0.003). TP53 expression was associated with an increased rate of recurrent disease (p = 0.0005). CD274 expression was associated with lymph node metastasis (p = 0.04). In 16 patients the CD274, CDKN2A, TP53, and EGFR expression changed between primary tumors, recurrences and lymph node metastases during tumor progression. In conclusion, a significant percentage of vulvar squamous cell carcinoma has a heterogeneous biomarker expression during tumor progression. We highlight the importance of some of these markers to be used as prognostic biomarkers. This data brings new light to future treatment using targeted therapy to EGFR or CD274 to include retesting such biomarkers in recurrence and lymph nodes metastases.

Role of Inflammatory Markers in Prognosis of Oral Squamous Cell Carcinoma.

Background:This estudie evaluated the immunostaining of cytokines in oral carcinoma, in tissue of margin of surgical resecate (MSR) and metastatic lymph nodes, as well as their role in patient prognosis. Methods:A retrospective study was carried out in patients with oral squamous cell carcinomas, and sociodemographic and clinical-pathological data were evaluated. In addition, surgical site analysis of the patients was conducted by immunohistochemistry, using a tissue microarray for inflammatory (Tumor Necrosis Factor-alpha, Interleukin-1beta, Interleukin-6, interleukin-10), transcription NF-kappa B and CD68 markers. Immunoexpression was assessed qualitatively and quantitatively using ImageJ software, and data were correlated with the prognostic factors and patient survival rates. Results:There was a greater immunoexpression of inflammatory and CD68 cytokines in primary tumour and lymph node metastasis than in MSR. In a multinomial logistic regression model, patients with low education (p = 0.041) and a high histoscore for TNF-α (p = 0.021) showed a survival rate of 15.64 (95% CI = 1.13-217.24) and 6.81 (95% CI = 1.02-105.96). Conclusion:Therefore, despite there is an increased immunoexpression of cytokines in the primary tumour, only TNF-α was the inflammatory cytokine that influenced the survival of patients with oral cancer.

Electrochemotherapy Associated with Calcium Electroporation in Metastatic Feline Cutaneous Malignant Melanoma

Background: Calcium electroporation (CaEP) is a novel therapeutic treatment that has been studied for cancer due to its selective killing cancer cells by necrosis and danger signals. Besides that, electrochemotherapy (ECT) is an effective local treatment that involves the administration of chemotherapeutic drugs followed by delivery of electrical pulses to the tumor. The combination with ECT and CaEP has been reported in literature suggesting that additional response of immune system could have been enhanced by electroporation with calcium. This case, report on the successful treatment with CaEP combined with ECT for treatment of a regional metastasis in a feline model of malignant melanoma.Case: A 9-year-old, mixed breed cat was referred to the veterinary clinic with a 2-month history of cutaneous peripalpebral plaque lesion (0.19 cm³) and a submandibular lymph node enlargement (0.5 cm³). Incisional biopsy of the cutaneous lesion and fine-needle aspiration of submandibular lymph node confirmed a cutaneous melanoma with submandibular lymph node metastasis. Tumor staging was set in T1N1M0 according to WHO staging criteria. ECT for the primary lesion and lymph node metastasis was proposed. For the ECT, bleomycin (15,000 UI/m²) application was performed intravenous followed by electroporation (8 pulses of 100 μs at 1000 V/cm, and 1 Hz) using a needle array electrode consisted of two parallel rows with six needles in each row. At 28-day post-ECT complete remission of the primary tumor and metastatic foci was achieved. However, 120 days after ECT, recurrence was observed in submandibular and retropharyngeal lymph nodes. A second ECT approach was performed adding to bleomycin the intra lymph nodal application of calcium gluconate. The dose of calcium gluconate was diluted in an isotonic 0.9% NaCl solution resulting in a low concentration at 9 mM, injected in both metastatic lymph nodes (submandibular total volume: 1.4 ml; retropharyngeal total volume: 0.5 mL) and pulses were delivered immediately after drug administration. No systemic adverse effects were observed. Local adverse effects were considered mild as transitory edema and ulceration post procedure. One-week post-ECT+CaEP, complete remission of local metastasis occurred. However, the patient achieved five months disease free interval, and died during a surgical approach, achieving nine months of survival time.Discussion: Feline cutaneous malignant melanoma is an aggressive disease with a short survival time for the patients with mean of 4.5 months. CaEP is a novel anticancer treatment that has been study in the past years due to its selective killing cancer cells by necrosis and danger signals. The CaEP induces supraphysiological calcium influx into neoplastic cells leading to acute ATP depletion and necrosis of tumor cells. This use could be an interesting therapeutic choice for both human and veterinary medicine. In this patient, it was demonstrated a good clinical response with its use, showing temporarily tumor remission from the case presented with disease free interval of five months when compared to other report of two months. This description showed that ECT associated with CaEP improved outcome of regional melanoma lymph node metastasis in a cat. However, further investigations are needed to understand the use of CaEP in patients for local control metastasis as well as evaluate the use of both modalities to determine its synergistic effect.

Minimally invasive surgery for laryngopharyngeal cancer: Multicenter feasibility study of a combination strategy involving transoral surgery and real-time indocyanine green fluorescence-navigated sentinel node navigation surgery.

Sentinel node navigation surgery using indocyanine green (ICG-SNNS) can be performed in the operation room. The combination of minimally invasive transoral surgery (TOS) with ICG-SNNS can provide functional preservation options for both primary lesions and lymph node (LN) metastasis. This multicenter feasibility study of this strategy was conducted in Japan. Patients with clinical T1 or T2, N0 oropharyngeal, hypopharyngeal, or supraglottic cancer were enrolled. The identification rate of sentinel nodes, delayed cervical LN metastasis in 2 years, and survival rate were assessed. Twenty-two patients (10 oropharynx, 8 hypopharynx, 4 supraglottic cancer) were enrolled. The identification rate was 100%. One case had delayed nodal metastasis. The accuracy was 95.5%, sensitivity was 75%, and specificity was 100%. The 5-year disease-specific survival was 100%, overall survival was 72.3%, and disease-free survival was 60.5%. The combination of TOS with ICG-SNNS is feasible as a minimally invasive strategy and has favorable oncological outcomes.

The influence factors of pathologic complete response of the ipsilateral supraclavicular lymph node of breast cancer after neoadjuvant chemotherapy

Objective: To investigate the factors affecting the pathologic complete response (PCR) of the ipsilateral supraclavicular lymph node (ISLN) of breast cancer after neoadjuvant chemotherapy (NAC). Methods: A total of 178 patients with breast cancer who had primary ipsilateral supraclavicular lymph node metastasis (ISLNM), receiving NAC and subsequent ISLN dissection, were retrospectively reviewed. The single factor and multi factor analysis were carried out by the chi square test and the Logistic regression model. Results: The enrolled patients were all female, 28 to 74 years old. The rate of PCR on the ISLN was 52.2%. Single factor analysis showed that KI67 expression level (χ(2)=7.717,P=0.005), breast PCR (bPCR) (χ(2)=33.564,P<0.001), and axillary PCR (aPCR) (χ(2)=31.750, P<0.001) were associated with the ISLN PCR. Multifactor analysis showed that KI67 expression level (OR=4.096, 95%CI: 1.176-14.263, P=0.027), bPCR (OR=4.452, 95%CI: 1.894-10.461, P<0.001) and aPCR (OR=5.183, 95%CI: 1.974-13.605, P<0.001) were independent predictors of ISLN PCR. The rate of PCR on the ISLN was 90.9% in the patients with KI67>30% and simultaneous breast and axilla PCR. Conclusions: The PCR rate of the ISLN after neoadjuvant chemotherapy is higher than that of the breast and axillary PCR. The expression level of KI67, the bPCR and the aPCR are independent predictors of the PCR on the ISLN.

Identification of differentially expressed genes between primary lung cancer and lymph node metastasis via bioinformatic analysis

Lung cancer (LC), with its high morbidity and mortality rates, is one of the most widespread and malignant neoplasms. Mediastinal lymph node metastasis (MLNM) severely affects postoperative survival of patients with LC. Additionally, the molecular mechanisms of LC with MLNM (MM LC) remain not well understood. To identify the key biomarkers in its carcinogenesis and development, the datasets GSE23822 and GSE13213 were obtained from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified, and the Database for Annotation, Visualization and Integrated Discovery was used to perform functional annotations of DEGs. Search Tool for the Retrieval of Interacting Genes and Cytoscape were utilized to obtain the protein-protein interaction (PPI) network, and to analyze the most significant module. Subsequently, a Kaplan-Meier plotter was used to analyze overall survival (OS). Additionally, one co-expression network of the hub genes was obtained from cBioPortal. A total of 308 DEGs were identified in the two microarray datasets, which were mainly enriched during cellular processes, including the Gene Ontology terms ‘cell’, ‘catalytic activity’, ‘molecular function regulator’, ‘signal transducer activity’ and ‘binding’. The PPI network was composed of 315 edges and 167 nodes. Its significant module had 11 hub genes, and high expression of actin β, MYC, arginine vasopressin, vesicle associated membrane protein 2 and integrin subunit β1, and low expression of NOTCH1, synaptojanin 2 and intersectin 2 were significantly associated with poor OS. In summary, hub genes and DEGs presented in the present study may help identify underlying targets for diagnostic and therapeutic methods for MM LC.

Abstract 4541: Malignant and non-malignant cells from primary tumor and lymph node metastasis ecosystems show different behavior for patient-associated protein signatures in head and neck cancer

Abstract Although lymph node metastasis is the main prognostic factor in patients with head and neck squamous cell carcinoma (HNSCC), the molecular signals involved in this process are poorly characterized and no molecular markers are currently used in clinical practice. Recently, cancer is being viewed as an ecosystem, in which tumor cells cooperate with each other and host cells in their microenvironment. Herein, we mapped the proteome of primary tumor and matched lymph node ecosystems, isolating malignant and non-malignant cells, to better understand the mechanisms underlying lymph node metastasis. For that, we used formalin-fixed paraffin-embedded (FFPE) primary tumors and paired lymph nodes from 27 HNSCC patients positive (N+, n=13) and negative (N0, n=14) for lymph node metastasis using laser microdissection combined with mass spectrometry-based proteomics. An average of 2,266 ± 216 proteins was identified in primary tumor and lymph node ecosystems. A hundred and five differentially abundant proteins from malignant cells in the primary site (N+ vs N0) were mainly involved in RNA stability and metabolism, while inflammation and splicing processes, respectively, were overrepresented in N+ vs N0 non-malignant cells signatures from primary tumor (N=33) and lymph node (N=13) sites (Student's t-test; P-value≤0.05), indicating particular lymph node metastasis signatures for each cell population. Interestingly, the protein pattern of malignant cells from the primary site and matched lymph nodes showed an individual patient-specific metastasis profile through hierarchical clustering analysis; however, the profile of non-malignant cells does not represent patient-specific subpopulations and grouped according to the site of isolation (primary tumor and lymph node microenvironment). Our data reveal potential protein signatures associated with prognosis in HNSCC, as well as a non-specificity of protein patterns from host non-malignant cells that can be related with the immunosurveillance failure. Financial support: FAPESP (Process number 2015/19191-6). Note: This abstract was not presented at the meeting. Citation Format: Ariane F. Busso-Lopes, César Rivera, Barbara P. Mello, Luisa L. Villa, Wilfredo González-Arriagada, Adriana F. Paes Leme. Malignant and non-malignant cells from primary tumor and lymph node metastasis ecosystems show different behavior for patient-associated protein signatures in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4541.

Abstract 2132: Next generation sequencing for screening of clinically actionable mutations in intestinal-type sinonasal adenocarcinoma

Abstract Introduction: Sinonasal ITAC is a rare tumor commonly related to occupational wood or leather dust exposure. Clinical management has improved,however, recurrences are frequent, and the overall 5-year survival rateremains poor (60%). Therefore, there is a clear need for new therapeutic options. The aim of this study was to identify genetic alterations as specific targets for personalized treatment. Experimental procedures: Next Generation Sequencing using SureSelect QXT (Ilumina) was performed on a panel of 120 genes against which drugs have been approved by the FDA. Tumor and germline DNA of 25 patients was sequenced, for one case also the recurrence and metastasis. Pools were sequenced in a MiSeq system with a minumum coverage of 500X. To select somatic and pathogenic variants, the raw sequencing data were manually curated as follows: all silent mutations and variants with allele frequency &amp;gt;5% were discarded, only variants with tumor frequency &amp;gt;10 % of readings were considered; by comparing with the germline sample, only variants that appeared exclusively in the tumor sample were selected, as well as homozygous changes in the tumor that were heterozygous in the germline. Results: Five tumors (20%) did not show any somatic alteration in the 120 genes tested.In the remaining 20 tumors, mutations were found in 33 genes. The most relevant were involved in the EGFR-PI3K-AKT-MTOR, the MAPK-ERK and the Wnt signaling pathways, indicating an important role in ITAC tumorigenesis. The most frequently affected genes were tyrosin-kinase receptors (44%), especially the ERBB family (20% of cases with mutations) and FGFR1 (20% of tumors with gene copy number amplifications). Thirty-six percent and 28% of tumors showed alterations in the MAPK-ERK and PI3K-AKT-MTOR cascades respectively. PI3K mutations were particulary frequent (7 different variants in 5 tumors.) With regard to the Wnt pathway, both APC and CTNBB1 genes appeared mutated in 3 cases. In one patient, the primary tumor, recurrence and lymph node metastasis were investigated. All somatic alterations observed in the primary tumor (mutations in ATM, BRC2, KMT2B and amplifications of MET, ATM and FGF1R) remained present in both recurrence and metastasis, excluding the possibility of a second primary tumor. Additional mutations in the recurrence (NTRK1, TSC2 and PIK3R2) were also seen in the metastasis, while no alterations occurred in the metastasis exclusively. Confirmatory studies are underway correlating the most relevant sequencing results with gene and protein expression data of the affected pathways. Conclusions: By sequencing 120 clinically actionable genes in ITAC, several alterations were found that could be used as targets for new, personalized therapies. Examples include Dovitinib against FGFR1 amplification, antibody and small molecule inhibitors of ERBB, BRAF and MTOR, and tankyrase inhibitors targeting the Wnt pathway. Citation Format: Cristina Riobello, Virginia N. Cabal, Paula Sánchez Fernández, Rocío García-Marín, Blanca Vivanco, Fernando López, José L Llorente, Mario Hermsen. Next generation sequencing for screening of clinically actionable mutations in intestinal-type sinonasal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2132.

Nomogram predicted disease free survival for colorectal liver metastasis patients with preoperative chemotherapy followed by hepatic resection

BackgroundHepatic resection is regarded to as a potentially curative option for colorectal cancer liver metastases (CRLM), but it is associated with a high rate of recurrence. The present study intended to establish an effective nomogram to predict disease free survival (DFS) and select candidates of hepatic resection. MethodsThe nomogram was based on a retrospective study on 447 CRLM patients treated with preoperative chemotherapy followed by hepatic resection using a multicentric database between January 1st, 2010 and December 31st, 2017. Results were validated using bootstrap resampling on 117 patients. The predictive accuracy and discriminative ability of the nomogram were determined by concordance index (C-index) and calibration curve. Overall survival, disease free survival, and local recurrence rate for patients with colorectal cancer were measured. ResultsBased on multivariate analysis of the primary cohort, independent factors for DFS included tumor size larger than 5 cm, multiple liver metastases(>1), RAS mutation, primary lymph node metastasis and tumor size increase after preoperative chemotherapy. These five factors were all considered in the nomogram. The C-index of the nomogram for predicting survival was 0.675. With external validation, the C-index of the nomogram for the prediction of the DFS was 0.77, which demonstrated that this model has a good level of discriminative ability. For the 382 patients (66.7%) who developed recurrence, the optimal cutoff point for early recurrence was determined to be 12 months after hepatic resection. ConclusionsThe proposed nomogram demonstrated accurate prognostic prediction of DFS for CRLM patients with preoperative chemotherapy followed by hepatic resection.

Analysis of the relationship between ultrasonographic features and cervical lymph node skip metastasis of papillary thyroid micro-carcinoma

Objective: To investigate the correlation between cervical lymph node skip metastasis with ultrasonographic characteristics of papillary thyroid micro- carcinoma (PTMC). Methods: We reviewed ultrasonographic features of 385 primary PTMC and cervical lymph node metastasis, confirmed by pathology in Tianjin Medical University Cancer Institute and Hospital, to evaluate the efficacy of ultrasonography in the diagnosis of cervical lymph node metastasis of PTMC patients. The relationship between ultrasonographic features of primary lesions and skip metastasis of cervical lymph nodes was analyzed by χ(2) test and multiple factor Cox regression. Results: Among the 385 cases of PTMC patients with cervical lymph node metastasis, 231 cases were central lymph node metastasis alone, 31 cases were lateral cervical lymph node metastasis alone, 123 cases were both central and lateral cervical lymph node metastasis. Among the 354 cases without skip metastasis of cervical lymph nodes, 48 cases were level Ⅱ, 92 cases were level Ⅲ, 83 cases were level Ⅳ, 9 cases were level Ⅴ, 354 cases were level Ⅵ. Among the 31 cases with skipping metastasis of cervical lymph nodes, 12 cases were level Ⅱ, 14 cases were level Ⅲ, 14 cases were level Ⅳ, 1 case was level Ⅴ. The sensitivity and specificity of preoperative ultrasonography in the diagnosis of central cervical lymph node metastasis were 46.3% and 66.7%, respectively, and those of lateral cervical lymph node were 91.0% and 87.8%, respectively. Univariate analysis showed that the abutment/perimeter, diameter and location of PTMC were significantly associated with skip metastasis (P<0.05), multivariate analysis showed that abutment/perimeter and location of PTMC were significantly associated with skip metastasis (P<0.05). Conclusions: The sensitivity and specificity of preoperative ultrasound diagnosis for lateral cervical lymph node metastasis of PTMC is higher than that of central metastasis. PTMC with abutment/perimeter ≥1/4 and upper portion location are prone to skip metastasis.