Biomarkers for Prognosis in Osteosarcoma: From Molecular Signatures to Clinical Applications

Increased expression of microRNA-191 as a potential serum biomarker for diagnosis and prognosis in human osteosarcoma.

Background: Loss-of-function mutations in genes encoding components of the homologous recombination DNA damage response (DDR) machinery, notably BRCA1/2, are associated with tumor sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi). Little is known about the potential contribution of tumor alterations in non-DDR genes to modulating sensitivity to PARP inhibitors in the clinic. In EMBRACA, the PARPi TALA improved progression-free survival (PFS) (HR [95% CI] 0.54 [0.41-0.71], P<0.001) vs chemotherapy (CT) in germline BRCA1/2-mutated HER2-negative locally advanced/metastatic breast cancer. Methods: Baseline tumor tissue from 308 pts (71%; intent-to-treat) was tested by FoundationOne®. To support exploratory identification of tumor gene alterations associated with best vs worst outcomes, pts were mapped into 2 groups: [1] BEST: Pts in TALA arm with OS ≥ 30 mo and duration of treatment ≥ 24 mo, Pts in CT arm with OS ≥ 30 mo; [2] WORST: Pts in TALA or CT arm with a PFS event (PD by Independent Radiological Facility or death) ≤ 12 wks. 2 pts had short PFS but long OS and were initially mapped to both groups but then assigned to BEST and excluded from WORST. Results: Exploratory heat map visualizations of known/likely pathogenic genetic alterations defined by the FoundationOne® gene panel were used to assess differences in genetic alterations between BEST and WORST in TNBC and non-TNBC subpopulations, leading to the identification of MYC and RAD21 as commonly altered genes of high interest. Based on these results showing imbalances in tumor amplification events between BEST and WORST outcome pts, Cox regression analysis was used to explore potential associations of MYC or RAD21 amplification with OS across the evaluable ITT population. In TNBC pts receiving TALA, OS was shorter with MYC amplification than without [HR (95% CI) 1.877 (1.102, 3.196)]. No such association was evident in TNBC pts receiving CT [HR (95% CI) 0.706 (0.303, 1.643)]. In contrast, for non-TNBC pts receiving TALA, no association with OS was evident for MYC amplification versus without [HR (95% CI) 0.603 (0.310, 1.173)], while for pts receiving CT OS was shorter with MYC amplification than without [HR (95% CI) 1.917 (1.037, 3.544)]. RAD21 amplification status was not associated with OS in either TNBC or non-TNBC patients for either treatment arm, although it should be noted that two RAD21 subgroups were very small (n=7 and 11; others had n ≥ 21). Conclusions: Based on these exploratory, retrospective analyses MYC amplification was associated with shorter OS in TNBC pts receiving TALA. This may reflect the role of MYC in positive regulation of genes involved in homologous recombination such as RAD51 (Carey et al, Cancer Res 2018;78(3):742-757). MYC and RAD21 are both located at 8q24 and frequently coamplified in breast cancer (eg, Annunziato et al, Nat Commun. 2019;10(1):397), hence the lack of association of RAD21 amplification with outcome in TNBC suggests that the association of MYC amplification with shorter OS seen in pts receiving TALA may be gene-specific. Further investigation is warranted. Funding: Pfizer TNBCNon-TNBCTALACTTALACTGene AlterationBest n=10Worst n=15Best n=7Worst n=16Best n=17Worst n=11Best n=20Worst n=11MYC amplification0 (0%)7 (47%)2 (29%)6 (38%)5 (29%)1 (9%)3 (15%)5 (46%)RAD21 amplification1 (10%)2 (13%)3 (43%)5 (31%)5 (29%)1 (9%)7 (35%)4 (36%) Citation Format: Johannes Ettl, A. Douglas Laird, Joanne L. Blum, Hope S. Rugo, Sara A. Hurvitz, Miguel Martín, Henri Roché, Young-Hyuck Im, Annabel Goodwin, Rinat Yerushalmi, Tiziana Usari, Silvana Lanzalone, Akos Czibere, Julia Hopkins, Lee A. Albacker, Lida A. Mina, Jennifer K. Litton. A retrospective analysis of association of MYC and RAD21 amplification with final overall survival (OS) data in the phase 3 EMBRACA study with talazoparib [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-07.

Background: The p53 tumor suppressor gene is important for cell cycle regulation and DNA repair. Somatic p53 gene mutations are associated with poor prognosis. Mutations in the p53 gene result in prolonged stability of the protein that can be detected by immunohistochemical (IHC) techniques. The impact of p53 protein status was investigated using the disease-free survival (DFS) rates and the efficacy of adjuvant chemotherapy (anthracycline+/-taxane) in relation to breast cancer subtypes. Methods: Primary invasive breast cancer patients who underwent treatment from January 2002 to December 2018 were enrolled in this retrospective study. A total of 4463 primary breast cancer cases were analyzed. The factors investigated included nodal status, tumor size, nuclear grade, ER/PgR and HER2 status, p53 overexpression (cutoff point: 50%), and the Ki-67 index value (cutoff point: 20%). Breast cancer subtypes were categorized based on the IHC data derived from ER/PgR, HER2 and Ki-67 values in invasive tumors. DFS was calculated using the Kaplan-Meier method and evaluated using the log-rank test. Cox's proportional hazard model was used to perform univariate and multivariate analyses of the factors related to DFS. Median follow-up period was 85 months. Results: 1. p53 overexpression rate was 18.1% (n=808) in all of the cases. The p53 overexpression significantly correlated with ER/PgR negativity, HER2 positivity, higher Ki-67 index values and nuclear grade, positive nodes and larger tumors. Patients with p53 overexpression received chemotherapy more often than those without overexpression in adjuvant settings. HER2 type and triple negative (TN) type had significantly higher rates of p53 overexpression, but luminal A and B types had lower p53 overexpression rates. 2. Patients with p53 overexpression had significantly worse DFS in luminal A and B types. However, p53 overexpression was not predictive for DFS in HER2 enriched, luminal HER2 and TN types. Multivariate analysis revealed that p53 was a significant factor for DFS in luminal A/B types. 3. The efficacy of chemotherapy for DFS was investigated according to the subtypes and p53 status. Chemotherapy did not affect the DFS in relation to p53 status in luminal A/B and HER2 types. However, in the TN type, patients with p53 overexpression had marginally worse DFS in cases without chemotherapy. On the other hand, there was no difference in DFS in the cases with chemotherapy. These findings suggest that chemotherapy is effective in improving the DFS in TN cases with p53 overexpression. Conclusion: p53 overexpression correlated with a higher grade of malignancy and unfavorable prognosis in patients with Luminal A/B subtypes. Although p53 status did not correlate with the prognosis in HER2 and TN subtypes, patients with p53 overexpression may benefit from chemotherapy in cases with TN subtype in adjuvant setting. Citation Format: Reiki Nishimura, Tomofumi Osako, Yasuhiro Okumura, Masahiro Nakano, Mamiko Fujisue, Nobuyuki Arima. An evaluation of p53 overexpression as a predictor of prognosis and chemotherapy benefit in relation to subtypes in primary invasive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-62.

10043 Background: Risk stratification in osteosarcoma relies on metastatic status and tumor necrosis after chemotherapy. Despite a complex genomic landscape, genomic biomarkers are not yet used for predicting therapy response. MYC amplification has previously been identified as a potential biomarker for chemotherapy resistance but studies of MYC expression are limited. This study evaluated the relationship between MYC amplification and protein expression and between these biologic features and survival in pediatric and young adult osteosarcoma. Methods: In a cohort of 93 patients with high-grade osteosarcoma, MYC copy number was evaluated using a targeted sequencing panel, and MYC protein expression was quantified via IHC with an H-Score. The relationship between copy number and protein expression were evaluated via spearman correlation. Amplification (AMP) was defined as > 7 copies, and high expression (EXP) as > 175 H-score. The primary outcome, overall survival (OS), was assessed using Kaplan Meier analysis (median [IQR]) for unadjusted models and cox proportional hazard analysis (HR±SE) with models adjusted for metastatic status at diagnosis. Results: Among the 93 patients, 64% were male, with a median age of 14 years [range: 4–29]. MYC AMP was present in 16 (17%) patients, high MYC EXP in 19 (20%), and both AMP + high EXP in 8 (9%) patients. Copy number status was positively correlated with expression (r = 0.57, p < 0.0001). Patients with AMP were more likely metastatic at diagnosis (75% vs 38%, p = 0.011) and more so for AMP + high EXP (100% vs 39%, p = 0.0009), but not high EXP alone (63% vs 39%, p = 0.11). OS was reduced for AMP compared to non-AMP (median OS: 1.3 [1.0, 2.6] vs 6.2 [2.8, 12.8] years, p < 0.0001; Adj HR 3.2±0.4, p = 0.001) and high EXP compared to low EXP (median OS: 1.5 [1.0, 3.1] vs. 6.2 [2.8, 12.8] years, p < 0.0001; Adj HR 5.6±0.4, p < 0.0001). A dose-response relationship was seen with higher copy number or expression linked to reduced OS. Compared to non-AMP + low EXP (median OS: 7.23 [3.1, 12.8] years) there was reduced OS for AMP-only/high EXP-only (median OS: 3.1 [2.0, 4.0] years, p = 0.01; Adj HR 2.8±0.4, p = 0.009) and further reduced OS for AMP + high EXP (median OS: 1.0 [0.5, 1.0] years, p < 0.0001; Adj HR 15.3±0.5, p < 0.0001). Metastasis at diagnosis predicted reduced OS compared to localized disease (median OS: 2.0 [1.0, 4.5] vs 6.2 [4.0, 12.8] years, p < 0.0001), but age, sex, and tumor necrosis were not associated with OS. Conclusions: MYC amplification and protein expression are positively correlated, and both independently and synergistically predict poor OS in osteosarcoma, even after adjusting for metastatic status at diagnosis. Incorporating MYC amplification and expression status into risk stratification may help identify patients with worse prognosis. These data also underscore the need for therapeutic approaches tailored to the genetic basis of osteosarcoma tumor biology.

Treatment Outcomes of Different Chemotherapy Sequences in N3 Stage Nasopharyngeal Carcinoma

Objective This study aimed to perform an updated meta-analysis to explore the clinical, diagnostic, and prognostic values of circRNAs in osteosarcoma. Methods : PubMed, Web of Science, EMBASE, Scopus, and Cochrane Library were systematically searched up to December 15, 2020. Eligible studies regarding the relationship between circRNAs levels and clinicopathological, diagnostic, and prognostic values in osteosarcoma were included for study. Results 31 studies involving 1979 osteosarcoma patients were enrolled, with 22 studies on clinicopathological parameters, eleven on diagnosis, and 23 on prognosis. For clinical parameters, overexpression of oncogenic circRNAs was intimately correlated with larger tumor size, advanced Enneking stage, poor differentiation, and distant metastasis (DM). In contrast, the downregulated circRNAs showed negative correlation with Enneking stage and DM. For the diagnostic values, the summary area under the curve of circRNA for the discriminative efficacy between osteosarcoma patients and non-cancer counterparts was estimated to be 0.87, with a weighted sensitivity of 0.79, specificity of 0.81, respectively. For the prognostic significance, oncogenic circRNAs had poor overall survival (OS) and disease-free survival, while elevated expression of tumor-suppressor circRNAs were closely related to longer OS. Conclusion This study showed that aberrantly expressed circRNA signatures could serve as potential biomarkers in diagnosis and prognosis in osteosarcoma.

Type II endometrial carcinoma mainly originates from p53 aberration. However, the detailed prognostic significance of p53 aberration in endometrial carcinoma remains to be clarified. In the present study, abnormal p53 accumulation was analyzed using immunohistochemical techniques in endometrial carcinoma samples derived from 221 consecutive patients. The expression levels of p53 were associated with clinicopathological parameters and patient survival. P53 overexpression was observed in 37/221 patients (17%), and was associated with non-endometrioid histology, post-menopause and advanced tumor stage (III/IV; P=0.0006, P=0.03 and P=0.025, respectively). Survival analysis indicated that patients with p53-overexpressing tumors exhibited poor overall survival (OS) compared with patients without p53 overexpression (P<0.000001). Univariate and multivariate analyses demonstrated that the parameters p53 overexpression, age ≥70, non-endometrioid histology and advanced stage were significant and independent prognostic factors for poor OS (P=0.00012, P=0.00048, P=0.0027 and P=0.0015, respectively). Additionally, adjuvant radiotherapy was associated with increased OS in patients without p53 overexpression. This finding was not observed for patients with adjuvant chemotherapy. In contrast to patients without p53 overexpression, patients with p53 overexpression exhibited no association with OS (P=0.02 vs. P=0.40). Notably, adjuvant radiotherapy was identified to be a significant prognostic factor for favorable OS in the subset of patients that did not exhibit p53 overexpression and received post-operative treatment (P=0.026). The findings suggested that abnormal p53 accumulation may influence patient survival via unfavorable biological tumor properties, including rapid progression and radioresistance. The present study offered valuable insights for the genome-directed management of endometrial carcinoma.

Objective To explore the clinicopathological characteristics, recurrence, metastasis and survival of triple negative breast cancer (TNBC) , and to analyze the correlation factors affecting the prognosis. Methods Data of 378 breast cancer patients treated from Jan. 2008 to Dec. 2011 were retrospectively analyzed. According to immunohistochemical staining of estrogen of receptor (ER) , progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) , they were divided into TNBC group (61 cases) and non-triple negative breast cancer group (non-TNBC, 317 cases) . The two groups were compared in terms of clinicopathological characteristics, prognosis and survival. Results Patients in TNBC group had significant differences in the following aspects: ratio of patients with newly diagnosed age <35 years old, patients with family history of breast cancer, the maximum diameter of tumor more than 5 cm, positive preoperative axillary lymph node status, tumor in clinical stage Ⅲ, histological grade of tumor in level III, Ki67 overexpression and P53 overexpression, while there was no statistical difference in the aspects of menstrual status, pathological type or surgical method between them. The local recurrence and distant metastasis rate were obviously higher in TNBC group than in non-TNBC group. 5-year disease-free survival (DFS) and 5-year overall survival (OS) were significantly lower in TNBC group than in non-TNBC group. Univariate analysis showed that factors related to 5-year DFS in TNBC group were: age, the maximum diameter of tumor, the preoperative axillary lymph node status, clinical staging of tumor, and P53 overexpression. The maximum diameter of tumor, the preoperative axillary lymph node status and clinical staging of tumor were recognized as the influence factors of 5-year OS. Independent factors affecting 5-year DFS in TNBC group were: the maximum diameter of tumor, and the preoperative axillary lymph node status. The maximum diameter of tumor and the preoperative axillary lymph node status were the independent factors influencing 5-year OS. Conclusions The clinicopathological characteristics of TNBC include: younger onset age, family clustering of breast cancer, the larger maximum of tumor diameter, larger portion of positive preoperative axillary lymph node, later clinical staging of tumor, higher histological grade of tumor, easier local recurrence and distant metastasis, lower 5-year DFS and 5-year OS. The factors of age, the maximum diameter of tumor, the preoperative axillary lymph node status, clinical staging of tumor, P53 overexpression especially the maximum diameter of tumor and the preoperative axillary lymph node status play the important clinical roles in judging the prognosis of TNBC. Key words: Triple negative breast cancer; Clinicalpathologic characteristics; Prognosis

Background-Aim: Breast cancer is a heterogeneous disease and despite recent scientific progress there is still need for the identification of biomarkers associated with risk for relapse, as well as for markers identifying patients who will benefit from specific treatments. The aim of the present study was to investigate the role of MYC, as a clinically meaningful biomarker, in the outcome of breast cancer subtypes. Patients and Methods: We have pooled the patients and the respective breast carcinomas from two randomized anthracycline-based adjuvant phase III trials, consecutively conducted by the Hellenic Cooperative Oncology Group (HE10/97 and HE10/00). The HE10/97 trial included a non-paclitaxel arm. Tissue microarrays were constructed from 1,060 formalin-fixed paraffin-embedded tumor tissue samples that were collected retrospectively in the first and prospectively in the second trial. MYC was evaluated by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in 986 cases. Results: In total 61.0% of the cases showed positive cytoplasmic MYC immunostaining, while 26.5% showed positive nuclear staining. 65-80% of the patients were characterized as non-amplified or loss/normal-low gain in all FISH cut-offs examined. A weak association was observed between FISH and nuclear protein expression of MYC. High histological grade was associated with MYC protein overexpression and gene amplification. In terms of disease-free survival (DFS), low (2.5-5 copies) and high (≥5 copies) gain of MYC was of adverse prognostic value compared to loss/normal (&amp;lt;2.5 copies) MYC (HR=1.50, 95% CI 1.13-1.98, Wald's p=0.004 and HR=1.45, 95% CI 1.07-1.97, p=0.016, respectively). Comparable results were observed for overall survival (OS) (HR=1.51, 95% CI 1.09-2.08, p=0.013 and HR=1.65, 95% CI 1.17-2.33, p=0.005, respectively). The comparison of neoplasms with CEP8 ratio ≥1.3 and polysomy 8 for MYC versus all others resulted in worse survival prognosis (HR=1.44, 95% CI 1.13-1.83, p=0.004), while tumors with nuclear protein overexpression were associated with better DFS (HR=0.77, 95% CI 0.60-0.99, p=0.039) and OS (HR=0.73, 95% CI 0.55-0.98, p=0.034). In HER2-enriched patients, MYC amplification was found to be an adverse prognostic factor for DFS (HR=2.11, 95% CI 1.09-4.07, p=0.026) and OS (HR=2.41, 95% CI 1.12-5.15, p=0.024). Treatment with paclitaxel was found to differentiate the effect of MYC: CEP8 ratio ≥1.3 and polysomy 8 in terms of DFS and OS in our total cohort. Among patients with CEP8 ratio ≥1.3 and polysomy 8, those treated with paclitaxel performed significantly better than those not treated, while among patients not treated with paclitaxel, those with CEP8 ratio ≥1.3 and polysomy 8 performed much worse than those with CEP8 ratio &amp;lt;1.3 or no polysomy 8. Conclusions: Our data suggest that MYC has prognostic and predictive value in patients with breast cancer. MYC amplification and MYC protein overexpression are detected in breast cancer patients and are of adverse prognostic value for DFS and OS. Polysomy 8 is also associated with worse prognosis. Treatment with paclitaxel in the adjuvant setting benefits breast cancer patients with MYC:CEP8 ratio ≥1.3 and polysomy 8. Citation Format: Batistatou A, Razis E, Bobos M, Tsolaki E, Timotheadou E, Alexopoulou Z, Goussia A, Gogas H, Koutras A, Karina M, Pentheroudakis G, Efstratiou I, Petraki K, Sotiropoulou M, Pavlakis K, Koletsa T, Kotoula V, Fountzilas G. Associations of MYC protein expression and gene status with breast cancer subtypes and outcome in patients treated with anthracycline-based adjuvant chemotherapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-50.

Adjuvant therapy for colon cancer is now a mature and widely accepted standard of care for patients with resected large bowel tumors: adjuvant therapy for stage III colon cancer has also been shown to be highly cost-effective. The cost of 5-FU/levamisole therapy for stage III colon cancer per year of life saved is less than $ 5,000, which represents a favorable cost-benefit relationship for a medical intervention. The clinician managing a patient with colon cancer at the present time has several options for therapy. In patients with stage III colon cancer, therapy with 5-FU-based regimens clearly increases overall and disease-free survival. It is also clear that the results that have been obtained are not perfect; therefore, the first option of therapy should always be an ongoing clinical trial. Many such trials are available, and Table 7 lists currently active studies in the United States. The clinician managing a patient with stage III colon cancer who is not in a clinical trial may choose a variety of regimens administered for durations of 6 to 12 months (Table 8). The preponderance of evidence suggests that 5-FU plus levamisole for 12 months is equal in efficacy to 5-FU plus leucovorin-based regimens given for a shorter period of time. A clinician may still choose the 5-FU plus levamisole regimen because of the decreased oral, myelosuppressive, and diarrheal toxicities associated with that regimen as opposed to the 5-FU/leucovorin regimens. Portal vein infusion of fluorinated pyrimidines still must be considered investigational. Finally, although we cannot be absolutely sure about the benefit of adjuvant therapy in patients with resected node-negative colon cancer, the NSABP data suggest that some benefit may be seen in these patients. It is known that patients with stage II cancers demonstrating high-grade bowel obstruction or bowel perforation have poor prognoses with surgery alone. Such patients may be good candidates for adjuvant therapy. Also, a major effort to define high risk and low risk for recurrence in patients with stage II colon cancer by analyzing molecular genetic factors (tumor ploidy and alternations in tumor suppressor genes) may lead to a selection of Dukes B patients definitely requiring adjuvant therapy.

Objective To investigate the clinical efficacy of radical resection for stage T3 gallbladder cancer and prognostic factors. Methods The retrospective case-control study was conducted. The clinico-pathological data of 87 patients with T3 gallbladder cancer who were admitted to Tianjin Medical University Cancer Institute and Hospital from January 2005 to June 2016 were collected. There were 44 males and 43 females, aged 29-79 years, with a median age of 61 years. According to the different preoperative pathological classification and intraoperative exploration of gallbladder cancer, corresponding surgeries were performed. Observation indicators: (1) surgical and postoperative conditions; (2) clinical efficacy of stage T3 gallbladder cancer and prognostic factors analysis; (3) clinical efficacy of stage T3 gallbladder adenocarcinoma and prognostic factors analysis; (4) clinical efficacy of stage T3 gallbladder adenosquamous carcinoma and prognostic factors analysis. Follow-up by outpatient examination or telephone interview was performed to detect the postoperative survival of patients up to June 2018. Measurement data with skewed distribution were represented as M (range), and count data were described as absolute numbers. Survival curve, survival time and survival rate were drawn and calculated by the Kaplan-Meier method. Survival analysis was performed by the Log-rank test. Univariate analysis was performed using the Log-rank test and multivariate analysis using the COX proportional hazard model. Results (1) Surgical and postoperative conditions: all the 87 patients underwent radical resection of gallbladder cancer, including 29 cases of hepatic wedge resection and 58 cases of extended hepatectomy. Of the 87 patients, 42 underwent standard lymph node dissection and 45 underwent enlarged lymph node dissection. There were 27 cases receiving extrahepatic bile duct reconstruction. The postoperative pathological results of 87 patients showed that 64 were diagnosed with gallbladder adenocarcinoma and 23 were diagnosed with gallbladder adenosquamous carcinoma. There were 59 cases comorbid with liver invasion and 3 cases comorbid with vascular invasion. The marginal histopathological examination showed negative margin in 63 cases and positive margin in 24 cases. The degree of tumor differentiation: there were 23 patients with highly differentiated tumor and 64 with poorly differentiated tumor. Of the 87 patients, 43 received postoperative adjuvant therapy and 44 didn′t receive adjuvant therapy. (2) Clinical efficacy of stage T3 gallbladder cancer and prognostic factors analysis. ① All the 87 patients were followed up for 1.8-128.0 months, with a median follow-up time of 26.3 months. All the 87 patients had survived for 1.1-82.7 months, with a median time of 20.1 months. The 2-year overall survival rate of patients was 59.8%, and the 2-year disease-free survival rate was 49.4%. ② Univariate analysis showed that preoperative alkaline phosphatase (ALP) level, tumor diameter, pathological type of tumor, lymph node metastasis, and range of hepatectomy were associated factors for the postoperative 2-year overall survival rate of patients (χ2=5.451, 4.900, 8.256, 4.419, 5.858, P 0.05), but a significant difference in the postoperative 2-year disease-free survival rate between them (56.3% vs. 30.4%, χ2=5.828, P<0.05). (3) Clinical efficacy of stage T3 gallbladder adenocarcinoma and prognostic factors analysis. ① Sixty-four patients with gallbladder adenocarcinoma had the median survival time of 23.1 months, with a range from 3.2 to 82.7 months. The postoperative 2-year overall survival rate was 68.8%, and the postoperative 2-year disease-free survival rate was 56.3%. ② For the 64 patients with T3 stage gallbladder adenocarcinoma, univariate analysis showed that preoperative CA19-9 level and range of lymph node dissection were associated factors for the postoperative 2-year overall survival rate (χ2=4.012, 8.837, P<0.05). The range of lymph node dissection was an associated factor for the postoperative 2-year disease-free survival rate (χ2=6.361, P<0.05). Multivariate analysis showed that range of lymph node dissection was an independant factor for both the postoperative 2-year overall survival rate and postoperative 2-year disease-free survival rate (HR=0.244, 0.382, 95%CI: 0.088-0.674, 0.176-0.831, P<0.05). ③ Survival analysis: range of lymph node dissection was an associated factor for both the postoperative 2-year overall survival rate and postoperative 2-year disease-free survival rate of patients. Of the 64 patients with T3 stage gallbladder adenocarcinoma, the postoperative 2-year overall survival rate and disease-free survival rate of patients undergoing enlarged lymph node dissection were 84.8% and 69.7%, versus 51.6% and 41.9% of the patients undergoing standard lymph node dissection (χ2=8.837, 6.361, P<0.05). (4)Clinical efficacy of stage T3 gallbladder adenosquamous carcinoma and prognostic factors analysis. ① Twenty-three patients with gallbladder adenosquamous carcinoma had the median survival time of 13.2 months, with a range from 1.1 to 70.3 months. The postoperative 2-year overall survival rate was 34.8%, and the postoperative 2-year disease-free survival rate was 30.4%. ② For the 23 patients with T3 stage gallbladder adenosquamous carcinoma, univariate analysis showed that preoperative ALP level, lymph node metastasis, range of hepatectomy, and extrahepatic bile duct reconstruction were associated factors for the postoperative 2-year overall survival rate of patients (χ2=5.288, 4.574, 12.960, 4.106, P<0.05). The lymph node metastasis and range of hepatectomy were associated factors for the postoperative 2-year disease-free survival rate of patients (χ2=7.364, 10.582, P<0.05). Multivariate analysis showed that range of hepatectomy was an independant factor for both the postoperative 2-year overall survival rate and postoperative 2-year disease-free survival rate (HR=0.102, 0.153, 95%CI: 0.012-0.880, 0.033-0.718, P<0.05). ③ Survival analysis: range of hepatectomy was an independant factor for both the postoperative 2-year overall survival rate and postoperative 2-year disease-free survival rate of patients. Of the 23 patients with T3 stage gallbladder adenosquamous carcinoma, the postoperative 2-year overall survival rate and postoperative 2-year disease-free survival rate of patients undergoing extended hepatectomy were 87.5% and 75.0%, versus 6.7% and 6.7% of the patients undergoing hepatic wedge resection (χ2=12.960, 10.528, P<0.05). Conclusions Lymph node metastasis is an independent factor influencing the postoperative 2-year overall survival rate and postoperative 2-year disease-free survival rate of patients with T3 stage gallbladder cancer. The range of lymph node dissection is an independent factor for the postoperative 2-year overall survival rate and postoperative 2-year disease-free survival rate of patients with stage T3 gallbladder adenocarcinoma. Range of hepatectomy is an independent factor for the postoperative 2-year overall survival rate and postoperative 2-year disease-free survival rate of patients with stage T3 gallbladder adenosquamous carcinoma. Patients with gallbladder adenocarcinoma should undergo enlarged lymph node dissection, and patients with gallbladder adenosquamous carcinoma need to undergo extended hepatectomy. Key words: Biliary neoplasms; Gallbladder cancer, stage T3; Gallbladder adenocarcinoma; Gallbladder adenosquamous carcinoma; Lymph node dissection; Prognostic analysis

Objecitve To evaluate the impact of adding methotrexate into DIA chemotherapy regimen (cisplatin, theraru-bicin and ifosfamide) on 3-year disease-free and overall survival of patients with IIB osteosarcoma in extremities. Methods Retro-spectively analyzed 34 cases of IIB limb osteosarcoma according to the Musculoskeletal Tumor Society (MSTS) surgical staging system with pathologically diagnosed concurrent chemotherapy and comprehensive surgical treatment from May 2008 to April 2014. There were 20 males and 14 females, with an average age of 19.5±7.7 years (ranged from 9 to 44 years). 28 patients underwent limb salvage surgery, and 6 patients received amputation. Tumor site included distal femur in 21 patients, proximal tibia in 9 patients, other sites in 4 patients. Seventeen patients underwent cisplatin, THP and ifosfamide (DIA) regimen of chemotherapy. The other 17 patients underwent cisplatin, THP, ifosfamide and methotrexate (MDIA) regimen. Patients charts were reviewed to get demographic data including age, gender, site, tumor size, surgery type, chemotherapy regimen, alkaline phosphatase, lactase dehydrogenase. The prognostic value of these factors on 3 years disease free survival and overall survival were calculated with Kaplan-Meier method and compared with Log-Rank test between different groups in univariate comparison and COX model for multivariate analysis was used to determine whether these demographic factors are independent prognostic factor for survival. Results Two patients had local recurrence, 7 patients had metastasis, and 2 patients had both local recurrence and metastasis. The 3-year survival of the whole group was 67.6% (23/34). Seven patients died during follow-up. The overall survival was 79.4% (27/34). The 3-year disease-free survival in DIA group and MDIA group was 70.6% (12/17) and 64.7% (11/17), respectively. The 3-year overall survival was 82.4% (14/17) and 76.5% (13/17) in DIA group and MDIA group, respectively. Univariate analysis showed that there was no significant difference in either 3-year disease-free survival (χ2=0.113, P=0.737) or overall survival (χ2=0.197, P=0.657) between DIA and MDIA. Age was significantly correlated with prognostic in univariate analysis (χ2=5.869, P=0.015), while gender, tumor site, tumor size, surgery type, ALP, LDH was not correlated with disease-free survival. Kaplan-meier analysis with Log-Rank test showed age (χ2=6.134, P=0.013) and tumor size (χ2=5.108, P=0.024) were statistically correlated with overall survival. Multivariate analysis with COX model showed that chemotherapy regimen was not an independent prognostic factor for 3-year disease-free survival or overall survival. Age was an independent prognostic factor for 3-year disease-free survival (HR=5.836, P=0.017). Conclusion The addition of MTX on the basis of DIA chemotherapy regimen did not significantly improve the 3-year disease-free survival and overall survival of stage IIB limb osteosarcoma. Key words: Osteosarcoma; Extremities; Antineoplastic combined chemotherapy protocols; Survival rate

Our study aimed to evaluate the expression pattern and prognostic value of long noncoding RNA HAGLROS (HAGLROS) in osteosarcoma. qRT-PCR was performed to detect the expression levels of HAGLROS in osteosarcoma tissues and matched normal bone tissues. The relationship between the expression of HAGLROS and the clinicopathological features was analyzed by chi-square test. The survival curves were calculated by the Kaplan-Meier method and the difference by the log-rank test. The Cox proportional hazards model for multivariate survival analysis was used to assess predictors related to survival. Herein, we showed that HAGLROS was frequently upregulated in osteosarcoma tissue and cell lines compared to normal human bone tissues (p < 0.01). In addition, HAGLROS upregulation more frequently occurred in osteosarcoma specimens with advanced TNM stage (p = 0.023), positively distant metastasis (p = 0.002) and poor differentiation (p = 0.021). Survival analysis showed that osteosarcoma patients with higher HAGLROS expression suffered poorer overall survival (p = 0.012) and disease-free survival (p = 0.003). In a multivariate Cox model, it was confirmed that HAGLROS up-regulation was an independent poor prognostic factor for both 5-year overall survival (HR=3.546, 95% CI: 1.273-5.326; p = 0.002) and 5-year disease-free survival (HR=3.854, 95% CI: 1.427-5.885; p = 0.001). We showed that HAGLROS was an independent predictor of unfavorable prognosis in osteosarcoma patients and may serve as a potential target.

Lung cancer is one of the cancers with the highest morbidity and mortality worldwide. Over the two past decades, although the treatment of non-small cell lung cancer (NSCLC) has been revolutionized by the use of tyrosine kinase inhibitor (TKI) and immune treatments, metastatic disease remains largely incurable and presents a 5-year survival rate of approximately 10% [1]. Metastasis is an evolutionary process that involves multiple stages, such as the spread of cancer cells from the primary tumor and then their intravasation into the blood or lymphatic system, survival in the bloodstream and/or lymphatic system, embedding into a distant organ, survival in a new environment, and formation of a new metastatic tumor [2]. A previous study demonstrated that this process could be intrinsically inefficient because the majority of cancer cells either die during circulation, become stuck in capillaries, or undergo apoptosis in the blood within 24 hours. After successful colonization of distal organs, only a subset of cancer cells will develop into macrometastatic tumors [3]. However, once metastases have been established, the tumor growth follows a 'big bang' model [4], and current treatments frequently may fail to provide durable responses. Each metastatic cell represents an evolutionary branch of its parental primary tumor and shares key driver changes. These cells are not under positive selection to metastasize. Rather, a set of key adaptations or hallmarks may be selected to obtain metastatic potential [5]. Herein, we focused on lung cancer metastasis from a genomic perspective, explored the differences in genomic events between metastatic and primary tumor samples, explored the evolutionary pattern and timing of lung cancer metastasis, and explored whether there are differences in evolutionary trajectories between different metastatic organs. During the process of metastasis, each step depends on the specific phenotypic characteristics of tumor cells and their interactions with the host microenvironment and immune system. Genetic alterations obtained from primary and metastatic tumor cells may explain these phenotypic and host interactions [2]. Targeted next-generation sequencing of 30 tumor specimens from 15 patients was performed in a previous study and demonstrated that recurrent genomic alterations between the primary tumor and matched metastasis presented a concordance rate of 94% whereas the likely passenger genomic alterations presented a concordance rate of 63% [6]. Furthermore, a 71.6% concordance rate in clonal somatic mutations was exhibited between the primary tumor and metastatic lymph nodes from 6 patients[7]. In 61 patients with brain metastases, mutations of major drivers, including EGFR, KRAS, TP53, and ALK, were highly concordant between the primary NSCLC and matched brain metastatic tumors (>80%) [8] (Figure 1). These results suggested that classic genomic drivers, such as EGFR and ALK, may be early clonal genomic events during the carcinogenesis of lung adenocarcinoma. For total genetic alterations, Tang et al. [9] revealed that the overall concordance between primary tumors (PT) and metastatic tumors (MT) was 45.6%, and it ranged from 7.3% to 80.6% per patient. Furthermore, metastatic site-specific differences were also observed. Among lymph node, bone, brain, and adrenal gland metastases, lymph node metastases exhibited the lowest proportion of primary tumors-metastatic tumors (PT-MT) shared alterations while adrenal gland metastases shared the highest proportion of genomic alterations with primary tumors (Figure 1). Therefore, inter-tumor genomic heterogeneity indicates the importance of comprehensively understanding the lung cancer gene landscape in clinical practice and could explain the heterogeneity of antitumor therapy efficacy. From the Genomics, Evidence, Neoplasia, Information, Exchange (GENIE) cohort [10], 1897 primary and 1133 unpaired metastatic samples were directly compared. The results showed a large degree of overlap in the frequency of commonly mutated genes, with only TP53 remaining significant after adjusting for false discovery rate (FDR). Furthermore, Shih et al. [11] performed whole-exome sequencing on 73 brain metastatic samples of lung cancer and compared them with 503 primary lung adenocarcinoma samples from The Cancer Genome Atlas (TCGA) database. By screening of Genomic Identification of Significant Targets in Cancer (GISTIC) score and validation of animal models, the authors found MYC, YAP1, and MMP13 amplification represented potential drivers of brain metastasis in lung adenocarcinoma (Figure 1). However, the above previous studies exploring the driver events of lung cancer were mainly based on nonpaired primary-metastatic specimens, possibly due to specimen limitations. Paired analysis between primary and metastatic tumors can reveal whether specific events are enriched at the metastatic sites within individual tumors, thus, distinguishing events that occurred before or after metastatic dissemination [5]. Tang et al. [9] performed single-region or multiple-region whole-exome sequencing on 54 paired primary-metastatic samples and found that the potential drivers of lung cancer metastasis were MYC amplification, NKX2-1 amplification, RICTOR amplification, arm 20p gain, and arm 11p loss (Figure 1). The above somatic and molecular events can break through the evolutionary bottleneck in the process of tumor evolution and continue to be maintained in metastatic tumors, therefore, they exhibited elevated mutational frequencies in metastases. These results may help to explain the mechanism of lung cancer metastasis and promote to development of new targeted drugs. At present, two main evolution models are available for describing the process of tumor dissemination: linear evolutionary model and parallel evolutionary model [2]. Both assume that the primary tumor and its metastasis are clonally related and the differentiation is based on the time when the metastatic cells began to spread at the primary site and the genomic heterogeneity (the number of independent single-nucleotide mutations (SNV) between the primary tumor and the metastatic tumor after the occurrence of a common ancestor) between the primary tumor and the metastatic tumor. Linear evolutionary model: metastatic cells with metastatic cloning appear in the late stage of tumorigenesis and begin to spread as soon as the primary lesion can be detected in clinical practice. Since metastasis is seeded by the most advanced primary clone or subclone, the heterogeneity of the PT-MT gene is small. Parallel evolution model: metastatic clones or subclones appear at the early stage of the development of the primary tumor, and the clones of the primary tumor and the metastatic tumor continue to evolve parallel under different pressures, resulting in significant genetic differences between the primary lesion and the metastatic lesion (Figure 2). Different types of cancer exhibit different evolutionary patterns. Breast cancer cells [12] have the ability to metastasize at a later stage of tumorigenesis. In contrast, Hu et al. [13] reported that in colorectal cancer, most metastatic cells usually appear in the early stage of the primary tumor. To quantitatively analyze the timing of dissemination for lung cancer metastasis, Tang et al.[9] applied a published tool, spatial computational inference of metastatic timing (SCIMET)[13] to estimate the cell population of the PT at dissemination (Nd) and the mutation rate (u) using a spatial tumor growth model. It indicated that most lung cancer metastases (33/54 cases; 61.1%) preferred late dissemination. However, lung cancer had site-specific metastatic timing, with lymph nodes showing susceptibility to early dissemination, while metastasis to the pleura, bone, adrenal gland and brain tended to occur during the late dissemination stage. Furthermore, before the primary tumor diagnosis, the time taken for tumor cells to migrate to lymph nodes was at an average of 4.26±0.74 years. However, for pleura metastases and distant metastases, the average dissemination time was only approximately 2.11±0.33 years before the detection of the PT. The results are consistent with those of a previous pan-cancer study including breast, colorectal and lung cancer, which indicated a seeding age of 3.6 years (interquartile range = 2.8-3.7) for overall lung cancer metastasis[14]. Overall, these results indicate the importance of early screening. Lymph node metastases likely occur earlier than distant metastases, and they needed earlier and more frequent follow-up. In colorectal cancer, Naxerova et al. [15] found that lymphatic and distant metastases shared common subclonal origin in only 35% of 17 cases, with most cases arising from independent subclones in the primary tumor. By mapping the routes of dispersal of lymphatic metastases in human colorectal cancer, Zhang et al. [16] identified three models of spreading, namely interlayer sequential spread, interlayer skip spread and intralayer spread, with 44.3% of the cases presenting interlayer sequential spread. For lung cancer metastasis, understanding the evolutionary relationship between lymph nodes and distant metastasis is of great clinical significance for preventing distant metastasis although limited currently available relevant data. A multicancer analysis of clonality with 457 paired primary tumor and metastatic samples from 136 patients with breast, colorectal and lung cancer [14] found that polyclonal seeding was common in untreated lymph node metastases and distant metastases but less frequent in treated distant metastases, suggesting that treatment exerts strong selection pressure during tumor evolution. Recently, by reconstructing tumor phylogenies, Tang et al. [9] found that most metastases (7/8) may be directly seeded by the primary tumor but not sequentially spread from lymph nodes. Tumor metastasis can spread through multiple routes and in different directions. However, larger sample sizes on lymph nodes and distant metastatic tumors as well as multiple distant metastatic tumors are needed to explore the evolutionary trajectories of lung cancer metastasis. To our knowledge, lung cancer genomic evolution is primarily considered to be limited to early or locally advanced lung cancer [17] and the evolutionary trajectories of lung cancer metastasis are poorly understood. Tumor metastasis is a highly non-random process [18], and lung cancer also exhibits organ-specific morbidity and overall survival [19]. However, the mechanisms underlying the organ-specific pattern of lung cancer metastasis remain unclear. Previous studies have found some potential metastasis drivers but the underlying mechanisms need to be further explored. A study on pan-cancer evolution [14] suggested that clonal seeding also exhibits organ specificity. However, due to the small lung cancer sample size and the restriction of metastatic sites to the lymph nodes and brain, clonal seeding models of lung cancer metastasis have not been clarified. Herein, a more comprehensive multiple omics landscape of lung cancer metastasis should be developed to better understand the biology and characteristics of lung cancer metastasis, to prevent lung cancer metastasis and improve the overall survival of these patients. Collectively, lung cancer metastasis mostly presents late dissemination and features moderate genomic heterogeneity and specific genomic drivers of metastasis. In addition, selective pressure for treatment could alter the clonal seeding patterns of lymph nodes and distant metastases. Finally, lung cancer metastasis exhibited organ-specific patterns, including genomic heterogeneity and timing of dissemination. Not applicable. All authors consent to publish the paper. Not applicable. This work was supported by the Zhongshan City People's Hospital Major Project (Top Youth Program) (Grant No. B2021003). Project of National Natural Science Foundation (Grant No. 81872510), High-Level Hospital Construction Project (Grant No. DFJH201801), Guangdong Provincial People's Hospital Young Talent Project (Grant No. GDPPHYTP201902), GDPH Scientific Research Funds for Leading Medical Talents and Distinguished Young Scholars in Guangdong Province (Grant No. KJ012019449), and Guangdong Basic and Applied Basic Research Foundation (No. 2019B1515130002). All authors indicated no conflicts of interest. All authors contributed to the conception and design of the study. WFT and RF drafted the manuscript. YL, JSL, ZBQ, YLW, and WZZ provided critical revisions of the manuscript. All authors read and approved the final manuscript. Not applicable.

Objective To investigate the assessment and treatment strategy of patients with renal cell carcinoma. Methods The clinical data of 43 patients with renal cell carcinoma and bone metastases admitted to the First Affiliated Hospital of Nanjing Medical University from January 2006 to December 2018 were retrospectively analyzed. The follow-up time was 6 years, with an average age of 55.4 years (21-87 years). There were 29 males, 14 females, 22 cases of limb bone metastasis, 12 cases of spinal metastasis, 9 cases of multiple bone metastasis, 21 cases of Fuhrman grade 1 and 2, 19 cases of T1, and 20 cases of N0. All patients were confirmed by postoperative pathological examination or imaging data suggesting that bone metastasis are from renal cell carcinoma. Forty-three patients underwent primary renal surgery, and molecular targeted therapy was used after the operation. The treatment process was smooth, no obvious discomfort, and postoperative pathology showed clear cell carcinoma.22 patients with limb bones metastasis and 12 patients with spinal metastasis included in the study all met the indications for secondary surgery after the disease assessment. After communicating with the patient, 13 patients with limbs metastasis and 6 patients with spinal metastasis received local treatment, including complete resection of the extremities and spinal fixation, the remaining 15 patients and 9 patients with multiple bone metastasis were treated conservatively. There were 19 patients in the local treatment group, 13 patients with limbs bone metastasis, 6 patients with spinal bone metastasis, the average age was 54.9 years, the average diameter of the primary tumor was 4.7 cm. There were 24 patients in the conservative treatment group, 9 patients with limbs metastasis, 6 patients with spinal metastases and 9 cases with multiple bone metastasis, with an average age of 56 years and a primary tumor diameter of 5.6 cm. Limb metastatic lesions were evaluated according to the patient's general condition, bone pain, fracture risk, and bone metastasis. Spinal lesions were evaluated according to Tokuhashi score, Harrington score, Tomita score, vertebral stability assessment, and molecular targeted therapy. Aminokinase inhibitors, conservative treatment with local radiotherapy and bisphosphonate treatment. Results During the follow-up period, the 1-year overall survival rate of the local treatment group was 100.0%, the 2-year overall survival rate was 89.4%, and the 5-year overall survival rate was 73.7%. The 1-year overall survival rate of the conservative treatment group was 87.5%, and the 2-year overall survival rate was 62.5%. The 5-year overall survival rate was 16.7%. The 2-year and 5-year survival rates of the local treatment group were statistically different (P=0.044, P=0.000) compared with the conservative treatment group. For patients with limb bone metastasis, the 5-year survival rate was significantly higher in patients receiving topical treatment than in the conservative treatment group (P=0.011). For spinal metastasis, spinal pain in the local treatment group was alleviated to varying degrees. No spinal instability and spasticity were observed after follow-up. In the spine patients who received conservative treatment, 3 patients developed paraplegia, which was statistically different from local treatment (P=0.046). Another 9 patients with multiple bone metastases did not undergo local surgery, and all died after multiple organ failure. Conclusions At the same time of molecular targeted therapy, according to the evaluation results, selective treatment of bone metastases with secondary surgical indications, including complete resection of the extremities and spinal fixation, can significantly improve the survival and quality of life of those patients. Key words: Carcinoma, renal cell; Bone metastasis; Condition evaluation; Treatment strategy; Complete resection; Spine fixation