Reduced Disease Free Survival Research Articles

Histological risk score and its role in predicting recurrence in early-stage oral squamous cell carcinomas.

Oral squamous cell carcinoma (OSCC) comprises more than 90% of oral cancers and is the most common carcinoma affecting the oral cavity. Early stage T1/T2 OSCC have a heterogeneous prognosis and a significant number of patients develop loco regional recurrence (LRR) and have reduced disease free survival (DFS) with increased disease related mortality. To assess the impact of the three parameters used in Brandwein-Gensler risk model along with lympho-vascular invasion (LVI), depth of invasion (DOI) and lymph node metastases in predicting LRR in early stage OSCC. This was a retrospective study on early stage T1/2 OSCC patients over a period of 2 years who received treatment by surgical resection and had follow-up data. LRR was assessed based on recurrence of OSCC at the initial site or in regional lymph nodes. Out of 1135 OSCC cases during our study period a total of 207 cases befitted our inclusion criteria. Recurrence was noted in 113 (54.6%) cases. Univariate analysis identified LVI (P < 0.00001), DOI (P < 0.00001), nodal involvement (P < 0.00001), worst pattern of invasion (WPOI) (P < 0.00001), lymphocytic host response (LHR) (P = 0.004), perineural invasion (PNI) (P = 0.012) as strong statistically significant risk factors for LRR. Adequate assessment of simple parameters on routine H and E by incorporating Brandwein-Gensler histological risk scoring model at the initial presentation can help prognosticate and predict LRR and select patients for post-surgical adjuvant therapy.

Disease Free Survival in Locally Advanced Breast Cancer: An Analysis of Clinical and Pathological Factors

Background: The TNM stage is the widely accepted tool to predict prognosis in breast cancer cases, but it deals with a limited range of factors and ignores patient-specific conditions, pathological or genotype characteristics and treatments. The purpose of this study was to evaluate if and how different clinical (tobacco chewing, age etc) and pathological (lymphovascular and perineural invasion etc) variables influence the disease free survival. Material and Methods: This study involves North Eastern Indian population. Data of 101 patients with locally advanced breast cancer diagnosed in the time period of January to December 2018 was probed and they were followed up till March 2020. Association between various clinicopathological risk factors and disease free survival was assessed using univariate and multivariate models. Survival estimates were computed using the Kaplan-Meier method and differences between survival times were assessed by means of the Log rank test. Multivariate analyses were carried out using Cox’s proportional hazards model. Results: During follow up period 14 patients developed metastasis. There was significantly reduced disease free survival seen trough Kaplan Meier plots in patients having tumor size &gt;6 cm, N2/ N3 stage, triple negative receptor status, tobacco chewing and positive perineural invasion. Multivariate analysis revealed that stage (N2, N3) and positive perineural invasion are two independent variables which adversely affect the survival. Conclusion: Bottom line of the study is that, disease free survival in LABC patients is significantly reduced with higher stage (N2, N3) and positive perineural invasion as revealed by multivariate analysis. Three other factors which adversely affect DFS are tobacco chewing, tumor size &gt; 6cm and triple negative hormone receptor status according to univariate analysis.

Angiogenesis, programmed death ligand 1 (PD-L1) and immune microenvironment association in laryngeal carcinoma.

In the specific field of laryngeal carcinoma (LSCC), evidence about the interaction between angiogenetic pathway and immune microenvironment has not yet been explored. Given the potential relevance of such an interaction for prognostic and therapeutic purposes, the main aim of this exploratory study was to investigate the existence of a correlation between angiogenesis (quantified through CD31 expression), programmed cell death ligand 1 (PD-L1) expression, and immune microenvironment. A secondary aim was to verify whether considering a combination of angiogenesis and immune microenvironment variables might improve prognostic accuracy compared to the traditional clinical-pathological prognostic tools. CD31-assessed micro-vessel density (MVD), PD-L1 in terms of combined positive score (CPS), and tumour infiltrating lymphocytes (TILs) were assessed on 45 consecutive cases of LSCC. Cox proportional hazards model revealed increasing CD31-assessed MVD values, PD-L1 CPS <1, and TILs count rate <30%, as predictive of reduced disease free survival (DFS). Multivariate analysis found that MVD (p<0.0001) and TILs (p=0.0420) retained their significant independent prognostic value. Spearman's correlation model disclosed a significant negative correlation between CD31-assessed MVD values and PD-L1 CPS (p=0.0040). PD-L1 CPS and TILs count rate were positively correlated (p<0.0001). DFS was significantly lower in the CD31-assessed MVD >7, PD-L1 CPS <1, TILs <30% group than in the MVD ≤7, PD-L1 CPS ≥1, TILs ≥30% group (p=0.0001). These data preliminarily support an integrated interpretation of the prognostic role or angiogenesis and immune microenvironment markers in LSCC. This is of potential clinical relevance suggesting a synergistic effect of the combination of anti-angiogenic drugs with programmed death-1/PD-L1 checkpoint inhibitors in advanced LSCC.

Is CD 44 really a marker of breast cancer metastasis or is nefarious in its nature

Aims and Objectives: To study the expression of immunomarker CD 44 and evaluate its diagnostic and prognostic significance and its correlation with clinicopathological variables in Breast Carcinoma. Materials and Methods: 50 diagnosed cases of breast carcinoma, presenting clinically with breast lumps were processed routinely for histopathological diagnosis of breast cancer and immunoexpression of CD 44 was performed. The immunoexpression of CD44 was detected mainly at the membrane of tumour cells and the scoring was performed. Results: 217 (98.2%) were females and 4(1.8%) males. Most common subtype involved was invasive carcinoma (NST) in 205(92.8%) cases. Out of 29 cases of grade 2 tumor, 07 (14.0%) showed 1+ score, 10(20.0 %) showed 2+ score and 12 (24.0%) cases showed 3+ immunoexpression. Out of 25 cases of T2 (2-5cm) size, 07(14.0%) showed 1+ score, 09(18.0%) case each showed 2+ score and 3+ immunoexpression. Out of 26 cases of stage III, 04(8.0%) showed 1+ score, 09(18.0%) showed 2+ score and 13(26.0%) cases showed 3+ immunoexpression. Out of 05 cases of N3, 02(4.0%) showed 2+ score and 03(6.0%) showed 3+ immunoexpression. Out of 21 cases of basal cell type, 03(6.0%) showed 1+ score, 07(14.0 %) showed 2+ score and 11(22.0%) cases showed 3+ immunoexpression. Out of 11 (50.0%) cases with weak to moderate expression of CD44, 7(31.8%) cases were stable and alive while 4(18.2%) died due to distant metastasis to various sites. Conclusions: CD 44 is associated with disease progression, recurrence, advanced metastasis and reduced disease free survival and can be used in future to predict early diagnosis of advanced disease. Keywords: Breast carcinoma, CD 44, Immunohistochemistry, Prognosis.

Prognostic impact of Notch1 receptor and clinicopathological High-Risk Predictors in lacrimal gland adenoid cystic carcinoma.

Adenoid Cystic Carcinoma (ACC) is an aggressive malignant lacrimal gland tumour associated with poor prognosis. Aberrant Notch signalling has been investigated in various tumours. However, very few studies on Notch signalling in lacrimal gland ACC are reported. The aim of the present study was to evaluate the status of Notch1 receptor and activated Notch1 (NICD1) in lacrimal gland ACC and to correlate it with high-risk clinicopathological features. A total of 23 cases of histopathologically proven lacrimal gland ACC, who underwent surgical treatment, were included in this study. Expression of Notch1 receptor and NICD1 was evaluated by immunohistochemistry on formalin fixed paraffin embedded tissues. The results obtained were correlated with clinicopathological high-risk features and survival of the patients. Kaplan-Meier survival and multivariate analysis was performed to determine the prognostic significance. Overexpression of Notch1 receptor and NICD1 was observed in 65% and 39% of lacrimal gland ACC cases, respectively. On Kaplan-Meier survival analysis, patients with Notch1 receptor overexpression had reduced disease free survival. On univariate analysis, male gender, bone erosion, perineural invasion, solid histologic pattern, intracranial extension and advanced tumour stage were also indicators of poor prognosis. On multivariate analysis bone erosion was the most significant poor prognostic indicator. Our study demonstrates that overexpression of Notch1 receptor plays a critical role in the biology and aggressive behaviour of lacrimal gland ACC. Bone erosion, solid histologic pattern, advanced T stage, perineural invasion and intracranial extension are other high-risk clinicopathological predictors of lacrimal gland ACC.

Abstract 2830: The impact of gut microbial toxins and PGE2 on IL-23 production and its role in obesity associated colorectal tumorigenesis

Abstract Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. Studies indicate that excessive intake of high-fat diet (HFD) promotes CRC by modulating gut microbiome-associated inflammatory mediators. Recent evidence suggests that interleukin-23 (IL-23) plays an important role in the impact of a HFD on obesity, the gut microbiome and colon carcinogenesis. Our study aimed to elucidate the role of IL-23 in obesity associated CRC and explore an anti-IL-23 approach for prevention and treatment. First we profiled human plasma (n=15/arm) for circulating IL-23 levels and found significantly higher levels (p&amp;lt;0.0001) in obese individuals (BMI&amp;gt;30). In addition, IL-23 expression was evaluated in colonic tumors from both humans and preclinical rodent models. Data from the Cancer Genome Atlas showed significant over expression (p&amp;lt;0.0001) of IL-23A in CRC tumors (n=262) compared to normal tissue (n=41). This overexpression correlated with reduced disease free survival. Levels of both IL-23 and its receptor (IL23R) were evaluated in matched human CRC tissues by western blot and IHC and confirmed overexpression in CRC. IL-23 was also upregulated in colonic tumors from both sporadic (AOM-induced rat CRC) and FAP (Apcmin/+ mouse) rodent models when compared to matched normal mucosa. These data reveal a clear association of IL-23 overexpression with both obesity and colonic tumorigenesis. To understand IL-23 production in the context of gut microbial toxins and prostaglandin (PG)E2 levels, we performed several mechanistic studies to mimic the tumor microenvironment. To elucidate PGE2-mediated regulation of IL-23 in colon tumorigenesis we studied both macrophages and dendritic cell lines in in vitro and ex vivo conditions. The immune cells treated with either PGE2 or bacterial toxins (LPA/LTA) showed a significant increase in IL-23 expression which associated with macrophage polarization and dendritic cell phenotypic changes. Interestingly, when CaCo2 cancer cells were co-cultured with PGE2/LTA-LPA-educated macrophages or dendritic cells, the cancer cells displayed enhanced IL-23 expression along with increased tumor cell proliferation and self-renewal. Upon addition of human recombinant IL-23 directly to cancer cells (CaCo2 and HCT116), the cells exhibited increased IL-23R expression along with enhanced self-renewal, cell migration and invasive properties as well as loss of epithelial barrier permeability. In ex-vivo experiments we found that treatment of rat colon tumors with PGE2 increased the expression of IL-23 compared to normal mucosa. Overall our study results demonstrate that IL-23 is associated with both obesity and poor survival of CRC patients and that PGE2 and gut microbe toxins play an important role in IL-23-associated colonic tumor progression. This newly identified nexus represents a potentially important target for prevention of obesity associated colorectal cancer. Citation Format: Janani Panneerselvam, Venkateshwar Madka, Rajani Rai, Parthasarathy Chandrakesan, Courtney W. Houchen, Dharambir K. Sanghera, Katherine T. Morris, Chinthalapally V. Rao. The impact of gut microbial toxins and PGE2 on IL-23 production and its role in obesity associated colorectal tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2830.

Clinical Relevance of +936 C>T VEGFA and c.233C>T bFGF Polymorphisms in Chronic Lymphocytic Leukemia.

Angiogenesis process contributes to the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) being the levels of VEGFA and bFGF higher in patients than in healthy controls. Our aim was to evaluate the implication of angiogenesis factors genetic variants in the predisposition to B-CLL and their association with clinical factors and survival. We performed a population-based case-control study in 224 Spanish B-CLL patients and 476 healthy randomly selected controls to evaluate susceptibility to developing B-CLL. Six polymorphisms were evaluated: rs1109324, rs1547651, rs3025039 (+936 C>T), rs833052 of the VEGFA gene, rs1449683 (c.233C>T) of the bFGF gene and (−710 C>T) of the VEGFR1 gene. The association between clinical parameters and patient outcome was analyzed. Carriers of the CT/TT variants of rs3025039 showed a significant protective effect against developing B-CLL. The CT/TT variants of rs1449683 show a tendency towards the development of the disease and the same variants associated significantly with higher genetic risk and with reduced disease free survival. Moreover, the association persisted in the early-stage disease subgroup. Our study provides evidence of the protective effect of the T/- rs3025039 VEGFA variant against B-CLL development and the association of CT/TT variants of the rs1449683 bFGF gene with genetic risk and an adverse survival.

TAK1 suppresses RIPK1-dependent cell death and is associated with disease progression in melanoma.

Melanoma cells are highly resistant to conventional genotoxic agents, and BRAFV600/MEK-targeted therapies as well as immunotherapies frequently remain inefficient. Alternative means to treat melanoma, in particular through the induction of programmed cell death modalities such as apoptosis or necroptosis, therefore still need to be explored. Here, we report that melanoma cell lines expressing notable amounts of RIPK1, RIPK3 and MLKL, the key players of necroptosis signal transduction, fail to execute necroptotic cell death. Interestingly, the activity of transforming growth factor β-activated kinase 1 (TAK1) appears to prevent RIPK1 from contributing to cell death induction, since TAK1 inhibition by (5Z)-7-Oxozeaenol, deletion of MAP3K7 or the expression of inactive TAK1 were sufficient to sensitize melanoma cells to RIPK1-dependent cell death in response to TNFα or TRAIL based combination treatments. However, cell death was executed exclusively by apoptosis, even when RIPK3 expression was high. In addition, TAK1 inhibitor (5Z)-7-Oxozeaenol suppressed intrinsic or treatment-induced pro-survival signaling as well as the secretion of cytokines and soluble factors associated with melanoma disease progression. Correspondingly, elevated expression of TAK1 correlates with reduced disease free survival in patients diagnosed with primary melanoma. Overall, our results therefore demonstrate that TAK1 suppresses the susceptibility to RIPK1-dependent cell death and that high expression of TAK1 indicates an increased risk for disease progression in melanoma.

Testes-specific protease 50 as an independent risk factor for poor prognosis in patients with non-small cell lung cancer.

Testes-specific protease 50 (TSP50) is normally expressed in the testes and is overexpressed in various types of human cancers, including breast cancer, colorectal carcinoma and laryngocarcinoma. However, little has been reported on the association between TSP50 and non-small cell lung cancer (NSCLC). The present study aimed to detect TSP50 expression in 198 strict follow-up cases of paired NSCLC and 15 cases of normal lung parenchymal specimens using immunohistochemical staining. The expression levels of TSP50 were then correlated with the clinicopathological factors of NSCLC to assess its potential diagnostic and prognostic value. The relationship between TSP50 expression and the clinicopathological parameters of NSCLC was evaluated using χ2 and Fisher's exact tests. Survival rates for the overall population (n=198) were calculated using the Kaplan-Meier method, and univariate and multivariate analyses were performed using the Cox's proportional hazards regression model. P<0.05 was considered to indicate a statistically significant difference. The expression of TSP50 was significantly increased in NSCLC tissue compared with in adjacent non-tumor or normal lung parenchymal tissue (P<0.001). A significant association was revealed between high expression levels of TSP50 and clinicopathological characteristics including tumor differentiation (P=0.012), late tumor status (P=0.004) and late tumor node metastasis stage (P=0.026), as well as a reduced disease free survival (P=0.009) and overall survival rate (P=0.002) in all patients with NSCLC. Multivariate analyses demonstrated that high TSP50 expression in tumor tissues was significantly associated with a shorter disease-free survival rate [hazard ratio (HR) =1.590, 95% confidence interval (CI): 1.035-2.441], and with a shorter overall survival rate (HR=1.814; 95% CI: 1.156-2.846). In conclusion, the present data demonstrated that increased TSP50 protein expression may be a potential predictor of early recurrence and poor prognosis in NSCLC, and that TSP50 expression levels possess the potential to be used as a biomarker and therapeutic target for the treatment of patients with NSCLC.

\u03b2-lapachone suppresses tumour progression by inhibiting epithelial-to-mesenchymal transition in NQO1-positive breast cancers

NQO1 is a FAD-binding protein that can form homodimers and reduce quinones to hydroquinones, and a growing body of evidence currently suggests that NQO1 is dramatically elevated in solid cancers. Here, we demonstrated that NQO1 was elevated in breast cancer and that its expression level was positively correlated with invasion and reduced disease free survival (DFS) and overall survival (OS) rates. Next, we found that β-lapachone exerted significant anti-proliferation and anti-metastasis effects in breast cancer cell lines due to its effects on NQO1 expression. Moreover, we revealed that the anti-cancer effects of β-lapachone were mediated by the inactivation of the Akt/mTOR pathway. In conclusion, these results demonstrated that NQO1 could be a useful prognostic biomarker for patients with breast cancer, and its bioactivatable drug, β-lapachone represented a promising new development and an effective strategy for indicating the progression of NQO1-positive breast cancers.

Treatment of locally advanced parotid malignancies with parotidectomy and temporal bone resection

PurposeIn this study we review our institution's experience and outcomes with temporal bone resection and parotidectomy in the treatment of advanced parotid malignancies. MethodsPatients undergoing lateral temporal bone resection and parotidectomy from 2007–2013 were identified in the EPIC electronic medical record. Primary tumor location, staging, surgical procedure, and patient demographic and outcome data were collected retrospectively. ResultsFifteen patients underwent combined temporal bone resection and parotidectomy for parotid malignancy. Carcinoma ex-pleomorphic and squamous cell carcinoma were the most common pathologies. Two year disease free survival was 40%. Distant metastases were the most common site of disease recurrence. Only nodal disease was predictive of reduced disease free survival, though pre-operative facial paralysis showed a trend towards significance. Margin status and operating for recurrent tumor did not influence outcome in our series. ConclusionLocal and regional tumor controls are attainable with combined skull base approaches to advanced parotid malignancies. Unfortunately these cases have a high rate of distant recurrence despite negative margins and local control.

Abstract P1-01-03: Dynamics of circulating tumor cells during the course of chemotherapy and prognostic relevance across molecular subtypes in high-risk early breast cancer patients – Results from the adjuvant SUCCESS A trial

Abstract Background: The presence of circulating tumor cells (CTCs) before chemotherapy is known to be associated with reduced disease free survival (DFS) and overall survival (OS) in early breast cancer (EBC). In addition, recent findings suggest that CTCs persisting after adjuvant chemotherapy indicate poor prognosis. In an explorative analysis of the SUCCESS A trial, we evaluated the prognostic relevance of changes in CTC counts during the course of adjuvant chemotherapy across molecular subtypes to assess whether the prognostic role of persisting CTCs varies according to tumor biology. Methods: The SUCCESS A trial is a phase III study, where patients with high-risk EBC (stage pN1-3 or pT2-4 or grade 3 or age ≤ 35 or hormone-receptor negative) were randomized to adjuvant chemotherapy with 3 cycles of epirubicin-fluorouracil-cyclophosphamide followed by either 3 cycles of docetaxel or 3 cycles of gemcitabine-docetaxel. CTC enumeration was performed before and after chemotherapy using the FDA-approved CellSearch® System (Janssen Diagnostics, LLC), and CTC positivity was defined as ≥ 1 CTC in 23 ml blood. Molecular subtypes were defined as luminal A like (hormone-receptor positive, grading 1 or 2), luminal B like (hormone-receptor positive, grading 3), triple-negative or HER2-positive. Patient outcome in terms of DFS and OS was analyzed using univariate log-rank tests and Cox regression models (median follow-up time 65.2 months). Results: Data on both molecular subtypes and CTC status before and after chemotherapy were available for 1485 (39.6%) of 3754 patients randomized. This cohort contained 577 (38.9%) luminal A like, 236 (15.9%) luminal B like, 379 (25.5%) HER2-positive and 293 (19.7%) triple negative tumors. Overall, 917 (61.8%) patients were CTC negative before and after chemotherapy (neg/neg), 260 (17.5%) patients had a negative CTC status before and a positive CTC status after chemotherapy (neg/pos), 229 (15.4%) patients converted from positive to negative CTC status (pos/neg), and 79 (5.3%) patients were positive for CTCs at both time points (pos/pos). There were significant differences in DFS and OS among these four groups in patients with luminal A like tumors (log rank test, both p &amp;lt; 0.003) and patients with luminal B like tumors (log rank test, both p &amp;lt; 0.001). In both patients with luminal A like or luminal B like tumors, persistently CTC positive patients had the worst outcome (relative to persistently CTC-negative patients) in terms of DFS and OS. In contrast to luminal-like tumors, no significant differences with regard to DFS or OS were found among the four groups (neg/neg, neg/pos, pos/neg, pos/pos) in patients with HER2-positive or triple-negative tumors (log rank test, all p &amp;gt; 0.13). Conclusion: The presence of CTCs both before and after adjuvant chemotherapy was associated with poor survival in luminal A like and luminal B like tumors, but not in HER2-positive or triple-negative tumors. Further research is needed to evaluate the effect of chemotherapy on CTC prevalence in different molecular subtypes of EBC. Citation Format: Tzschaschel MLJ, Rack B, Andergassen U, Friedl TWP, Schneeweiss A, Mueller V, Tanja F, Pantel K, Gade J, Lorenz R, Rezai M, Tesch H, Soeling U, Polasik A, Alunni-Fabbroni M, Trapp EK, Mahner S, Schindlbeck C, Lichtenegger W, Beckmann MW, Fasching PA, Janni W. Dynamics of circulating tumor cells during the course of chemotherapy and prognostic relevance across molecular subtypes in high-risk early breast cancer patients – Results from the adjuvant SUCCESS A trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-03.

P2.02-047 Association of FDG PET, Complete Pathological Response and Overall Survival in Patients with Pancoast Tumors Treated with Trimodality Therapy

Induction chermoradiotherapy (CRT) and surgical resection is considered standard care for treatment of node negative Pancoast tumors. However, not all patients benefit from this approach and there are no well-defined preoperative parameters to identify patients for whom addition of surgery may be unnecessary due to local control with CRT alone. We investigated whether baseline FDG positron emission tomography (PET) scan parameters or changes post induction may predict complete pathological response (pCR) to CRT and hence obviate the need for subsequent resection. We conducted a retrospective review of our prospectively maintained single institution database with supplemental chart review to evaluate: PET, histopathological, and clinical outcome parameters in consecutive patients undergoing curative intent trimodality treatment of Pancoast tumors from 2001 to 2015. Metabolic parameters based on the standardized uptake values (SUV) were calculated including SUVmax (maximum SUV value), SUVPTL (peak tumor-to-liver ratio), and TGV (total glycolytic volume, mean SUV x tumor volume). Two pathologists independently reviewed specimens to assess percentage viable tumor in resected tumors. Nineteen patients (10 females), median age 61 (42-75) yrs completed trimodality treatment with Cisplatin, Etoposide and 45Gy in the majority of cases. Histopathological was data available for all patients. Of the 19 patients baseline PET was available in 15 and post induction PET in 13. Baseline SUVmax < 9.4 was associated with pCR in 4/4 vs. 4/11 patients (p=0.03). A trend towards improved locoregional control with cPR did not reach significance. No PET measured parameter was associated with locoregional control. Baseline TGV > 441 was associated with reduced disease free survival (DFS) 5.7(0.7-10.5) vs. NR months (p=0.002), and overall survival (OS) 15.9 (3.1-28.8) vs. NR months (p=0.04). No change in PET parameter measured post induction was associated with cPR, local recurrence, DFS or OS. In our series, low baseline SUVmax was associated with complete pathological response. If confirmed in a larger cohort, including multivariate analysis of other prognostic and predictive factors, this may allow patients at high perioperative risk to be better stratified to either definitive CRT or trimodality therapy. A larger series may be able to identify an association between metabolic response on FDG-PET and CPR, DFS or overall survival.

Prognostic relevance of SAMSN1 expression in gastric cancer.

The prognosis for patients with advanced gastric cancer (GC) remains poor. The identification of biomarkers relevant to the recurrence and metastasis of GC is advantageous for stratifying patients and proposing novel molecular targets. In the present study the oncological roles of SAM domain, SH3 domain and nuclear localization signals 1 (SAMSN1), a mediator of B-cell function, were elucidated in GC. The expression and methylation status of SAMSN1 were investigated in a panel of 11 GC cell lines. Immunohistochemical staining was performed to determine the pattern of SAMSN1 protein expression in gastric tissues. The prognostic impact of SAMSN1 expression was determined by analyzing 175 pairs of surgically resected gastric tissues. A marked decrease in the level of SAMSN1 mRNA was detected in 8/11 GC cell lines as compared with that in a non-transformed intestinal epithelium cell line (FHs 74) without promoter methylation. The mean expression level of SAMSN1 mRNA was reduced in GC tissues compared with normal adjacent tissues, an observation that was independent of tumor differentiation. The pattern of SAMSN1 protein expression was significantly correlated with that of SAMSN1 mRNA. Low SAMSN1 mRNA expression was significantly associated with tumor size (>60 mm; P=0.026) and shorter overall survival times (P=0.004). Multivariate analysis identified low SAMSN1 mRNA expression as an independent prognostic factor for poor overall survival (hazard ratio, 1.80; 95% confidence interval, 1.07-3.05; P=0.025). The difference in survival between the low and high SAMSN1 expression groups was more marked in patients with stage II/III GC compared to those with stage IV GC. In patients with stage II/III GC who underwent curative surgery, low SAMSN1 expression was associated with reduced disease free survival times. The results of the present study indicate that downregulation of SAMSN1 transcription may affect the progression and recurrence of GC, and therefore may represent a novel biomarker of GC.

Hepatic vascular inflow occlusion is associated with reduced disease free survival following resection of colorectal liver metastases

BackgroundHepatic vascular inflow occlusion (VIO) can be applied during resection of colorectal liver metastases (CRLM) to control intra-operative blood loss, but has been linked to accelerated growth of micrometastases in experimental models. This study aimed to investigate the effects of hepatic VIO on disease-free and overall survival (DFS and OS) in patients following resection for CRLM. MethodsAll patients who underwent liver resection for CRLM between January 2006 and September 2015 at our center were analyzed. Hepatic VIO was performed if deemed indicated by the operating surgeon and severe ischemia was defined as ≥20 min continuous or ≥45 min cumulative intermittent VIO. Cox regression analysis was performed to identify predictive factors for DFS and OS. ResultsA total of 208 patients underwent liver resection for CRLM. VIO was performed in 64 procedures (31%), and fulfilled the definition of severe ischemia in 40 patients. Patients with severe ischemia had inferior DFS (5-year DFS 32% vs. 11%, P < 0.01), and inferior OS (5-year OS 37% vs. 64%, P < 0.01). At multivariate analysis, a high clinical risk score (Hazard ratio (HR) 1.60 (1.08–2.36)) and severe ischemia (HR 1.89 (1.21–2.97)) were independent predictors of worse DFS. Severe ischemia was not an independent predictor of OS. ConclusionThe present cohort study suggests that prolonged hepatic VIO during liver resection for CRLM was associated with reduced DFS. A patient-tailored approach seems advisable although larger studies should confirm these findings.

Nested quantitative PCR approach for urinary cell-free EZH2 mRNA and its potential clinical application in bladder cancer.

EZH2 is overexpressed in bladder cancer (BC) and plays important roles in tumor development and progression. Recent studies show cell free (cf) RNAs released from cancer cells can reflect tissues changes and are stable and detectable in urine. Although conventional quantitative real-time PCR (qPCR) is highly sensitive, low abundances of urinary cf-RNAs usually result in false-negatives. Thus, this study develops a nested qPCR (nqPCR) approach to quantify cf-EZH2 mRNA in urine and further assess its clinical significance for BC. Forty urine samples were first selected to evaluate feasibility of nqPCR. Then, levels of urinary cf-EZH2 mRNA were detected using developed method in an independent cohort of subjects with 91 healthy, 81 cystitis, 169 nonmuscle invasive BC (NMIBC) and 103 muscle-invasive BC (MIBC). In cf-EZH2 mRNA detection, nqPCR method was significantly associated with qPCR, but it could detect more urine samples and increase detection limit three orders of magnitude. Based on nqPCR method, cf-EZH2 mRNA levels have been found to be increased in urine of NMIBC and MIBC patients (p < 0.001). Compared with cytology, cf-EZH2 mRNA showed higher diagnostic ability for MIBC (p < 0.001) while not for NMIBC (p > 0.05). Moreover, it also could distinguish MIBC from NMIBC, with AUC of 0.787. For MIBC patients, high expression of cf-EZH2 mRNA associated with advanced stage and was an independent predictor of reduced disease free survival or overall survival. In conclusion, detection of cf-EZH2 mRNA in urine by nqPCR is a sensitive and noninvasive approach and may be used for diagnosis and prognosis prediction of MIBC.

End Binding 1 (EB1) overexpression in oral lesions and cancer: A biomarker of tumor progression and poor prognosis

IntroductionOral squamous cell carcinoma (OSCC) patients are at high risk of loco-regional recurrence and despite the improvement in treatment strategy, 5-year survival rates are about 50%. Identification of patients at high risk of recurrence may enable rigorous personalized post-treatment management. In an earlier proteomics study we observed overexpression of End Binding Protein (EB1) in OSCC. In the present study we investigated the diagnostic and prognostic significance of alterations in expression of EB1 in oral cancer. MethodsIn this retrospective study, the expression of EB1 protein was evaluated in 259 OSCCs, 41 dysplasia, 166 hyperplasia and 126 normal tissues using immunohistochemistry and correlated with clinical-pathological parameters and prognosis of OSCC patients over a follow-up period of up to 91months. ResultsSignificantly higher expression of cytoplasmic EB1 was observed in hyperplasia [p<0.001, OR=7.2, 95% CI=4.1–12.8], dysplasia (p<0.001, OR=21.8, CI=8.8–50.2) and OSCCs (p<0.001, OR=10.1, CI=5.8–17.4) in comparison with normal mucosa. Univariate analysis revealed cytoplasmic EB1 association with tumor grade, tumor size and recurrence of the disease. Kaplan Meier survival analysis of EB1 expression showed significantly reduced disease free survival (DFS) (p=0.003). Notably, OSCC patients showing cytoplasmic EB1 overexpression demonstrated significantly reduced DFS (p=0.004, HR=2.1). ConclusionEB1 overexpression is an early event in oral tumorigenesis and cytoplasmic EB1 accumulation is associated with poor prognosis and tumor recurrence in OSCC patients.

Role of PRL-3, Snail, Cytokeratin and Vimentin expression in epithelial mesenchymal transition in breast carcinoma.

To study epithelial mesenchymal transition (EMT) in breast cancer, molecules such as PRL-3, Snail, Cytokeratin and Vimentin involved in EMT were evaluated. In this study, m-RNA expression of PRL3 and Snail by RT PCR, protein expression of PRL-3, Snail, Cytokeratin and Vimentin by immunohistochemistry were evaluated on paraffin-embedded tissue sections of 100 patients with breast cancer. PRL3 m-RNA expression (above cut off level > 2487301.00) and PRL-3 protein expression was noted in 52% and 70% of breast carcinoma patients, respectively. The higher incidence of PRL3 protein than m-RNA expression could be due to post translation modification. Further, Snail m-RNA expression (above cut off level > 1285142.00) and Snail protein expression was noted in 53% and 54% of breast cancer patients respectively and Snail protein expression was found significantly higher in patients with pre-menopausal status. The loss of cytokeratin expression in 32% and gain of vimentin expression in 17% was noted in these patients. Vimentin expression was found significantly higher in patients with stage IV disease, BR score 4 and PR negativity. In multivariate survival analysis, Vimentin expression found as strong indicator of biologically aggressive breast cancer predicting reduced disease free survival (DFS) and overall survival (OS). In our study reveals that Vimentin expression emerged as significant biomarker for predicting reduced DFS and OS in breast cancer. The study proposes routine evaluation of Vimentin with other predictive parameters can allow use of EMT inhibitors with conventional therapy to revert EMT in breast cancer.

Abstract P4-01-07: Different prognostic value of circulating and disseminated tumor cells in primary breast cancer. Influence of bisphosphonate intake?

Abstract Background: Disseminated tumor cells (DTCs) in the bone marrow and circulating tumor cells (CTCs) in blood of breast cancer patients (pts) were shown to provide independent prognostic information and can be regarded as an early indicator of therapy failure. Here we demonstrate a different prognostic value of DTCs and CTCs and explain these findings by early bisphosphonate intake. Patients and Methods: 10 ml blood and two bone marrow aspirates of 488 pts with first diagnosis of breast cancer between Aug 2006 and Dec 2010 were studied for DTCs and CTCs. CTCs were detected using the AdnaTest BreastCancer (AdnaGen AG, Hannover, Germany) for the detection of EpCAM, MUC-1 and HER2 transcripts. DTCs were analyzed by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. In addition to chemo-, radio- and anti-hormonal therapy, bisphosphonates were given to DTC-pos pts [clodronate (2×520 mg per day) for at least two years] and 69 DTC-neg, postmenopausal pts [zoledronic acid (4 mg, twice a year) for three years]. Results: After a median follow-up time of 48 months (range: 5 to 87 months), the overall survival rate was 93% (456/488 pts) and relapses occurred in 11% (56/488 pts) of cases. CTCs were detected in 109/488 pts (22%) and significantly correlated with reduced disease free survival (DFS; p=0.02). Negative prognostic relevance was predominantly related to the lobular subtype (p=0.0047), the ER-pos subtype, (pER+/Her2=0.01; pER+/Her2+=0.25; pER-/Her2+=0.5) and pts who had received chemotherapy (p=0.015) or radiation therapy (p=0.0083) but not to pts with anti-hormonal regimens (p=0.06). DTCs were detected in 162/488 pts (33%). In contrast to CTCs, no prognostic significance could be shown with regard to DFS (p=0.49), all clinical parameters as well as treatment regimens. In contrast, only in CTC-pos pts, the presence of DTCs significantly correlated with DFS (p=0.04). We further investigated whether this lack in prognostic significance was due to bisphosphonate intake in case of DTC-positivity. Predominantly, in the CTC-neg group, bisphosphonate treatment significantly influenced DFS in both histological subtypes (pCTC-/ductal=0.04; pCTC-/lobular=0.0021). However, in the CTC-pos group of pts, no such effect was observed (pCTC+/ductal=0.76; pCTC+/lobular=0.51).Conclusion: Here we show, that CTCs, rather than DTCs were significantly prognostic for early relapse. The ER-pos subtype was mostly affected which is in concordance with our previous studies, demonstrating that CTCs show EMT and tumor stem cell characteristics and, thus, down regulate ER which probably makes anti-hormonal treatment less effective. In addition, the presence of these CTC-subtypes might also explain the negative prognostic impact of CTCs in pts receiving chemo- or radiation therapy which we could verify in a subgroup of our pts (n=238) showing a significant correlation of CTCs and EMT/stem cell like CTCs (p=0.017; data not shown). The lack of prognostic significance of DTCs can be related to bisphosphonate intake in all DTC-pos pts, a subgroup of DTC-neg pts as well as in DTC-pos/CTC-neg pts, underlining our assumption that CTCs might be a high risk indicator for already ongoing metastasis not limited to bone metastasis. Citation Format: Sabine Kasimir-Bauer, Bahriye Aktas, Siegfried Hauch, Rainer Kimmig, Oliver Hoffmann. Different prognostic value of circulating and disseminated tumor cells in primary breast cancer. Influence of bisphosphonate intake? [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-07.

PDGFRβ and FGFR2 mediate endothelial cell differentiation capability of triple negative breast carcinoma cells

Triple negative breast cancer (TNBC) is a very aggressive subgroup of breast carcinoma, still lacking specific markers for an effective targeted therapy and with a poorer prognosis compared to other breast cancer subtypes.In this study we investigated the possibility that TNBC cells contribute to the establishment of tumor vascular network by the process known as vasculogenic mimicry, through endothelial cell differentiation.Vascular-like functional properties of breast cancer cell lines were investigated in vitro by tube formation assay and in vivo by confocal microscopy, immunofluorescence or immunohistochemistry on frozen tumor sections. TNBCs express endothelial markers and acquire the ability to form vascular-like channels in vitro and in vivo, both in xenograft models and in human specimens, generating blood lacunae surrounded by tumor cells. Notably this feature is significantly associated with reduced disease free survival. The impairment of the main pathways involved in vessel formation, by treatment with inhibitors (i.e. Sunitinib and Bevacizumab) or by siRNA-mediating silencing, allowed the identification of PDGFRβ and FGFR2 as relevant players in this phenomenon. Inhibition of these tyrosine kinase receptors negatively affects vascular lacunae formation and significantly inhibits TNBC growth in vivo.In summary, we demonstrated that TNBCs have the ability to form vascular-like channels in vitro and to generate blood lacunae lined by tumor cells in vivo. Moreover, this feature is associated with poor outcome, probably contributing to the aggressiveness of this breast cancer subgroup. Finally, PDGFRβ and FGFR2-mediated pathways, identified as relevant in mediating this characteristic, potentially represent valid targets for a specific therapy of this breast cancer subgroup.