Transcriptomics-based liquid biopsy panel for early non-invasive identification of peritoneal recurrence and micrometastasis in locally advanced gastric cancer.

BACKGROUND: This study aimed to develop a novel six-gene expression biomarker panel to enhance the early detection and risk stratification of peritoneal recurrence and micrometastasis in locally advanced gastric cancer (LAGC). METHODS: We used genome-wide transcriptome profiling and rigorous bioinformatics to identify a six-gene expression biomarker panel. This panel was validated across multiple clinical cohorts using both tissue and liquid biopsy samples to predict peritoneal recurrence and micrometastasis in patients with LAGC. RESULTS: Through genome-wide expression profiling, we identified six mRNAs and developed a risk prediction model using 196 samples from a surgical specimen training cohort. This model, incorporating a 6-mRNA panel with clinical features, demonstrated high predictive accuracy for peritoneal recurrence in gastric cancer patients, with an AUC of 0.966 (95% CI: 0.944-0.988). Transitioning from invasive surgical or endoscopic biopsy to noninvasive liquid biopsy, the model retained its predictive efficacy (AUC = 0.963; 95% CI: 0.926-1.000). Additionally, the 6-mRNA panel effectively differentiated patients with or without peritoneal metastasis in 95 peripheral blood specimens (AUC = 0.970; 95% CI: 0.936-1.000) and identified peritoneal micrometastases with a high efficiency (AUC = 0.941; 95% CI: 0.874-1.000). CONCLUSIONS: Our study provides a novel gene expression biomarker panel that significantly enhances early detection of peritoneal recurrence and micrometastasis in patients with LAGC. The RSA model's predictive capability offers a promising tool for tailored treatment strategies, underscoring the importance of integrating molecular biomarkers with clinical parameters in precision oncology.

Outcomes in Premenopausal Patients with HR+/HER2- Breast Cancer and Lymph Node Micrometastasis Based on the 21-Gene Recurrence Score.

BACKGROUND: Postmenopausal patients with hormone receptor positive, HER2-negative (HR+/HER2-) early breast cancer (EBC) and 21-gene OncotypeDX (ODX) recurrence scores (RS) <26 do not benefit from chemoendocrine therapy ("CET") compared to endocrine monotherapy ("E"), regardless of nodal status. In premenopausal patients, nodal status is significant in interpretation of RS. However, guidelines are not explicit in recommendations for patients with micrometastasis ("pN1mi" staging). METHODS: A cohort of patients aged <50 years with HR+/HER2- EBC who underwent ODX testing was identified within the National Cancer Database 2004-2019 dataset. We confirmed the prognostic value of ODX in pN1mi disease with multivariate Cox regression for overall survival (OS). We explored how patterns of practice differed by nodal status in cases of low RS (<26) with chi-squared testing. Finally, we performed Kaplan-Meier models comparing OS for those with RS <26 receiving E versus CET, controlling for nodal status. RESULTS: Of 72 068 patients aged <50 years with HR+/HER2- EBC, 6.1% (n = 4402) had micrometastasis. Multivariate Cox regression confirmed prognostic value of ODX in this pN1mi cohort (P < .001). In the context of RS <26, CET was used most commonly in patients with 1-3 involved lymph nodes ("pN1a-c" disease), less frequently in pN1mi disease, and least in node-negative ("pN0") disease. A benefit in OS was observed in cases with RS <26 and pN1a-c receiving CET vs. E (P = .017), but not in pN1mi (P = .49) or pN0 (P = .57) disease. CONCLUSION: Our large registry analysis found CET was associated with improved OS in pN1a-c, but not in pN1mi or pN0 disease.

Combination of CEACAM5, EpCAM and CK19 gene expressions in mediastinal lymph node micrometastasis is a prognostic factor for non-small cell lung cancer.

BACKGROUND: Lung cancer is known as the most common and highly metastatic form of cancer worldwide. Tumour node metastasis (TNM) staging is the gold standard classification system for the decision-making process for appropriate treatment. Particularly N status has the most important prognostic value in the absence of distant metastasis. Traditional diagnostic methods are capable of detecting metastasis; however, they may fail to detect micrometastasis, which plays a role in disease recurrence and patients' long-term survival. Occult micrometastasis can change the tumour's TNM staging and, consequently, the patient's treatment regimen. METHODS: The median number of three lymph node tissues were collected from 30 patients who underwent surgery for non-small cell lung cancer. Lymph node tissues were collected from different lymph node stations according to the location of the patient's tumour. CK19, EpCAM and CEACAM5 gene expressions were analysed in tissues using quantitative real-time polymerase chain reaction to detect micrometastasis in distant lymph nodes. RESULTS: Triple positivity was seen in 26 out of 30 patients which 19 patients were upstaged from N0 to N2. While survival was not significantly affected between upstaged and non-upstaged patients, patients upstaged with multiple-station N2 had a significantly higher recurrence and lower survival compared to single-station N2. CONCLUSION: A combination of CK19, EpCAM and CEACAM5 gene expressions in lymph nodes can be used to identify micrometastasis which postoperatively may be used as a tool to predict patients' recurrence and survival.

Curative-intent Metastasis-directed Therapies for Molecularly-defined Oligorecurrent Prostate Cancer: A Prospective Phase II Trial Testing the Oligometastasis Hypothesis.

BACKGROUND: The hypothesis of a curable oligometastatic prostate cancer (PCa) state remains to be clinically-proven. Conventional imaging often fails to localize early recurrences, hampering the potential for radical approaches. OBJECTIVE: We hypothesize that prostate-specific membrane antigen (PSMA)-targeted PET-MR/CT allows for earlier detection and localization of oligorecurrent-PCa, unveiling a molecularly-defined state amenable to curative-intent metastasis-directed treatment (MDT). DESIGN/SETTING/PARTICIPANTS: Single-institution single-arm phase-two study. Patients with rising PSA (0.4-3.0 ng/mL) after maximal local therapy (radical prostatectomy and post-operative radiotherapy), negative conventional staging, and no prior salvage hormonal therapy (HT) were eligible. INTERVENTIONS: All patients underwent [18F]DCFPyL PET-MR/CT. Patients with molecularly-defined oligorecurrent-PCa had MDT (stereotactic ablative body radiotherapy [SABR] or surgery) without HT. OUTCOME MEASUREMENTS/STATISTICAL ANALYSIS: Primary endpoint was biochemical response (complete, i.e. biochemical 'no evidence of disease' [bNED], or partial response [100% or ≥50% PSA decline from baseline, respectively]) after MDT. Simon's two-stage design was employed (null and alternate hypotheses <5% and >20% response rate, respectively), with α and ß of 0.1. RESULTS: Seventy-two patients were enrolled (May/2017-July/2019). Thirty-eight (53%) had PSMA-detected oligorecurrent-PCa amenable for MDT. Thirty-seven (51%) agreed to MDT: 10 and 27 underwent surgery and SABR, respectively. Median follow-up was 15.9 months (IQR 9.8-19.1). Of patients receiving MDT, the overall response rate was 60%, including 22% rendered bNED. One (2.7%) grade 3 toxicity (intra-operative ureteric injury) was observed. CONCLUSIONS: PSMA-defined oligorecurrent-PCa can be rendered bNED, a necessary step towards cure, in 1 of 5 patients receiving MDT alone. Randomized trials are justified to determine if MDT +/- systemic agents can expand the curative therapeutic armamentarium for PCa. PATIENT SUMMARY: We studied men treated for prostate cancer with rising PSA. We found PSMA imaging detected recurrent cancer in three-quarters of patients, and targeted treatment to these areas significantly decreased PSA in half of patients.

Transcriptomic Profiling Identifies a Risk Stratification Signature for Predicting Peritoneal Recurrence and Micrometastasis in Gastric Cancer.

PURPOSE: Gastric cancer peritoneal carcinomatosis is fatal. Delay in detection of peritoneal metastases contributes to high mortality, highlighting the need to develop biomarkers that can help identify patients at high risk for peritoneal recurrence or metastasis. EXPERIMENTAL DESIGN: We performed a systematic discovery and validation for the identification of peritoneal recurrence prediction and peritoneal metastasis detection biomarkers by analyzing expression profiling datasets from 249 patients with gastric cancer, followed by analysis of 426 patients from three cohorts for clinical validation. RESULTS: Genome-wide expression profiling identified a 12-gene panel for robust prediction of peritoneal recurrence in patients with gastric cancer (AUC = 0.95), which was successfully validated in a second dataset (AUC = 0.86). Examination of 216 specimens from a training cohort allowed us to establish a six gene-based risk-prediction model [AUC = 0.72; 95% confidence interval (CI): 0.66-0.78], which was subsequently validated in an independent cohort of 111 patients with gastric cancer (AUC = 0.76; 95% CI: 0.67-0.83). In both cohorts, combining tumor morphology and depth of invasion further improved the predictive accuracy of the prediction model (AUC = 0.84). Thereafter, we evaluated the performance of the identical six-gene panel for its ability to detect peritoneal metastasis by analyzing 210 gastric cancer specimens (prior 111 patients plus additional 99 cases), which discriminated patients with and without peritoneal metastasis (AUC = 0.72). Finally, our biomarker panel was also remarkably effective for identifying peritoneal micrometastasis (AUC = 0.72), and its diagnostic accuracy was significantly enhanced when depth of invasion was included in the model (AUC = 0.85). CONCLUSIONS: Our novel transcriptomic signature for risk stratification and identification of high-risk patients with peritoneal carcinomatosis might serve as an important clinical decision making in patients with gastric cancer.

Impact of 21-Gene Recurrence Score on Chemotherapy Decision in Invasive Ductal Carcinoma of Breast with Nodal Micrometastases.

PURPOSE: The purpose of this study was to investigate the effect of 21-gene recurrence score (RS) on predicting prognosis and chemotherapy decision in node micrometastases (N1mi) breast invasive ductal carcinoma (IDC). Methods Patients with stage T1-2N1mi and estrogen receptor-positive IDC diagnosed between 2004 and 2015 were included. The associations of 21-gene RS with breast cancer-specific survival (BCSS), chemotherapy decision, and benefit of chemotherapy were analyzed. RESULTS: We identified 4,758 patients including 1,403 patients (29.5%) treated with adjuvant chemotherapy. In the traditional RS cutoffs, 2,831 (59.5%), 1,634 (34.3%), and 293 (6.2%) patients were in the low-, intermediate-, and high-risk RS groups, respectively. In 3,853 patients with human epidermal growth factor receptor-2 (HER2) status available, most patients were HER2-negative disease (98.3%). A higher RS was independently related to chemotherapy receipt, and 14.0%, 47.7%, and 77.8% of patients in the low-, intermediate-, and high-risk RS groups received chemotherapy, respectively. The multivariate analysis indicated that a higher RS was related to worse BCSS (p < 0.001). The 5-year BCSS rates were 99.3%, 97.4%, and 91.9% in patients with low-, intermediate-, and high-risk RS groups, respectively (p < 0.001). However, chemotherapy receipt did not correlate with better BCSS in low-, intermediate-, or high-risk RS groups. There were similar trends using Trial Assigning Individualized Options for Treatment RS cutoffs. CONCLUSION: The 21-gene RS does predict outcome and impact on chemotherapy decision of N1mi breast IDC. Large cohort and long-term outcomes studies are needed to identify the effects of chemotherapy in N1mi patients by different 21-gene RS groups.

A Nomogram for Predicting the Oncotype DX Recurrence Score in Women with T1-3N0-1miM0 Hormone Receptor‒Positive, Human Epidermal Growth Factor 2 (HER2)‒Negative Breast Cancer.

PURPOSE: This preliminary study was conducted to evaluate the association between Oncotype DX (ODX) recurrence score and traditional prognostic factors. We also developed a nomogram to predict subgroups with low ODX recurrence scores (less than 25) and to avoid additional chemotherapy treatments for those patients. MATERIALS AND METHODS: Clinicopathological and immunohistochemical variables were retrospectively retrieved and analyzed from a series of 485 T1-3N0-1miM0 hormone receptor-positive, human epidermal growth factor 2‒negative breast cancer patients with available ODX test results at Asan Medical Center from 2010 to 2016. One hundred twenty-seven patients (26%) had positive axillary lymph node micrometastases, and 408 (84%) had ODX recurrence scores of ≤25. Logistic regression was performed to build a nomogram for predicting a low-risk subgroup of the ODX assay. RESULTS: Multivariate analysis revealed that estrogen receptor (ER) score, progesterone receptor (PR) score, histologic grade, lymphovascular invasion (LVI), and Ki-67 had a statistically significant association with the low-risk subgroup. With these variables, we developed a nomogram to predict the low-risk subgroup with ODX recurrence scores of ≤25. The area under the receiver operating characteristic curve was 0.90 (95% confidence interval [CI], 0.85 to 0.96). When applied to the validation group the nomogram was accurate with an area under the curve = 0.88 (95% CI, 0.83 to 0.95). CONCLUSION: The low ODX recurrence score subgroup can be predicted by a nomogram incorporating five traditional prognostic factors: ER, PR, histologic grade, LVI, and Ki-67. Our nomogram, which predicts a low-risk ODX recurrence score, will be a useful tool to help select patients who may or may not need additional ODX testing.

Molecular Staging of Patients with Colon Cancer. The C-Closer-II Study: A Multicentre Study in Portugal.

INTRODUCTION: Approximately 20% - 30% of histological lymph node-negative patients with colorectal cancer relapse at five years after surgical treatment. This recurrence is likely due to occult nodal disease undetected by standard histopathological practice which has implications in terms of the clinical management of patients. MATERIAL AND METHODS: Lymph nodes were collected from colectomy specimens. A central section from each lymph node was histologically examined following haematoxylin-eosin staining and the remaining tissue was subjected to OSNA - one step nucleic acid amplification analysis. RESULTS: A total of 1046 lymph nodes from 59 pN0 patients were assessed. Of these, 753 lymph nodes were examined by both methods. The median number of lymph nodes assessed with OSNA - one step nucleic acid amplification was 12 (IQR: 7;16). Among pN0 patients, 17 had OSNA - one step nucleic acid amplification-positive lymph nodes, resulting in a positive molecular staging rate of 28.8% (95% CI: 17.8 - 42.1). Among these patients, 12 (70.59%) were molecular-staged as pN1 and 5 (29.41%) were molecular staged as pN2. The tumour burden of lymph nodes assessed with OSNA - one step nucleic acid amplification ranged from 270 to 17 000 cytokeratin 19 mRNA copies/µL. Most of these patients (88.2%) were found to have lymph nodes with micrometastases only (250 - 4999 copies/µL). DISCUSSION: We provide the results from the first study of the use of the OSNA - one step nucleic acid amplification assay in colorectal cancer patients in Portugal. Our results are in-line with other international studies, showing the improvement on patients' staging by molecular examination of lymph nodes. CONCLUSION: In our study, 28.8% of patients with histologically negative lymph nodes were found to have metastatic lymph nodes using OSNA - one step nucleic acid molecular assessment. OSNA - one step nucleic acid assay allows a more accurate staging of patients with colorectal cancer and standardizes lymph node assessment.

Introdução: Cerca de 20% - 30% dos doentes com cancro colo-rectal, com gânglios linfáticos regionais negativos por histologia têm recidiva do carcinoma colo-rectal, após cinco anos do tratamento cirúrgico. Esta recorrência é provavelmente devida à presença de metástases ganglionares ocultas, não detetadas no exame anatomo-patológico standard. Material e Métodos: Os gânglios linfáticos foram obtidos a partir de espécimes de colectomia. Uma secção central de cada gânglio linfático foi analisada histologicamente com a coloração de hematoxilina-eosina e o tecido restante foi sujeito a análise de OSNA - one step nucleic acid amplification. Resultados: Um total de 1046 gânglios linfáticos de 59 doentes pN0 foram avaliados. Foram examinados 753 gânglios linfáticos por ambos os métodos. A mediana de gânglios linfáticos avaliados com OSNA - one step nucleic acid amplification foi de 12 (IQR:7; 16). Entre os doentes pN0, 17 tinham gânglios linfáticos positivos para OSNA - one step nucleic acid amplification, resultando numa taxa de estadiamento molecular positiva de 28,8% (95% CI: 17,8 - 42,1). De entre esses doentes, 12 (70,59%) apresentaram-se molecularmente pN1 e cinco (29,41%) pN2. A carga tumoral dos gânglios linfáticos avaliada com OSNA - one step nucleic acid amplification variou de 270 a 17 000 cópias/µL de ARNm de citoqueratina 19. A maioria desses doentes (88,2%) apresentou gânglios linfáticos com micrometástases (250 - 4999 cópias/µL). Discussão: Apresentamos os resultados do primeiro estudo do ensaio OSNA - one step nucleic acid amplification levado a cabo, em pacientes com cancro colo-rectal, em Portugal. Os nossos resultados estão em linha com outros estudos internacionais, demostrando uma melhoria no estadiamento dos doentes, pelo exame molecular dos gânglios linfáticos. Conclusão: Verificou-se que 28,8% dos doentes com gânglios linfáticos histologicamente negativos apresentavam doença ganglionar metastática, usando a avaliação molecular de OSNA - one step nucleic acid amplification. O ensaio OSNA - one step nucleic acid amplification permite um estadiamento mais preciso de doentes com carcinoma do colon e padroniza a avaliação dos gânglios linfáticos.

Laparoscopic surgery for early cervical squamous cell carcinoma and its effect on the micrometastasis of cancer cells.

The aim of this study was to evaluate the effect of laparoscopic radical hysterectomy on expressions of circulating tumor cells (CTCs) of cytokeratin 19 (CK19), cytokeratin 20 (CK20), and squamous cell carcinoma antigen (SCC-Ag) mRNA.We collect 78 patients with stage IA2-IIA1 cervical cancer who underwent radical hysterectomy by laparotomy or laparoscopy in our study, and 34 uterine fibroids patients and 32 healthy subjects were recruited as the positive control group and negative control group, respectively. Blood samples were taken from early-stage primary cervical squamous cell carcinoma patients. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to amplify peripheral blood CK19, CK20 and SCC-Ag from total RNA. We measured the expression of CK19, CK20, and SCC-Ag before laparoscopic radical hysterectomy, 24 hours and 30 days after surgery. Meanwhile, the expression of these markers was compared between laparoscopic and laparotomy groups.The expressions of CK19, CK20, and SCC-Ag in the experimental group before surgery were (0.0035 ±â€Š0.0018), (1.06 ±â€Š0.49), and (1.48 ±â€Š0.46), respectively, and the positive rates were 32.1%, 33.3%, and 35.9%, respectively. The expression levels of CK19, CK20, and SCC-Ag in the experimental group before surgery was significantly higher than the positive and negative control groups, and there were no significant differences between the positive and negative control groups. The expressions and positive rates of CK19, CK20, and SCC-Ag before laparoscopic radical hysterectomy were significantly lower than the stage at 24 hours after surgery (P < .05), but higher than the stage at 30 days after surgery (P > .05). There were no significant differences in CK19, CK20, and SCC-Ag expressions before surgery, 24 hours and 30 days after surgery between laparoscopic group and laparotomy group (P > .05).Both laparotomy and laparoscopic radical mastectomy tend to increase the expression of CTCs in peripheral blood, and the expressions have no differences between these 2 groups. So, the use of CK19, CK20, and SCC-Ag expression levels from peripheral blood from early stage cervical cancer radical patients before hysterectomy can aid to overcome the lack of radiographic examination and tumor markers measurement, and provide clues for postoperative treatment and prognosis determination.

Chromosomal Aberrations Associated with Sequential Steps of the Metastatic Cascade in Colorectal Cancer Patients.

BACKGROUND: Genomic information can help to identify colorectal tumors with high and low metastatic potential, thereby improving prediction of benefit of local and/or systemic treatment. Here we investigated chromosomal aberrations in relation to the different stages of the metastatic cascade: dissemination of tumor cells into the mesenteric vein, metastatic outgrowth in the liver, intravasation of the peripheral blood circulation, and development of further distant metastasis. METHODS: Peripheral and mesenteric blood from colorectal cancer patients (n = 72) were investigated for circulating tumor cells, and DNA extracted from their primary tumors was subjected to array comparative genomic hybridization profiling. The results were validated with an independent set of primary colorectal tumors (n = 53) by quantitative reverse transcription PCR. RESULTS: Mesenteric intravasation and liver metastasis were correlated with losses of chromosomes 16p (72%), 16q (27%), and 19 (54%), gain along 1q31 (45%) and 20q (60%), tumor cell infiltration into the peripheral blood circulation, and further distant metastasis with gain of chromosome 8q (59%) and 12 (47%, P < 0.01). Chromosome 12 gain was associated with poor overall survival in the initial (2.8 vs >7 years) and validation cohort (3.3 vs >6 years). The prospective study presented here is a hypothesis-generating study and confirmation with larger cohorts is required. CONCLUSIONS: This is the first study that investigated colorectal cancer in its different stages of metastasis in correlation with copy number changes of the primary tumor. This information might be helpful to identify patients with limited metastatic spread who may profit from liver metastasis resection and may lead to the discovery of new therapeutic targets.Microarray data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE82228.

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited. We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2Rγnull (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

Signal transducer and activator of transcription 3 overexpression promotes lymph node micrometastasis in early-stage non-small cell lung cancer.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in several malignancies. Here, we define the correlation between STAT3 expression and lymph node micrometastasis of early-stage non-small cell lung cancer. Then we highlight some possibilities associated with developing a way to detect tumor micrometastasis and an anticancer drug that might therapeutically inhibit the STAT3 signaling pathway. METHODS: The samples were collected from 50 patients with early-stage non-small cell lung cancer and 50 patients with benign lung tumors. Mucin 1 mRNA expression was evaluated to determine lymph node micrometastasis status. STAT3 mRNA, STAT3 protein, and phosphorylated STAT3 protein expression were evaluated through reverse transcription polymerase chain reaction, western blot, and immunohistochemistry, respectively. Measurement data was represented as mean ± standard deviation, and the t-rest or F-test were used. The χ2 -test was used in enumeration data. Logistic regression analysis was carried out to determine the independent risk factors influencing lymph node micrometastasis. RESULTS: STAT3 mRNA and proteins expression were correlated with lymph node micrometastasis (P < 0.05). Logistic regression analysis revealed STAT3 protein overexpression and the differentiation degree of tumors were independent risk factors for lymph node micrometastasis. CONCLUSION: Overexpression of STAT3 might promote lymphatic micrometastasis of early-stage non-small cell lung cancer and might be a clinical predictor of lymph node micrometastasis.

TBL1XR1 predicts isolated tumor cells and micrometastasis in patients with TNM stage I/II colorectal cancer.

BACKGROUND AND AIM: A considerable number of early-stage colorectal cancer (CRC) patients may develop cancer relapse or metastasis after curative surgery. Isolated tumor cells (ITC) and micrometastasis in lymph nodes (LNMM), which are undetectable by conventional pathological examination, may be one primary reason. Detection of ITC/LNMM is time-consuming and cost-ineffective; we aimed to find biomarkers in primary CRC tissues to help predicting ITC/LNMM status. METHODS: We enrolled 137 node-negative patients with early-stage CRC in this study. Existence of ITC/LNMM was identified by immunohistological staining with cytokeratin 20 in resected lymph nodes. Expression of transducin (ß)-like 1 X-linked receptor 1 (TBL1XR1) in primary CRC tissues was also investigated. Chi-squared test was performed to reveal the correlations between ITC/LNMM and clinicopathological characteristics. Univariate and multivariate analyses were used to determine independent prognostic factors. Knockdown experiment together with proliferation and invasion assays were carried out to explore molecular mechanisms between TBL1XR1 and ITC/LNMM. RESULTS: About 29.2% (40/137) patients were identified as ITC/LNMM positive, and most of them (32/40 cases, 80%) showed high TBL1XR1 expression in primary CRC tissues. Both ITC/LNMM and TBL1XR1 expression were independent prognostic factors for disease relapse or metastasis. In vitro experiments demonstrated that TBL1XR1 can regulate the expression of vascular endothelial growth factor C and epithelial-mesenchymal transition proteins, thus mediate the process of lymph node metastasis. CONCLUSIONS: Identification of ITC/LNMM is significant in evaluating clinical outcome and guiding adjuvant chemotherapy for early-stage CRC patients. TBL1XR1 overexpression in CRC tissues can serve as an efficient biomarker to predict the status of ITC/LNMM.

Modulated Expression of Specific tRNAs Drives Gene Expression and Cancer Progression.

Transfer RNAs (tRNAs) are primarily viewed as static contributors to gene expression. By developing a high-throughput tRNA profiling method, we find that specific tRNAs are upregulated in human breast cancer cells as they gain metastatic activity. Through loss-of-function, gain-of-function, and clinical-association studies, we implicate tRNAGluUUC and tRNAArgCCG as promoters of breast cancer metastasis. Upregulation of these tRNAs enhances stability and ribosome occupancy of transcripts enriched for their cognate codons. Specifically, tRNAGluUUC promotes metastatic progression by directly enhancing EXOSC2 expression and enhancing GRIPAP1-constituting an "inducible" pathway driven by a tRNA. The cellular proteomic shift toward a pro-metastatic state mirrors global tRNA shifts, allowing for cell-state and cell-type transgene expression optimization through codon content quantification. TRNA modulation represents a mechanism by which cells achieve altered expression of specific transcripts and proteins. TRNAs are thus dynamic regulators of gene expression and the tRNA codon landscape can causally and specifically impact disease progression.

Cathepsin L in tumor angiogenesis and its therapeutic intervention by the small molecule inhibitor KGP94.

A significant proportion of breast cancer patients harbor clinically undetectable micrometastases at the time of diagnosis. If left untreated, these micro-metastases may lead to disease relapse and possibly death. Hence, there is significant interest in the development of novel anti-metastatic agents that could also curb the growth of pre-established micrometastases. Like primary tumor, the growth of metastases also is driven by angiogenesis. Although the role of cysteine protease Cathepsin L (CTSL) in metastasis associated tumor cell functions such as migration and invasion is well recognized, its role in tumor angiogenesis remains less explored. The present study examines the contribution of CTSL to breast cancer angiogenesis and evaluates the anti-angiogenic efficacy of CTSL inhibitor KGP94. CTSL semi-quantitative RT-PCR analysis on breast tissue panels revealed significant upregulation of CTSL in breast cancer patients which strongly correlated with increased relapse and metastatic incidence and poor overall survival. Preclinically, CTSL ablation using shRNA or KGP94 treatment led to a significant reduction in MDA-MB-231 tumor cell induced angiogenesis in vivo. In-vitro assessments demonstrated a significant decrease in various angiogenic properties such as endothelial cell sprouting, migration, invasion, tube formation and proliferation in the presence of KGP94. Microarray analyses revealed a significant upregulation of cell cycle related genes by CTSL. Western blot analyses further confirmed upregulation of members of the cyclin family by CTSL. Collectively, these data indicate that CTSL is an important contributor to tumor angiogenesis and that the CTSL inhibition may have therapeutic utility in the treatment of breast cancer patients.

Breast cancer upregulating genes in stromal cells by LINE-1 hypermethylation and micrometastatic detection.

UNLABELLED: Changes in the methylation level of genes containing LINE-1 alter host gene regulation. AIM: This study demonstrates that paracrine signaling of breast cancer influences the epigenetic regulation of stromal cells. METHODS: We proved in vitro and in vivo breast cancer promoted LINE-1 methylation exists exclusively in female stromal cells. RESULTS: Genes containing LINE-1 of breast cancer stromal cells were upregulated. Furthermore, one of the genes, MUC-1, was demonstrated to have expression in plasma cells from the lymph nodes of patients with lymph node metastasis or micrometastasis. CONCLUSION: Breast cancer sends a paracrine signal to stroma cells causing LINE-1 epigenetic regulation. Moreover, the regulated genes in stroma cells are potential biomarkers for detecting breast cancer micrometastasis.

Model-based genotype-phenotype mapping used to investigate gene signatures of immune sensitivity and resistance in melanoma micrometastasis.

In this paper, we combine kinetic modelling and patient gene expression data analysis to elucidate biological mechanisms by which melanoma becomes resistant to the immune system and to immunotherapy. To this end, we systematically perturbed the parameters in a kinetic model and performed a mathematical analysis of their impact, thereby obtaining signatures associated with the emergence of phenotypes of melanoma immune sensitivity and resistance. Our phenotypic signatures were compared with published clinical data on pretreatment tumor gene expression in patients subjected to immunotherapy against metastatic melanoma. To this end, the differentially expressed genes were annotated with standard gene ontology terms and aggregated into metagenes. Our method sheds light on putative mechanisms by which melanoma may develop immunoresistance. Precisely, our results and the clinical data point to the existence of a signature of intermediate expression levels for genes related to antigen presentation that constitutes an intriguing resistance mechanism, whereby micrometastases are able to minimize the combined anti-tumor activity of complementary responses mediated by cytotoxic T cells and natural killer cells, respectively. Finally, we computationally explored the efficacy of cytokines used as low-dose co-adjuvants for the therapeutic anticancer vaccine to overcome tumor immunoresistance.

Gene promoter hypermethylation is found in sentinel lymph nodes of breast cancer patients, in samples identified as positive by one-step nucleic acid amplification of cytokeratin 19 mRNA.

We analysed the promoter methylation status of five genes, involved in adhesion (EPB41L3, TSLC-1), apoptosis (RASSF1, RASSF2) or angiogenesis (TSP-1), in intraoperative sentinel lymph node (SLN) biopsy samples from patients with breast cancer, that had been processed by the one-step nucleic acid amplification (OSNA) technique. SLN resection is performed to estimate the risk of tumour cells in the clinically negative axilla, to avoid unnecessary axillary lymph node dissection. OSNA is currently one of the eligible molecular methods for detecting tumour cells in SLNs. It is based on the quantitative evaluation of cytokeratin 19 mRNA which allows distinguishing between macrometastasis, micrometastasis and isolated tumour cells, on the basis of the quantity of tumour cells present. There have been no prior studies on the question whether or not samples processed by OSNA can be used for further molecular studies, including epigenetic abnormalities which are some of the most important molecular alterations in breast cancer. Genomic DNA was extracted from samples obtained from 50 patients diagnosed with primary breast cancer. The content of tumour cells in SLNs was evaluated by OSNA, and the promoter methylation status of the selected genes was analysed by methylation-specific PCR. All were found to be hypermethylated to a variable degree, and RASSF1 hypermethylation was significantly associated with macrometastasis, micrometastasis and isolated tumour cells (p = 0.002). We show that samples used for OSNA are suitable for molecular studies, including gene promoter methylation. These samples provide a new source of material for the identification of additional biomarkers.