Association of Biological Age with Tumor Microenvironment in Patients with Esophageal Adenocarcinoma.

INTRODUCTION: Esophageal cancer is the seventh most common cancer worldwide and typically tends to manifest at an older age. Marked heterogeneity in time-dependent functional decline in older adults results in varying grades of clinically manifest patient fitness or frailty. The biological age-related adaptations that accompany functional decline have been shown to modulate the non-malignant cells comprising the tumor microenvironment (TME). In the current work, we studied the association between biological age and TME characteristics in patients with esophageal adenocarcinoma. METHODS: We comparatively assessed intratumoral histologic stroma quantity, tumor immune cell infiltrate, and blood leukocyte and thrombocyte count in 72 patients stratified over 3 strata of biological age (younger <70 years, fit older ≥70 years, and frail older adults ≥70 years), as defined by a geriatric assessment. RESULTS: Frailty in older adults was predictive of decreased intratumoral stroma quantity (B = -14.66% stroma, p = 0.022) relative to tumors in chronological-age-matched fit older adults. Moreover, in comparison to younger adults, frail older adults (p = 0.032), but not fit older adults (p = 0.302), demonstrated a lower blood thrombocyte count at the time of diagnosis. Lastly, we found an increased proportion of tumors with a histologic desert TME histotype, comprising low stroma quantity and low immune cell infiltration, in frail older adults. CONCLUSION: Our results illustrate the stromal-reprogramming effects of biological age and provide a biological underpinning for the clinical relevance of assessing frailty in patients with esophageal adenocarcinoma, further justifying the need for standardized geriatric assessment in geriatric cancer patients.

Tumour-stroma ratio (TSR) in breast cancer: comparison of scoring core biopsies versus resection specimens.

PURPOSE: Tumour-stroma ratio (TSR) is an important prognostic and predictive factor in several tumour types. The aim of this study is to determine whether TSR evaluated in breast cancer core biopsies is representative of the whole tumour. METHOD: Different TSR scoring methods, their reproducibility, and the association of TSR with clinicopathological characteristics were investigated in 178 breast carcinoma core biopsies and corresponding resection specimens. TSR was assessed by two trained scientists on the most representative H&E-stained digitised slides. Patients were treated primarily with surgery between 2010 and 2021 at Semmelweis University, Budapest. RESULTS: Ninety-one percent of the tumours were hormone receptor (HR)-positive (luminal-like). Interobserver agreement was highest using 100 × magnification (κcore = 0.906, κresection specimen = 0.882). The agreement between TSR of core biopsies and resection specimens of the same patients was moderate (κ = 0.514). Differences between the two types of samples were most frequent in cases with TSR scores close to the 50% cut-off point. TSR was strongly correlated with age at diagnosis, pT category, histological type, histological grade, and surrogate molecular subtype. A tendency was identified for more recurrences among stroma-high (SH) tumours (p = 0.07). Significant correlation was detected between the TSR and tumour recurrence in grade 1 HR-positive breast cancer cases (p = 0.03). CONCLUSIONS: TSR is easy to determine and reproducible on both core biopsies and in resection specimens and is associated with several clinicopathological characteristics of breast cancer. TSR scored on core biopsies is moderately representative for the whole tumour.

LncRNA LITATS1 suppresses TGF-ß-induced EMT and cancer cell plasticity by potentiating TßRI degradation.

Epithelial cells acquire mesenchymal phenotypes through epithelial-mesenchymal transition (EMT) during cancer progression. However, how epithelial cells retain their epithelial traits and prevent malignant transformation is not well understood. Here, we report that the long noncoding RNA LITATS1 (LINC01137, ZC3H12A-DT) is an epithelial gatekeeper in normal epithelial cells and inhibits EMT in breast and non-small cell lung cancer cells. Transcriptome analysis identified LITATS1 as a TGF-ß target gene. LITATS1 expression is reduced in lung adenocarcinoma tissues compared with adjacent normal tissues and correlates with a favorable prognosis in breast and non-small cell lung cancer patients. LITATS1 depletion promotes TGF-ß-induced EMT, migration, and extravasation in cancer cells. Unbiased pathway analysis demonstrated that LITATS1 knockdown potently and selectively potentiates TGF-ß/SMAD signaling. Mechanistically, LITATS1 enhances the polyubiquitination and proteasomal degradation of TGF-ß type I receptor (TßRI). LITATS1 interacts with TßRI and the E3 ligase SMURF2, promoting the cytoplasmic retention of SMURF2. Our findings highlight a protective function of LITATS1 in epithelial integrity maintenance through the attenuation of TGF-ß/SMAD signaling and EMT.

Differential optineurin expression controls TGFß signaling and is a key determinant for metastasis of triple negative breast cancer.

Triple-negative breast cancer (TNBC) is the most challenging breast cancer subtype to treat due to its aggressive characteristics and low response to the existing clinical therapies. Distant metastasis is the main cause of death of TNBC patients. Better understanding of the mechanisms underlying TNBC metastasis may lead to new strategies of early diagnosis and more efficient treatment. In our study, we uncovered that the autophagy receptor optineurin (OPTN) plays an unexpected role in TNBC metastasis. Data mining of publicly available data bases revealed that the mRNA level of OPTN in TNBC patients positively correlates with relapse free and distance metastasis free survival. Importantly, in vitro and in vivo models demonstrated that OPTN suppresses TNBC metastasis. Mechanistically, OPTN inhibited the pro-oncogenic transforming growth factor-ß (TGFß) signaling in TNBC cells by interacting with TGFß type I receptor (TßRI) and promoting its ubiquitination for degradation. Consistent with our experimental findings, the clinical TNBC samples displayed a negative correlation between OPTN mRNA expression and TGFß gene response signature and expression of proto-typic TGFß target genes. Altogether, our study demonstrates that OPTN is a negative regulator for TGFß receptor/SMAD signaling and suppresses metastasis in TNBC.

Longitudinal Serum Protein Analysis of Women with a High Risk of Developing Breast Cancer Reveals Large Interpatient Versus Small Intrapatient Variations: First Results from the TESTBREAST Study.

The prospective, multicenter TESTBREAST study was initiated with the aim of identifying a novel panel of blood-based protein biomarkers to enable early breast cancer detection for moderate-to-high-risk women. Serum samples were collected every (half) year up until diagnosis. Protein levels were longitudinally measured to determine intrapatient and interpatient variabilities. To this end, protein cluster patterns were evaluated to form a conceptual basis for further clinical analyses. Using a mass spectrometry-based bottom-up proteomics strategy, the protein abundance of 30 samples was analyzed: five sequential serum samples from six high-risk women; three who developed a breast malignancy (cases) and three who did not (controls). Serum samples were chromatographically fractionated and an in-depth serum proteome was acquired. Cluster analyses were applied to indicate differences between and within protein levels in serum samples of individuals. Statistical analyses were performed using ANOVA to select proteins with a high level of clustering. Cluster analyses on 30 serum samples revealed unique patterns of protein clustering for each patient, indicating a greater interpatient than intrapatient variability in protein levels of the longitudinally acquired samples. Moreover, the most distinctive proteins in the cluster analysis were identified. Strong clustering patterns within longitudinal intrapatient samples have demonstrated the importance of identifying small changes in protein levels for individuals over time. This underlines the significance of longitudinal serum measurements, that patients can serve as their own controls, and the relevance of the current study set-up for early detection. The TESTBREAST study will continue its pursuit toward establishing a protein panel for early breast cancer detection.

Characteristics of tumour stroma in regional lymph node metastases in colorectal cancer patients: a theoretical framework for future diagnostic imaging with FAPI PET/CT.

PURPOSE: The recently developed fibroblast activation protein inhibitor (FAPI) tracer for PET/CT, binding tumour-stromal cancer-associated fibroblasts, is a promising tool for detection of positive lymph nodes. This study provides an overview of features, including sizes and tumour-stromal content, of lymph nodes and their respective lymph node metastases (LNM) in colorectal cancer (CRC), since literature lacks on whether LNMs contain sufficient stroma to potentially allow FAPI-based tumour detection. METHODS: Haematoxylin and eosin-stained tissue slides from 73 stage III colon cancer patients were included. Diameters and areas of all lymph nodes and their LNMs were assessed, the amount of stroma by measuring the stromal compartment area, the conventional and total tumour-stroma ratios (TSR-c and TSR-t, respectively), as well as correlations between these parameters. Also, subgroup analysis using a minimal diameter cut off of 5.0 mm was performed. RESULTS: In total, 126 lymph nodes were analysed. Although positive correlations were observed between node and LNM for diameter and area (r = 0.852, p < 0.001 and r = 0.960, p < 0.001, respectively), and also between the LNM stromal compartment area and nodal diameter (r = 0.612, p < 0.001), nodal area (r = 0.747, p < 0.001) and LNM area (r = 0.746, p < 0.001), novel insight was that nearly all (98%) LNMs contained stroma, with median TSR-c scores of 35% (IQR 20-60%) and TSR-t of 20% (IQR 10-30%). Moreover, a total of 32 (25%) positive lymph nodes had a diameter of < 5.0 mm. CONCLUSION: In LNMs, stroma is abundantly present, independent of size, suggesting a role for FAPI PET/CT in improved lymph node detection in CRC.

Standardization of the tumor-stroma ratio scoring method for breast cancer research.

PURPOSE: The tumor-stroma ratio (TSR) has repeatedly proven to be correlated with patient outcomes in breast cancer using large retrospective cohorts. However, studies validating the TSR often show variability in methodology, thereby hampering comparisons and uniform outcomes. METHOD: This paper provides a detailed description of a simple and uniform TSR scoring method using Hematoxylin and Eosin (H&E)-stained core biopsies and resection tissue, specifically focused on breast cancer. Possible histological challenges that can be encountered during scoring including suggestions to overcome them are reported. Moreover, the procedure for TSR estimation in lymph nodes, scoring on digital images and the automatic assessment of the TSR using artificial intelligence are described. CONCLUSION: Digitized scoring of tumor biopsies and resection material offers interesting future perspectives to determine patient prognosis and response to therapy. The fact that the TSR method is relatively easy, quick, and cheap, offers great potential for its implementation in routine diagnostics, but this requires high quality validation studies.

RNF12 is regulated by AKT phosphorylation and promotes TGF-ß driven breast cancer metastasis.

Transforming growth factor-ß (TGF-ß) acts as a pro-metastatic factor in advanced breast cancer. RNF12, an E3 ubiquitin ligase, stimulates TGF-ß signaling by binding to the inhibitory SMAD7 and inducing its proteasomal degradation. How RNF12 activity is regulated and its exact role in cancer is incompletely understood. Here we report that RNF12 was overexpressed in invasive breast cancers and its high expression correlated with poor prognosis. RNF12 promoted breast cancer cell migration, invasion, and experimental metastasis in zebrafish and murine xenograft models. RNF12 levels were positively associated with the phosphorylated AKT/protein kinase B (PKB) levels, and both displayed significant higher levels in the basal-like subtype compared with the levels in luminal-like subtype of breast cancer cells. Mechanistically, AKT-mediated phosphorylation induced the nuclear localization of RNF12, maintained its stability, and accelerated the degradation of SMAD7 mediated by RNF12. Furthermore, we demonstrated that RNF12 and AKT cooperated functionally in breast cancer cell migration. Notably, RNF12 expression strongly correlated with both phosphorylated AKT and phosphorylated SMAD2 levels in breast cancer tissues. Thus, our results uncovered RNF12 as an important determinant in the crosstalk between the TGF-ß and AKT signaling pathways during breast cancer progression.

Tumor-stroma ratio is associated with Miller-Payne score and pathological response to neoadjuvant chemotherapy in HER2-negative early breast cancer.

The tumor-stroma ratio (TSR) has proven to be a strong prognostic factor in breast cancer, demonstrating better survival for patients with stroma-low tumors. Since the role of the TSR as a predictive marker for neoadjuvant chemotherapy outcome is yet unknown, this association was evaluated for HER2-negative breast cancer in the prospective DIRECT and NEOZOTAC trials. The TSR was assessed on 375 hematoxylin and eosin-stained sections of pre-treatment biopsies. Associations between the TSR and chemotherapy response according to the Miller-Payne (MP) grading system, and between the TSR and pathological response were examined using Pearson's chi-square, Cochran-Armitage test for trend and regression analyses. A stroma-low tumor prior to neoadjuvant chemotherapy was significantly associated with a higher MP score (P = .005). This relationship remained significant in the estrogen receptor (ER)-negative subgroup (P = .047). The univariable odds ratio (OR) of a stroma-low tumor on pathological complete response (pCR) was 2.46 (95% CI 1.34-4.51, P = .004), which attenuated to 1.90 (95% CI 0.85-4.25, P = .119) after adjustment for relevant prognostic factors. Subgroup analyses revealed an OR of 5.91 in univariable analyses for ER-negativity (95% CI 1.19-29.48, P = .030) and 1.48 for ER-positivity (95% CI 0.73-3.01, P = .281). In conclusion, a low amount of stroma on pre-treatment biopsies is associated with a higher MP score and pCR rate. Therefore, the TSR is a promising biomarker in predicting neoadjuvant treatment outcome. Incorporating this parameter in routine pathological diagnostics could be worthwhile to prevent overtreatment and undertreatment.

Lessons Learned from Setting Up a Prospective, Longitudinal, Multicenter Study with Women at High Risk for Breast Cancer.

Women identified with an increased risk of breast cancer due to mutations in cancer susceptibility genes or a familial history of breast cancer undergo tailored screening with the goal of detecting tumors earlier, when potential curative interventions are still possible. Ideally, screening would identify signs of carcinogenesis even before a tumor is detectable by imaging. This could be achieved by timely signaling of altered biomarker levels for precancerous processes in liquid biopsies. Currently, the Nipple Aspirate Fluid (NAF) and the Trial Early Serum Test BREAST cancer (TESTBREAST), both ongoing, prospective, multicenter studies, are investigating biomarkers in liquid biopsies to improve breast cancer screening in high-risk women. The NAF study focuses on changes over time in miRNA expression levels both in blood and NAF samples, whereas the TESTBREAST study analyzes changes in protein levels in blood samples at sequential interval timepoints. These within-subject changes are studied in relation to later occurrence of breast cancer using a nested case-control design. These longitudinal studies face their own challenges in execution, such as hindrances in logistics and in sample processing that were difficult to anticipate. This article offers insight into those challenges and concurrently aims to provide useful strategies for the set-up of similar studies.See related commentary by Sauter, p. 429.