Survival-associated heterogeneity of marker-defined perivascular cells in colorectal cancer

Perivascular cells (PC) were recently implied as regulators of metastasis and immune cell activity. Perivascular heterogeneity in clinical samples, and associations with other tumor features and outcome, remain largely unknown. Here we report a novel method for digital quantitative analyses of vessel characteristics and PC, which was applied to two collections of human metastatic colorectal cancer (mCRC). Initial analyses identified marker-defined subsets of PC, including cells expressing PDGFR-beta or alpha-SMA or both markers. PC subsets were largely independently expressed in a manner unrelated to vessel density and size. Association studies implied specific oncogenic mutations in malignant cells as determinants of PC status. Semi-quantitative and digital-image-analyses-based scoring of the NORDIC-VII cohort identified significant associations between low expression of perivascular PDGFR-alpha and -beta and shorter overall survival. Analyses of the SPCRC cohort confirmed these findings. Perivascular PDGFR-alpha and -beta remained independent factors for survival in multivariate analyses. Overall, our study identified host vasculature and oncogenic status as determinants of tumor perivascular features. Perivascular PDGFR-alpha and -beta were identified as novel independent markers predicting survival in mCRC. The novel methodology should be suitable for similar analyses in other tumor collections

5-FU resistant colorectal cancer cells possess improved invasiveness and βIII-tubulin expression

Purpose: Elevated bIII-tubulin levels are associated with resistance to a broad spectrum of drugs in different carcinomas and are associated with poor prognosis of different epithelial cancers. 5-Fluorouracil (5-FU) is a widely used standard drug in chemotherapeutic regimens for colorectal cancer treatment, although the resistance to 5-FU is a major obstacle to successful therapy. The aim of the study was to compare the invasive and adhesion properties and the expression levels of bIII-tubulin in a 5-Fluorouracil (5-FU)-resistant colorectal cancer (CRC) cell line HCT116 and parental cells.Methods: The 5-FU-resistant cell line was established by continuous stepwise selection with increasing concentrations of 5-FU. Cell viability and properties were evaluated using MTT, adhesion and transwell invasion assays respectively. The expression of bIII-tubulin was revealed by immunoblot and immunofluorescence.Results: The derivative line is 25-fold resistant to 5-FU and characterized by altered cell morphology, twice as many cells of the 5-FU-resistant line fail to adhere than is the case for the parental cell line and were characterized by enhanced invasiveness, accompanied by increased bIII-tubulin expression. In addition, we found that loss of bIII-tubulin expression was correlated with loss of 5-FU resistance.Conclusion: Our results indicate that even though 5-FU does not target microtubules, there appears to be a correlation between bIII-tubulin expression and resistance to 5-FU and that this is particularly important with regard to invasiveness. These findings indicate a possible contribution of bIII-tubulin to 5-FU resistance in vivo.

Inter- and intra-tumoral relationships between vasculature characteristics, GLUT1 and budding in colorectal carcinoma

Vascular characteristics, hypoxia and tumor budding are features that have been implied in the biology and prognosis of colorectal cancer. Internal relationships and the inter- and intra-tumoral variation of these tumor properties remain to be determined. In the current study we have characterized blood vessel status in different areas of CRC and in the peritumoral fibroblastic stroma. Analyses of these characteristics have been supplemented by characterization of budding and hypoxia. Analyses revealed significantly lower values of vessel perimeter (VP) and vessel lumen area (VL) at the invasive front and surrounding stroma as compared to the tumor center. Also, the number of vessels (VN) in the peritumoral stroma was higher than in the center. Thus, tumor center displays larger and fewer vessels as compared to the tumor periphery. GLUT1 expression was correlated directly with VN (r=0.351, p=0.028) and inversely with VL and VP (r=- 0.432, p=0.006 and r=-0.484, p=0.002) at the invasive front. Moreover, GLUT1 expression, VP at the invasive front, and VN in the surrounding peritumoral stroma, were associated with budding score (r=0.574, p<0.000, r=-0.340, p=0,034 and r=-0,389, p=0.025 respectively). Furthermore, GLUT1, budding score, vessel number in peritumoral stroma, and vessel size in the invasive front, were significantly different in tumors with or without lymph node metastasis. This study reports previously unrecognized relationships between localization-specific vascular characteristics, hypoxia and tumor budding. The findings suggest potential functional relationships, which should be further explored, and also highlight the inter-tumoral variations in vasculature, which is highly relevant for ongoing efforts to identify vessel-based biomarkers

Over-Expression of βII-Tubulin and Especially Its Localization in Cell Nuclei Correlates with Poorer Outcomes in Colorectal Cancer

Tubulin is a heterodimer of &alpha; and &beta; subunits, both existing as isotypes differing in amino acid sequence encoded by different genes. Specific isotypes of tubulin have associations with cancer that are not well understood. Previous studies found that &beta;II-tubulin is expressed in a number of transformed cells and that this isotype is found in cell nuclei in non-microtubule form. The association of &beta;II expression and its nuclear localization with cancer progression has not previously been addressed. We here used a monoclonal antibody to &beta;II to examine patients with colorectal cancer and found that patients whose tumors over-express &beta;II have a greatly decreased life expectancy which is even shorter in those patients with nuclear &beta;II. Our results suggest that &beta;II-tubulin may facilitate cancer growth and metastasis and, to accomplish this, may not need to be in microtubule form. Furthermore, &beta;II expression and localization could be a useful prognostic marker. We also found that &beta;II appears in the nuclei of otherwise normal cells adjacent to the tumor. It is possible therefore that cancer cells expressing &beta;II influence nearby cells to do the same and to localize &beta;II in their nuclei by an as yet uncharacterized regulatory pathway

Image analysis-derived metrics of histomorphological complexity predicts prognosis and treatment response in stage II-III colon cancer

The complexity of tumor histomorphology reflects underlying tumor biology impacting on natural course and response to treatment. This study presents a method of computer-aided analysis of tissue sections, relying on multifractal (MF) analyses, of cytokeratin-stained tumor sections which quantitatively evaluates of the morphological complexity of the tumor-stroma interface. This approach was applied to colon cancer collection, from an adjuvant treatment randomized study. Metrics obtained with the method acted as independent markers for natural course of the disease, and for benefit of adjuvant treatment. Comparative analyses demonstrated that MF metrics out-performed standard histomorphological features such as tumor grade, budding and configuration of invasive front. Notably, the MF analyses-derived "alpha(max)" -metric constitutes the first response-predictive biomarker in stage II-III colon cancer showing significant interactions with treatment in analyses using a randomized trial-derived study population. Based on these results the method appears as an attractive and easy-to-implement tool for biomarker identification

CD30 expression in neoplastic T cells of follicular T cell lymphoma is a helpful diagnostic tool in the differential diagnosis of Hodgkin lymphoma

Follicular T cell lymphoma is derived from follicular T-helper cells. In many cases, neoplastic T cells form rosettes around Hodgkin\u2013Reed\u2013Sternberg-like cells, which can lead to the misdiagnosis of classical Hodgkin lymphoma. The aim of the present study was to obtain a better understanding of this rosetting phenomenon and to recognize features that are helpful in the differential diagnosis of classical Hodgkin lymphoma. Sixteen mostly elderly follicular T cell lymphoma patients (mean 66 years) were analyzed. Fifteen of the 16 follicular T cell lymphoma cases presented with Hodgkin\u2013Reed\u2013Sternberg-like cells, which were CD20-positive in 27% of the cases and Epstein\u2013Barr virus-infected in nearly all cases. Frequently, the immunophenotype of rosetting neoplastic T cells differed from the bulk neoplastic cells with less numerous T-follicular helper cell markers expressed, suggesting a modulation of T-follicular helper cell marker expression in the neoplastic T cells. In 75% of the cases, variable CD30 expression was encountered in the neoplastic T cells, likely reflecting an activation state in these cells. Hodgkin\u2013Reed\u2013Sternberg-like cells were positive for CCL17, and follicular T cell lymphoma tumor cells expressed its receptor CCR4 at variable intensity, thus potentially explaining the phenomenon of the tumor cells\u2019 rosetting around Hodgkin\u2013Reed\u2013Sternberg-like cells. In summary, this study confirms the presence of Hodgkin\u2013Reed\u2013Sternberg-like cells in a high number of cases of follicular T cell lymphoma, suggesting that Hodgkin\u2013Reed\u2013Sternberg-like cells may contribute to the development of this lymphoma. Hodgkin\u2013Reed\u2013Sternberg-like cells in follicular T cell lymphoma cannot reliably be differentiated from the Hodgkin\u2013Reed\u2013Sternberg cells of classical Hodgkin lymphoma based on their immunophenotype. In contrast, demonstration of a T-follicular helper cell phenotype with CD10 and frequent CD30 expression in the neoplastic T cell population can help to establish the diagnosis of follicular T cell lymphoma, and may even indicate CD30 as a therapeutic target for these patients

Treatment-related survival associations of claudin-2 expression in fibroblasts of colorectal cancer

Claudin-2 is a trans-membrane protein-component of tight junctions in epithelial cells. Elevated claudin-2 expression has been reported in colorectal cancer (CRC). The aim of this study was to investigate the expression patterns of claudin-2 in human CRC samples and analyze its association with clinical characteristics and treatment outcome. TMAs of primary tumors from two cohorts of metastatic CRC (mCRC) were used. Claudin-2 IHC staining was evaluated in a semi-quantitative manner in different regions and cell types. Claudin-2 expression was also analyzed by immunofluorescence in primary cultures of human CRC cancer-associated fibroblasts (CAFs). Initial analyses identified previously unrecognized expression patterns of claudin-2 in CAFs of human CRC. Claudin-2 expression in CAFs of the invasive margin was associated with shorter progression-free survival. Subgroup analyses demonstrated that the survival associations occurred among cases that received 5-FU+oxaliplatin combination treatment, but not in patients receiving 5-FU +/- irinotecan. The finding was validated by analyses of the independent cohort. In summary, previously unreported stromal expression of claudin-2 in CAFs of human CRC was detected together with significant association between high claudin-2 expression in CAFs and shorter survival in 5-FU+oxaliplatin-treated mCRC patients

An immune score reflecting pro- and anti-tumoural balance of tumour microenvironment has major prognostic impact and predicts immunotherapy response in solid cancers

Background: Cancer immunity is based on the interaction of a multitude of cells in the spatial context of the tumour tissue. Clinically relevant immune signatures are therefore anticipated to fundamentally improve the accuracy in predicting disease progression. Methods: Through a multiplex in situ analysis we evaluated 15 immune cell classes in 1481 tumour samples. Single-cell and bulk RNAseq data sets were used for functional analysis and validation of prognostic and predictive associations. Findings: By combining the prognostic information of anti-tumoural CD8+ lymphocytes and tumour supportive CD68+CD163+ macrophages in colorectal cancer we generated a signature of immune activation (SIA). The prognostic impact of SIA was independent of conventional parameters and comparable with the state-of-art immune score. The SIA was also associated with patient survival in oesophageal adenocarcinoma, bladder cancer, lung adenocarcinoma and melanoma, but not in endometrial, ovarian and squamous cell lung carcinoma. We identified CD68+CD163+ macrophages as the major producers of complement C1q, which could serve as a surrogate marker of this macrophage subset. Consequently, the RNA-based version of SIA (ratio of CD8A to C1QA) was predictive for survival in independent RNAseq data sets from these six cancer types. Finally, the CD8A/C1QA mRNA ratio was also predictive for the response to checkpoint inhibitor therapy. Interpretation: Our findings extend current concepts to procure prognostic information from the tumour immune microenvironment and provide an immune activation signature with high clinical potential in common human cancer types. Funding: Swedish Cancer Society, Lions Cancer Foundation, Selanders Foundation, P.O. Zetterling Foundation, U-CAN supported by SRA CancerUU, Uppsala University and Region Uppsala