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.

Nuclear ßII-Tubulin and its Possible Utility in Cancer Diagnosis, Prognosis and Treatment.

Microtubules are organelles that usually occur only in the cytosol. Walss et al. (1999) discovered the ßII isotype of tubulin, complexed with α, in the nuclei of certain cultured cells, in non-microtubule form. When fluorescently labeled tubulins were microinjected into the cells, only αßII appeared in the nucleus, and only after one cycle of nuclear disassembly and reassembly. It appeared as if αßII does not cross the nuclear envelope but is trapped in the nucleus by the re-forming nuclear envelope in whose reassembly ßII may be involved. ßII is present in the cytoplasm and nuclei of many tumor cells. With some exceptions, normal tissues that expressed ßII rarely had ßII in their nuclei. It is possible that ßII is involved in nuclear reassembly and then disappears from the nucleus. Ruksha et al. (2019) observed that patients whose colon cancer cells in the invasive front showed no ßII had a median survival of about 5.5 years, which was more than halved if they had cytosolic ßII and further lessened if they had nuclear ßII, suggesting that the presence and location of ßII in biopsies could be a useful prognostic indicator and also that ßII may be involved in cancer progression. Yeh and Ludueña. (2004) observed that many tumors were surrounded by non-cancerous cells exhibiting cytosolic and nuclear ßII, suggesting a signaling pathway that causes ßII to be synthesized in nearby cells and localized to their nuclei. ßII could be useful in cancer diagnosis, since the presence of ßII in non-cancerous cells could indicate a nearby tumor. Investigation of this pathway might reveal novel targets for chemotherapy. Another possibility would be to combine αßII with CRISPR-Cas9. This complex would likely enter the nucleus of a cancer cell and, if guided to the appropriate gene, might destroy the cancer cell or make it less aggressive; possible targets will be discussed here. The possibilities raised here about the utility of ßII in cancer diagnosis, prognosis, biology and therapy may repay further investigation.

Stroma-normalised vessel density predicts benefit from adjuvant fluorouracil-based chemotherapy in patients with stage II/III colon cancer.

BACKGROUND: Identification of biomarkers associated with benefit of adjuvant chemotherapy in stage II/III colon cancer is an important task. METHODS: Vessel density (VD) and tumour stroma were analysed in a randomised-trial-derived discovery cohort (n = 312) and in a stage II/III group of a population-based validation cohort (n = 85). VD was scored separately in the tumour centre, invasive margin and peritumoral stroma compartments and quantitated as VD/total analysed tissue area or VD/stroma area. RESULTS: High stroma-normalised VD in the invasive margin was associated with significantly longer time to recurrence and overall survival (OS) (p = 0.002 and p = 0.006, respectively) in adjuvant-treated patients of the discovery cohort, but not in surgery-only patients. Stroma-normalised VD in the invasive margin and treatment effect were significantly associated according to a formal interaction test (p = 0.009). Similarly, in the validation cohort, high stroma-normalised VD was associated with OS in adjuvant-treated patients, although statistical significance was not reached (p = 0.051). CONCLUSION: Through the use of novel digitally scored vessel-density-related metrics, this exploratory study identifies stroma-normalised VD in the invasive margin as a candidate marker for benefit of adjuvant 5-FU-based chemotherapy in stage II/III colon cancer. The findings, indicating particular importance of vessels in the invasive margin, also suggest biological mechanisms for further exploration.

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

Tubulin is a heterodimer of α and ß 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 ßII-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 ßII expression and its nuclear localization with cancer progression has not previously been addressed. We here used a monoclonal antibody to ßII to examine patients with colorectal cancer and found that patients whose tumors over-express ßII have a greatly decreased life expectancy which is even shorter in those patients with nuclear ßII. Our results suggest that ßII-tubulin may facilitate cancer growth and metastasis and, to accomplish this, may not need to be in microtubule form. Furthermore, ßII expression and localization could be a useful prognostic marker. We also found that ßII appears in the nuclei of otherwise normal cells adjacent to the tumor. It is possible therefore that cancer cells expressing ßII influence nearby cells to do the same and to localize ßII in their nuclei by an as yet uncharacterized regulatory pathway.

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-Reed-Sternberg-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-Reed-Sternberg-like cells, which were CD20-positive in 27% of the cases and Epstein-Barr 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-Reed-Sternberg-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' rosetting around Hodgkin-Reed-Sternberg-like cells. In summary, this study confirms the presence of Hodgkin-Reed-Sternberg-like cells in a high number of cases of follicular T cell lymphoma, suggesting that Hodgkin-Reed-Sternberg-like cells may contribute to the development of this lymphoma. Hodgkin-Reed-Sternberg-like cells in follicular T cell lymphoma cannot reliably be differentiated from the Hodgkin-Reed-Sternberg 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.

Proceedings From the 1st International Conference 'Current Concepts and Controversies in Gynecologic and Urologic Oncology'.

The N.N. Alexandrov National Cancer Center of Belarus organized a collaborative international conference entitled 'Current Concepts and Controversies in Gynecologic and Urologic Oncology' with the International Gynecologic Cancer Society and the United States National Cancer Institute. International, regional, and national experts presented recent developments and local conditions in the treatment of gynecologic cancers. Findings were reviewed with the intent of optimizing the management of women with gynecologic cancers across the Commonwealth of Independent States region. At the end of the conference, a resolution was adopted to identify areas for improvement and future collaborations.

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.

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 "αmax" -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.

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-ß or α-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-α and -ß and shorter overall survival. Analyses of the SPCRC cohort confirmed these findings. Perivascular PDGFR-α and -ß 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-α and -ß were identified as novel independent markers predicting survival in mCRC. The novel methodology should be suitable for similar analyses in other tumor collections.

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.

Tissue transglutaminase expression in celiac mucosa: an immunohistochemical study.

Tissue transglutaminase (tTG) constitutes the main autoantigen in celiac disease (CD). The aim of the study was to clarify weather celiac disease is associated with changes in tTG expression in duodenal mucosa. Tissue transglutaminase was assessed immunohistochemically (clone CUB 7402) in duodenal biopsy specimens from 22 untreated CD patients, ten normal controls (NC) with unremarkable duodenal mucosa, and nine disease nonceliac controls (DC). In 15 CD patients duodenal biopsy specimens were repeatedly assessed after these patients had been prescribed gluten-free diet. Positive pixel count algorithm of ImageScope was used for quantitative evaluation of immunohistochemistry. Tissue transglutaminase expression in superficial epithelium differed significantly between the three groups (p < 0.001). It was increased in DC in relation to NC (p < 0.001) and in CD--in relation to NC (p < 0.001) and DC (p = 0.003). In CD and DC, cryptal epithelium was stained more intensively than in NC (p < 0.001), but there was no difference between CD and DC (p = 0.507). The same pattern was seen in lamina propria. A significant decrease in tTG expression in all the compartments was seen in repeatedly assessed samples. Untreated CD is associated with tTG overexpression, which is reversible. Tissue transglutaminase up-regulation does not seem to be specific for CD and can appear in other pathological conditions.

Phagocytosing neutrophils down-regulate the expression of chemokine receptors CXCR1 and CXCR2.

CXC chemokines play a central role in regulation of neutrophil activation and chemotaxis. Because the chemotactic responses of neutrophils are impaired after phagocytosis, we explored the effect of phagocytic stimuli on the expression of interleukin-8 (IL-8) receptors, CXCR1 and CXCR2, in human neutrophils. After phagocytosis of opsonized yeast, the expression of CXCR1 and CXCR2 was substantially down-regulated and was accompanied by reduced Ca(++) responses to corresponding ligands, IL-8 and neutrophil-activating peptide-2 (NAP-2). The levels of CXCR1 and CXCR2 mRNA were constant during phagocytic stimulation of neutrophils. Confocal microscopy revealed that CXCR reduction was not via internalization. Metalloproteinase inhibitor, 1,10-phenantroline, prevented the reduction of CXCRs induced by phagocytosis, indicating that proteolytic degradation may be responsible for down-regulation. These observations suggest that down-regulation of CXCR expression may substantially reduce the responsiveness of phagocytosing neutrophils to CXC chemokines.