Tumour-infiltrating lymphocytes predict response to definitive chemoradiotherapy in head and neck cancer

Background:

We aimed to investigate the prognostic value of tumour-infiltrating lymphocytes'' (TILs) expression in pretreatment specimens from patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive chemoradiotherapy (CRT).

Methods:

The prevalence of CD3+, CD8+, CD4+ and FOXP3+ TILs was assessed using immunohistochemistry in tumour tissue obtained from 101 patients before CRT and was correlated with clinicopathological characteristics as well as local failure-free- (LFFS), distant metastases free- (DMFS), progression-free (PFS) and overall survival (OS). Survival curves were measured using the Kaplan–Meier method, and differences in survival between the groups were estimated using the log-rank test. Prognostic effects of TIL subset density were determined using the Cox regression analysis.

Results:

With a mean follow-up of 25 months (range, 2.3–63 months), OS at 2 years was 57.4% for the entire cohort. Patients with high immunohistochemical CD3 and CD8 expression had significantly increased OS (P=0.024 and P=0.028), PFS (P=0.044 and P=0.047) and DMFS (P=0.021 and P=0.026) but not LFFS (P=0.90 and P=0.104) in multivariate analysis that included predictive clinicopathologic factors, such as age, sex, T-stage, N-stage, tumour grading and localisation. Neither CD4 nor FOXP3 expression showed significance for the clinical outcome. The lower N-stage was associated with improved OS in the multivariate analysis (P=0.049).

Conclusion:

The positive correlation between a high number of infiltrating CD3+ and CD8+ cells and clinical outcome indicates that TILs may have a beneficial role in HNSCC patients and may serve as a biomarker to identify patients likely to benefit from definitive CRT.     

Tumour-associated CD66b+ neutrophil count is an independent prognostic factor for recurrence in localised cervical cancer

Background:

The prognostic impact of tumour-promoting immune cells in cervical cancer is unclear.

Methods:

Federation of Gynaecology and Obstetrics (FIGO) stage IB and IIA cervical cancer patients (N=101) were assessed for tumour-associated CD66b⁺ neutrophils and CD163⁺ macrophages by immunohistochemistry in whole tissue sections using stereology. Results were correlated with previous results on tumour-infiltrating CD3⁺, CD4⁺, and CD8⁺ lymphocytes in the same cohort with recurrence-free survival (RFS) as end point.

Results:

The highest densities of CD66b⁺ neutrophils and CD163⁺ macrophages were observed in the peritumoural compartment (median 53.1 cells mm⁻² and 1.3% area fraction, respectively). Above median peritumoural and stromal CD66b⁺ neutrophils and peritumoural CD163⁺ macrophages were significantly associated with short RFS. Multivariate analysis identified high peritumoural neutrophils (HR 2.27; 95% CI 1.09–4.75; P=0.03), low peritumoural CD8⁺ lymphocytes (HR 3.67; 95% CI 1.63–8.25; P=0.002), and lymph node metastases (HR 2.70; 95% CI 1.26–5.76; P=0.01) as independent prognostic factors for short RFS, whereas CD163⁺ macrophages were not significant. An index of combined intratumoral and peritumoral CD66b⁺ neutrophils to CD8⁺ lymphocytes had good discriminatory power for each quartile with 5-year RFS of 92%, 80%, 62%, and 44% (P=0.001).

Conclusion:

Tumour-associated neutrophil count is an independent prognostic factor for short RFS in localised cervical cancer. Combining CD66b and CD8 may further improve prognostic stratification. These findings require prospective validation.     

Low stromal Foxp3+ regulatory T-cell density is associated with complete response to neoadjuvant chemoradiotherapy in rectal cancer

Background:

Foxp3⁺ regulatory T cells (Tregs) play a vital role in preventing autoimmunity, but also suppress antitumour immune responses. Tumour infiltration by Tregs has strong prognostic significance in colorectal cancer, and accumulating evidence suggests that chemotherapy and radiotherapy efficacy has an immune-mediated component. Whether Tregs play an inhibitory role in chemoradiotherapy (CRT) response in rectal cancer remains unknown.

Methods:

Foxp3⁺, CD3⁺, CD4⁺, CD8⁺ and IL-17⁺ cell density in post-CRT surgical samples from 128 patients with rectal cancer was assessed by immunohistochemistry. The relationship between T-cell subset densities and clinical outcome (tumour regression and survival) was evaluated.

Results:

Stromal Foxp3⁺ cell density was strongly associated with tumour regression grade (P=0.0006). A low stromal Foxp3⁺ cell density was observed in 84% of patients who had a pathologic complete response (pCR) compared with 41% of patients who did not (OR: 7.56, P=0.0005; OR: 5.27, P=0.006 after adjustment for presurgery clinical factors). Low stromal Foxp3⁺ cell density was also associated with improved recurrence-free survival (HR: 0.46, P=0.03), although not independent of tumour regression grade.

Conclusions:

Regulatory T cells in the tumour microenvironment may inhibit response to neoadjuvant CRT and may represent a therapeutic target in rectal cancer.     

Tumour-infiltrating CD4+ and CD8+ lymphocytes as predictors of clinical outcome in glioma

Background:

T lymphocyte infiltration has been detected in glioma, although its significance remains unclear. The purpose of the present study was to explore the prognostic value of CD4⁺ and CD8⁺ tumour-infiltrating lymphocytes (TILs) in glioma, and the prognostic value of infiltrating Forkhead box protein 3 (FoxP3⁺) regulatory T cells were also investigated.

Methods:

CD4⁺, FoxP3⁺ and CD8⁺ TILs were assessed by immunohistochemical staining of tissue microarray cores from 284 gliomas. Kaplan–Meier analysis and Cox proportional hazards models were used to examine the survival function of these TILs in 90 glioblastoma patients.

Results:

The number of CD8⁺ TILs was inversely correlated with tumour grade (P=0.025), whereas the number of CD4⁺ TILs was positively correlated with tumour grade (P=0.002). FoxP3⁺ TILs were only observed in glioblastomas, but not in low-grade astrocytomas or oligodendroglial tumours. Among patients with glioblastoma, none of CD4⁺ TILs, FoxP3⁺ TILs and CD8⁺ TILs alone was significantly associated with patient prognosis. However, the presence of high CD4⁺ and low CD8⁺ TIL levels was an independent predictor of poor progress-free survival (multivariate hazard ratio (HR) 1.618, 95% confidence interval (CI) 1.245–2.101, P<0.001) and poor overall survival (multivariate HR 1.508, 95% CI 1.162–1.956, P=0.002). Moreover, pseudoprogression was more often found in patients with high CD4⁺ TILs and high CD8⁺ TILs.

Conclusions:

The combination of CD4⁺ and CD8⁺ TILs is a predictor of clinical outcome in glioblastoma patients, and a high level of CD4⁺ TILs combined with low CD8⁺ TILs was associated with unfavourable prognosis.     

Intratumoral regulatory T cells upregulate immunosuppressive molecules in head and neck cancer patients

Background:

Although regulatory T cells (Treg) are highly enriched in human tumours compared with peripheral blood, expression of the immune-checkpoint receptors, immunosuppressive molecules and function of Treg in these two sites remains undefined.

Methods:

Tumour-infiltrating lymphocytes and peripheral blood lymphocytes were isolated from a cohort of head and neck squamous cell carcinoma (HNSCC) patients. The immunosuppressive phenotypes and function of intratumoral Treg were compared with those of peripheral blood Treg.

Results:

The frequency of immune-checkpoint receptor-positive cells was higher on intratumoral FOXP3⁺CD25^hi Treg compared with circulating Treg (CTLA-4, P=0.002; TIM-3, P=0.002 and PD-1, P=0.002). Immunosuppressive effector molecules, LAP and ectonucleotidase CD39 were also upregulated on intratumoral FOXP3⁺ Treg (P=0.002 and P=0.004, respectively). CTLA-4 and CD39 were co-expressed on the majority of intratumoral FOXP3⁺CD4⁺ Treg, suggesting that these molecules have a key role in regulatory functions of these cells in situ. Notably, intratumoral Treg exhibited more potently immunosuppressive activity than circulating Treg.

Conclusion:

These results indicate that intratumoral Treg are more immunosuppressive than circulating Treg and CTLA-4 and CD39 expressed can be potential target molecules to inhibit suppressive activities of intratumoral Treg in situ.     

PD-1+ CD8+ T cells are exhausted in tumours and functional in draining lymph nodes of colorectal cancer patients

Background:

The blockade of PD-1–PD-L1 pathway is emerging as an effective therapeutic strategy for several advanced cancers. But the immune regulatory role of PD-1–PD-L1 pathway is not clear in colorectal cancer (CRC) patients. This study aims to evaluate the role of PD-1–PD-L1 pathway in CD8⁺ T-cell functions in tumour-draining lymph nodes (TDLNs) and tumours of CRC patients.

Methods:

PD-1 expression on CD8⁺ T cells was examined by flow cytometry, and PD-L1 expression in TDLNs and tumour tissues were examined by immunohistochemistry. Production of IFN-γ, IL-2 and expression of granzyme B, perforin in CD8⁺ T cells were detected by intracellular staining.

Results:

PD-1 expression is markedly upregulated on CD8⁺ T cells in TDLNs and tumours compared with that in peripheral blood. PD-1-expressing CD8⁺ T cells are competent for production of cytokine (IL-2 and IFN-γ) and perforin in the tumour-free lymph nodes (TFLNs), but exhibit exhausted phenotypes in tumours. In addition, PD-L1 is highly expressed in tumours rather than TFLNs, which is closely correlated with the impairment of IFN-γ production of tumour-infiltrating PD-1⁺ CD8⁺ T cells.

Conclusions:

Our findings suggest a suppressive effect of PD-1 on CD8⁺ T-cell function in tumours, but not in TFLNs.     

Automated prognostic pattern detection shows favourable diffuse pattern of FOXP3+ Tregs in follicular lymphoma

Background:

Histopathological prognostication relies on morphological pattern recognition, but as numbers of biomarkers increase, human prognostic pattern-recognition ability decreases. Follicular lymphoma (FL) has a variable outcome, partly determined by FOXP3 Tregs. We have developed an automated method, hypothesised interaction distribution (HID) analysis, to analyse spatial patterns of multiple biomarkers which we have applied to tumour-infiltrating lymphocytes in FL.

Methods:

A tissue microarray of 40 patient samples was used in triplex immunohistochemistry for FOXP3, CD3 and CD69, and multispectral imaging used to determine the numbers and locations of CD3⁺, FOXP3/CD3⁺ and CD69/CD3⁺ T cells. HID analysis was used to identify associations between cellular pattern and outcome.

Results:

Higher numbers of CD3⁺ (P=0.0001), FOXP3/CD3⁺ (P=0.0031) and CD69/CD3⁺ (P=0.0006) cells were favourable. Cross-validated HID analysis of cell pattern identified patient subgroups with statistically significantly different survival (35.5 vs 142 months, P=0.00255), a more diffuse pattern associated with favourable outcome and an aggregated pattern with unfavourable outcome.

Conclusions:

A diffuse pattern of FOXP3 and CD69 positivity was favourable, demonstrating ability of HID analysis to automatically identify prognostic cellular patterns. It is applicable to large numbers of biomarkers, representing an unsupervised, automated method for identification of undiscovered prognostic cellular patterns in cancer tissue samples.     

High levels of circulating CD34+ cells at autologous stem cell collection are associated with favourable prognosis in multiple myeloma

Background:

High-dose chemotherapy with autologous stem cell transplantation is a cornerstone in the first-line treatment of multiple myeloma patients. However, only few factors have been identified affecting the outcome in such patients. We hypothesised that varying levels of mobilised CD34+ cells confer prognostic information in myeloma patients undergoing high-dose chemotherapy.

Methods:

We determined circulating CD34+ cells at the day of peripheral stem cell collection in 158 consecutive myeloma patients between January 2001 and August 2010. Patients were stratified into two groups (super vs normal mobilisers) with a cutoff of 100 000 peripheral CD34+ cells per ml.

Results:

We found that patients with more than 100 000 peripheral CD34+ cells per ml had a better overall survival (P=0.005) and a prolonged time to progression (P=0.0398) than patients with CD34+ cell counts below 100 000 CD34+ cells per ml. High levels of CD34+ cells were an independent marker for better overall survival and time to progression in a multivariate analysis that included disease stage, response at transplant, light-chain subtype, age, sex, and height.

Conclusion:

Our results suggest that high levels of mobilised peripheral CD34+ cells are associated with favourable outcome in myeloma patients undergoing autologous transplantation.     

The intratumoural subsite and relation of CD8+ and FOXP3+ T lymphocytes in colorectal cancer provide important prognostic clues

Background:

To find improved tools for prognostic evaluation in patients with colorectal cancer (CRC), we have analysed how infiltration of cytotoxic T lymphocytes (CD8⁺) and regulatory T lymphocytes (FoxP3⁺) correlates to prognosis, not only according to quantity and relation, but also to subsite within tumours of different molecular characteristics (microsatellite instability and CpG island methylator phenotype status).

Methods:

CD8 and FOXP3 expression was evaluated by immunohistochemistry in 426 archival tumour tissue samples from patients surgically resected for CRC. The average infiltration of CD8⁺ and FOXP3⁺ cells was assessed along the tumour invasive front, in the tumour centre and within the tumour epithelium (intraepithelial).

Results:

We found that infiltration of CD8⁺ T lymphocytes within the tumour epithelium provided the strongest prognostic information (P<0.001). At the tumour invasive front and tumour centre, FOXP3 expression withheld the strongest association to prognosis (P<0.001), suggesting FOXP3⁺ T-lymphocyte infiltration to be a better prognostic tool than CD8⁺ T lymphocytes at these intratumoural subsites. We further analysed the possible prognostic impact of the relation between these T-cell subsets, finding that a high intraepithelial CD8 expression was associated with a better patient outcome, independent of FOXP3 infiltration. In groups of low intraepithelial CD8 expression, however, a high infiltration rate of FOXP3⁺ cells at the tumour invasive front, significantly improved prognosis.

Conclusions:

Analyses of intraepithelial infiltration of CD8⁺ T lymphocytes, infiltration of FOXP3⁺ T lymphocytes at the tumour front or centre, and the relation between these subsets, may be a valuable tool for predicting prognosis in colon cancer.     

Clinical value of circulating endothelial cells and of soluble CD146 levels in patients undergoing surgery for non-small cell lung cancer

Background:

Previous studies indicate that endothelial injury, as demonstrated by the presence of circulating endothelial cells (CECs), may predict clinical outcome in cancer patients. In addition, soluble CD146 (sCD146) may reflect activation of angiogenesis. However, no study has investigated their combined clinical value in patients undergoing resection for non-small cell lung cancer (NSCLC).

Methods:

Data were collected from preoperative blood samples from 74 patients who underwent resection for NSCLC. Circulating endothelial cells were defined, using the CellSearch Assay, as CD146+CD105+CD45−DAPI+. In parallel, sCD146 was quantified using an ELISA immunoassay. These experiments were also performed on a group of 20 patients with small-cell lung cancer, 60 healthy individuals and 23 patients with chronic obstructive pulmonary disease.

Results:

The CEC count and the plasma level of sCD146 were significantly higher in NSCLC patients than in the sub-groups of controls (P<0.001). Moreover, an increased CEC count was associated with higher levels of sCD146 (P=0.010). Both high CEC count and high sCD146 plasma level at baseline significantly correlated with shorter progression-free survival (P<0.001, respectively) and overall survival (P=0.005; P=0.009) of NSCLC patients.

Conclusions:

The present study provides supportive evidence to show that both a high CEC count and a high sCD146 level at baseline correlate with poor prognosis and may be useful for the prediction of clinical outcome in patients undergoing surgery for NSCLC.     

Mismatch repair status in patients with primary operable colorectal cancer: associations with the local and systemic tumour environment

Background:

Mismatch repair-deficient (dMMR) colorectal cancer (CRC) is associated with a conspicuous local immune infiltrate; however, its relationship with systemic inflammatory responses remains to be determined. The present study aims to examine the relationships and prognostic value of assessment of the local and systemic environment in the context of MMR status in patients with CRC.

Methods:

The relationship between MMR status, determined using immunohistochemistry, and the local inflammatory cell infiltrate, differential white cell count, neutrophil : platelet score (NPS), neutrophil : lymphocyte ratio and modified Glasgow Prognostic Score (mGPS), and cancer-specific survival was examined in 228 patients undergoing resection of stage I–III CRC.

Results:

Thirty-five patients (15%) had dMMR CRC. Mismatch repair deficiency was associated with a higher density of CD3⁺, CD8⁺ and CD45R0⁺ T lymphocytes within the cancer cell nests and an elevated mGPS (mGPS2: 23% vs 9%, P=0.007) and NPS (NPS2: 19% vs 3%, P=0.001). CD3⁺ density (P<0.001), mGPS (P=0.01) and NPS (P=0.042) were associated with survival independent of MMR status (P=0.367) and stratified 5-year survival of patients with MMR-competent CRC from 94% to 67%, 83% to 46% and 78% to 60% respectively.

Conclusions:

Mismatch repair deficiency was associated with local and systemic environments, and in comparison with their assessment, dMMR had relatively poor prognostic value in patients with primary operable CRC. In addition to MMR status, local and systemic inflammatory responses should be assessed in these patients.     

CD8+ lymphocytes/?tumour-budding index: an independent prognostic factor representing a ‘pro-/anti-tumour' approach to tumour host interaction in colorectal cancer

Background:

The tumour-host interaction at the invasive front of colorectal cancer, including the epithelial–mesenchymal transition and its hallmark ‘tumour budding'', is an important area of investigation in terms of prognosis. The aim of this study was to determine the prognostic impact of a ‘pro-/anti-tumour'' approach defined by an established ‘pro-tumour'' (tumour budding) and host-related ‘anti-tumour'' factor of the adaptive immunological microenvironment (CD8+ lymphocytes).

Methods:

Double immunostaining for CK22/CD8 on whole tissue sections (n=279; Cohort 1) and immunohistochemistry for CD8+ using tissue microarrays (n=191; Cohort 2) was carried out. Tumour buds, CD8+ and CD8+ T-lymphocytes : tumour buds indices were evaluated per high-power field.

Results:

In Cohort 1, a low-CD8+/ buds index was associated with lymph node metastasis (P<0.001), vascular invasion (P=0.009), worse survival in univariate (P<0.001) and multivariable (P<0.001) analysis, and furthermore in lymph node-negative patients (P=0.002). In Cohort 2, the CD8+/ buds index was associated with T stage (P<0.001), N stage (P=0.041), vascular invasion (P=0.005) and survival in patients with TNM stage II (P=0.019), stage III (P=0.004), and adjuvantly untreated (P=0.009) and treated patients (P<0.001).

Conclusion:

The CD8+ lymphocyte : tumour-budding index is an independent prognostic factor in colorectal cancer and a promising approach for a future prognostic score for patients with this disease.     

Prospective neoadjuvant analysis of PET imaging and mechanisms of resistance to Trastuzumab shows role of HIF1 and autophagy

Background:

Although Trastuzumab has improved survival of HER2+ breast cancer patients, resistance to the agent pre-exists or develops through the course of therapy. Here we show that a specific metabolism and autophagy-related cancer cell phenotype relates to resistance of HER2+ breast cancer to Trastuzumab and chemotherapy.

Methods:

Twenty-eight patients with locally advanced primary breast cancer were prospectively scheduled to received one cycle of Trastuzumab followed by a new biopsy on day 21, followed by taxol/Trastuzumab chemotherapy for four cycles before surgery. FDG PET/CT scan was used to monitor tumour response. Tissue samples were immunohistochemically analysed for metabolism and autophagy markers.

Results:

In pre-Trastuzumab biopsies, the LC3A+/HER2+ cell population was correlated with HIF1α expression (P=0.01), while GLUT1 and LC3B expression were correlated with Ki67 proliferation index (P=0.01 and P=0.01, respectively). FDG PET tumour dimensions before therapy were correlated with LC3B expression (P=0.005). Administration of Trastuzumab significantly reduced clinical and PET-detected tumour dimensions (P<0.01). An inverse association of tumour response with the percentage of cells expressing HIF1α at baseline was documented (P=0.01). Administration of Trastuzumab resulted in a decrease of the proliferation index (P=0.004), GLUT1 (P=0.04) and HER2 (P=0.01) expression. In contrast, the percentage of LC3A+/HER2+ cells was increased (P=0.01). High baseline HIF1α expression was the only parameter associated with poorer pathological response to preoperative chemotherapy (P=0.001).

Conclusions:

As the HER2+/LC3A+ phenotype, which often overexpresses HIF1α, is a major subpopulation increasing after therapy with Trastuzumab, LC3A- and HIF1α-targeting therapies should be investigated for the augmentation of anti-HER2 therapy efficacy.     

Brain metastasis in renal cancer patients: metastatic pattern,tumour-associated macrophages and chemokine/chemoreceptor expression

Background:

The mechanisms of brain metastasis in renal cell cancer (RCC) patients are poorly understood. Chemokine and chemokine receptor expression may contribute to the predilection of RCC for brain metastasis by recruitment of monocytes/macrophages and by control or induction of vascular permeability of the blood–brain barrier.

Methods:

Frequency and patterns of brain metastasis were determined in 246 patients with metastatic RCC at autopsy. Expression of CXCR4, CCL7 (MCP-3), CCR2 and CD68⁺ tumour-associated macrophages (TAMs) were analysed in a separate series of 333 primary RCC and in 48 brain metastases using immunohistochemistry.

Results:

Fifteen percent of 246 patients with metastasising RCC had brain metastasis. High CXCR4 expression levels were found in primary RCC and brain metastases (85.7% and 91.7%, respectively). CCR2 (52.1%) and CCL7 expression (75%) in cancer cells of brain metastases was more frequent compared with primary tumours (15.5% and 16.7%, respectively; P<0.0001 each). The density of CD68⁺ TAMs was similar in primary RCC and brain metastases. However, TAMs were more frequently CCR2-positive in brain metastases than in primary RCC (P<0.001).

Conclusion:

Our data demonstrate that the monocyte-specific chemokine CCL7 and its receptor CCR2 are expressed in tumour cells of RCC. We conclude that monocyte recruitment by CCR2 contributes to brain metastasis of RCC.     

CD133+ circulating haematopoietic progenitor cells predict for response to sorafenib plus erlotinib in non-small cell lung cancer patients

Background:

Blood-based biomarkers may be particularly useful for patient selection and prediction of treatment response for angiogenesis inhibitors. Circulating endothelial cells (CECs) and haematopoietic progenitor cells (HPCs) might have a role in tumour angiogenesis and in tumour growth. Measurement of CECs and HPCs in the blood of patients could be a simple, non-invasive way to monitor or predict responses to treatment.

Methods:

(VEGFR2⁺) CECs_, (CD133⁺) HPCs, plasma vascular endothelial growth factor (VEGF) and erythropoietin were measured in blood from 25 non-small cell lung cancer (NSCLC) patients before and during treatment with sorafenib plus erlotinib (SO/ER). In order to assess the drug specificity of changes in CECs and HPCs, 18 patients treated with bevacizumab plus erlotinib (BV/ER) and 10 patients with erlotinib (ER) monotherapy were studied. Response was measured in all patient groups by Response Evaluation Criteria in Solid Tumors (RECIST).

Results:

At day 7, SO/ER-treated patients showed a three-fold increase in CECs (P<0.0001) comparable to BV/ER-treated patients (P<0.01), and the CECs did not change with erlotinib treatment (P=0.8). At day 7, CD133⁺/HPCs decreased with SO/ER treatment (P<0.0001). HPC numbers did not change with either BV/ER or erlotinib. In SO/ER-treated patients pre-treatment CD133⁺/HPCs were significantly lower in responders (P=0.01) and pre-treatment CD133⁺/HPC numbers lower than the median correlated with a longer time-to-progression (TTP) (P=0.037).

Conclusion:

Pre-treatment CD133⁺/HPCs are a promising candidate biomarker to further explore for use in selecting NSCLC patients who might benefit from SO/ER treatment.     

The in situ local immune response,tumour senescence and proliferation in colorectal cancer

Background:

Immune cell infiltrates are important determinants of colorectal cancer (CRC) outcome. Their presence may be driven by tumour or host-specific factors. From previous studies in mice, senescence, a state of cell cycle arrest, may moderate tumour progression through upregulation of antitumour immune responses. The relationships between senescence and immune infiltrates have not previously been studied in humans. We explore whether a marker of senescence (p16^ink4a) in combination with low level expression of a proliferation marker (ki-67) relate to T cell infiltrates in CRC, and whether p16^ink4a, Ki-67 and immune infiltrates have similar prognostic value.

Methods:

Immunostaining of p16^inka and Ki-67 was performed within a CRC tissue microarray. Nuclear p16^inka and Ki-67 were categorised as high/low. T-cell markers, CD3, CD45RO, CD8 and FOXP3 were scored separately as high/low grade in three areas of the tumour: the invasive margin (IM), tumour stroma and cancer cell nests (CCNs).

Results:

Two hundred and thirty stage I–III cancers were studied. High nuclear p16^ink4a was expressed in 63% and high proliferation (Ki-67 >15%) in 61%. p16^ink4a expression was associated with reduced CD45RO+ cells at the IM (P<0.05) and within the stroma (P<0.05) and reduced CD8+ cells at the IM (P<0.01). A low Ki-67 proliferative index was associated with reduced density of CD3+ cells in CCNs (P<0.01), reduced CD45RO+ cells at the IM (P<0.05) and within the CCNs (P<0.001), reduced FOXP3+ cells at the IM (P<0.001), within the stroma (P=0.001) and within CCNs (P<0.001) and reduced CD8+ cells at the IM (P<0.05) and within the CCNs (P<0.05). Tumours with both a low proliferative index and expression of p16^ink4a demonstrated similar consistent relationships with reduced densities of T-cell infiltrates. On multivariate analysis, TNM stage (P<0.001), low CD3 cells at the IM (P=0.014), low CD8 cells at the IM (P=0.037), low proliferation (Ki-67; P=0.013) and low senescence (p16^ink4a; P=0.002) were independently associated with poorer cancer survival.

Conclusion:

Senescence, proliferation and immune cell infiltrates are independent prognostic factors in CRC. Although related to survival, p16^ink4a-associated senescence is not associated with an upregulation of antitumour T-cell responses.     

Prognostic impact of tumour-infiltrating immune cells on biliary tract cancer

Background:

Biliary tract cancers (BTC) are relatively rare malignant tumours with poor prognosis. It is known from other solid neoplasms that antitumour inflammatory response has an impact on tumour behaviour and patient outcome. The aim of this study was to provide a comprehensive characterisation of antitumour inflammatory response in human BTC.

Methods:

Tumour-infiltrating T lymphocytes (CD4+, CD8+, and Foxp3+), natural killer cells (perforin+), B lymphocytes (CD20+), macrophages (CD68+) as well as mast cells (CD117+) were assessed by immunohistochemistry in 375 BTC including extrahepatic (ECC; n=157), intrahepatic (ICC; n=149), and gallbladder (GBAC; n=69) adenocarcinomas. Overall and intraepithelial quantity of tumour-infiltrating immune cells was analysed. Data were correlated with clinicopathological variables and patient survival.

Results:

The most prevalent inflammatory cell type in BTC was the T lymphocyte. Components of the adaptive immune response decreased, whereas innate immune response components increased significantly in the biliary intraepithelial neoplasia – primary carcinoma – metastasis sequence. BTC patients with intraepithelial tumour-infiltrating CD4+, CD8+, and Foxp3+ T lymphocytes showed a significantly longer overall survival. Number of total intraepithelial tumour-infiltrating Foxp3+ regulatory T lymphocytes (HR: 0.492, P=0.002) and CD4+ T lymphocytes (HR: 0.595, P=0.008) were tumour grade- and UICC-stage-independent prognosticators. The subtype-specific evaluation revealed that the tumour-infiltrating lymphocytic infiltrate is a positive outcome predictor in ECC and GBAC but not in ICC.

Conclusion:

Our findings characterise the immune response in cholangiocarcinogenesis and identify inflammatory cell types that influence the outcome of BTC patients. Further, we show that BTC subtypes show relevant differences with respect to density, quality of inflammation, and impact on patient survival.     

CD44s signals the acquisition of the mesenchymal phenotype required for anchorage-independent cell survival in hepatocellular carcinoma

Background:

Circulating tumour cells (CTCs) have an important role in metastatic processes, but details of their basic characteristics remain elusive. We hypothesised that CD44-expressing CTCs show a mesenchymal phenotype and high potential for survival in hepatocellular carcinoma (HCC).

Methods:

Circulating CD44⁺CD90⁺ cells, previously shown to be tumour-initiating cells, were sorted from human blood and their genetic characteristics were compared with those of tumour cells from primary tissues. The mechanism underlying the high survival potential of CD44-expressing cells in the circulatory system was investigated in vitro.

Results:

CD44⁺CD90⁺ cells in the blood acquired epithelial–mesenchymal transition, and CD44 expression remarkably increased from the tissue to the blood. In Li7 and HLE cells, the CD44^high population showed higher anoikis resistance and sphere-forming ability than did the CD44^low population. This difference was found to be attributed to the upregulation of Twist1 and Akt signal in the CD44^high population. Twist1 knockdown showed remarkable reduction in anoikis resistance, sphere formation, and Akt signal in HLE cells. In addition, mesenchymal markers and CD44s expression were downregulated in the Twist1 knockdown.

Conclusions:

CD44s symbolises the acquisition of a mesenchymal phenotype regulating anchorage-independent capacity. CD44s-expressing tumour cells in peripheral blood are clinically important therapeutic targets in HCC.