Carsten T. Viehl

High frequency of tumor-infiltrating FOXP3+ regulatory T cells predicts improved survival in mismatch repair-proficient colorectal cancer patients†

Regulatory T cells (Treg) inhibit the generation of host‐versus‐tumor immunity via suppression of tumor‐specific effector T‐cell responses and development of immune tolerance to neoplastic cells. The transcription factor forkhead box P3 (FOXP3) is an intracellular key molecule for Treg development and function and is considered to represent the most specific Treg cell marker. The aim of this study was to analyze the frequency and prognostic impact of tumor‐infiltrating FOXP3+ Treg in colorectal cancer (CRC) stratified by mismatch‐repair (MMR) status. Using the tissue microarray technique, 1,420 tumor samples were immunohistochemically stained for FOXP3 and stratified into 1,197 MMR‐proficient and 223 MMR‐deficient CRCs. Additionally, the 1,197 MMR‐proficient CRCs were randomized into 2 subgroups (Test Groups 1 and 2; n = 613 and 584, respectively). In both MMR‐proficient CRC subgroups high frequency tumor‐infiltrating FOXP3+ Treg was associated with early T stage (p = 0.001 and <0.001), tumor location (p = 0.01 and 0.045) and increased 5‐year survival rate (p = 0.004 and <0.001), whereas in MMR‐deficient CRCs an association between FOXP3+ Treg and absence of lymph node involvement (p = 0.023), absence of vascular invasion (p = 0.023) and improved 5‐year survival rate (p = 0.029) could be detected. In a multivariable analysis including age, gender, T stage, N stage, tumor grade, vascular invasion, and tumor border configuration, a high FOXP3+ Treg frequency was an independent prognostic factor in both MMR‐proficient CRC subsets (p = 0.019 and p = 0.007), but not in the MMR‐deficient CRCs (p = 0.13). Therefore, high frequency of tumor‐infiltrating FOXP3+ Treg is associated with early T stage and independently predicts improved disease‐specific survival in MMR‐proficient CRC patients.

Clinical impact of programmed cell death ligand 1 expression in colorectal cancer.

BACKGROUND Programmed cell death 1 (PD-1) receptor triggering by PD ligand 1 (PD-L1) inhibits T cell activation. PD-L1 expression was detected in different malignancies and associated with poor prognosis. Therapeutic antibodies inhibiting PD-1/PD-L1 interaction have been developed. MATERIALS AND METHODS A tissue microarray (n=1491) including healthy colon mucosa and clinically annotated colorectal cancer (CRC) specimens was stained with two PD-L1 specific antibody preparations. Surgically excised CRC specimens were enzymatically digested and analysed for cluster of differentiation 8 (CD8) and PD-1 expression. RESULTS Strong PD-L1 expression was observed in 37% of mismatch repair (MMR)-proficient and in 29% of MMR-deficient CRC. In MMR-proficient CRC strong PD-L1 expression correlated with infiltration by CD8(+) lymphocytes (P = 0.0001) which did not express PD-1. In univariate analysis, strong PD-L1 expression in MMR-proficient CRC was significantly associated with early T stage, absence of lymph node metastases, lower tumour grade, absence of vascular invasion and significantly improved survival in training (P = 0.0001) and validation (P = 0.03) sets. A similar trend (P = 0.052) was also detectable in multivariate analysis including age, sex, T stage, N stage, tumour grade, vascular invasion, invasive margin and MMR status. Interestingly, programmed death receptor ligand 1 (PDL-1) and interferon (IFN)-γ gene expression, as detected by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in fresh frozen CRC specimens (n = 42) were found to be significantly associated (r = 0.33, P = 0.03). CONCLUSION PD-L1 expression is paradoxically associated with improved survival in MMR-proficient CRC.

Increased prevalence of regulatory T cells (Treg) is induced by pancreas adenocarcinoma.

We reported earlier that patients with breast or pancreas cancer have an increased prevalence of regulatory T cells (Treg) in the blood and tumor draining lymph nodes (TDLNs) compared with healthy individuals. In the current study, we tested the hypothesis that tumor cells promote the prevalence of Treg. The transforming growth factor-β (TGF-β) secreting murine pancreas adenocarcinoma, Pan02 cell line was injected into syngeneic C57BL/6 mice and the prevalence of Treg in the TDLNs and tumor spleen was measured weekly. Compared with control mice, the prevalence of CD25+CD4+ cells in TDLNs and in tumor spleen increased with tumor growth. Analysis of these CD25+CD4+ T cells in vitro confirmed expression of the Treg marker, Foxp3. In addition, their functional activity resembled that of Treg, as evidenced by a poor proliferative capacity; suppression of proliferation of CD25−CD4 or CD8T cells and inhibition of interferon-γ release by CD25−CD4+ T cells. Reconstitution of Pan02-bearing Rag−/− mice with naive syngeneic CD25−CD4+ T cells induced CD25+CD4+Foxp3+ T cells in TDLNs, but not in the spleen. In contrast, Foxp3 was not detected in unreconstituted Pan02-bearing Rag−/− mice, or reconstituted mice bearing a TGF-β–negative esophageal tumor. Furthermore, administration of neutralizing anti–TGF-β antibody blocked the induction of Foxp3 in reconstituted Pan02-bearing Rag−/− mice. These results mimic earlier in vitro studies showing induction of Foxp3 through CD3 plus CD28 stimulation in the presence of TGF-β. We conclude that Pan02 tumor promotes the prevalence of Treg, in part through the secretion of TGF-β, which may result in immune evasion.

Depletion of CD4+CD25+ regulatory T cells promotes a tumor-specific immune response in pancreas cancer-bearing mice

BackgroundPancreas cancer–bearing mice have an increased prevalence of immunosuppressive CD4+CD25+ regulatory T cells (Treg). Depletion of Treg results in smaller tumors and prolonged host survival. The objective of this study was to evaluate the tumor-specific immune response after depletion of Treg alone or in combination with a cancer vaccine.MethodsFour groups of C57BL/6 mice were challenged with pancreas adenocarcinoma cells (Pan02). The mice received four combinations of antibody-mediated Treg depletion and whole tumor cell vaccination: (1) no treatment, (2) Treg depletion only, (3) vaccination only, or (4) Treg depletion and vaccination. Splenocytes and lymphocytes from tumor-draining lymph nodes were analyzed for tumor-specific release of interferon γ by enzyme-linked immunosorbent spot assay.ResultsIn Treg-depleted and vaccinated mice, a strong statistical trend toward smaller tumors (P = .05) and longer survival (P = .054) was found compared with untreated mice. Treg-depleted mice showed significantly more tumor-specific cells than undepleted mice (P = .02). The number of tumor-specific cells was significantly higher in tumor-draining lymph nodes than in the spleen (P = .002). Similarly, significantly more tumor-specific cells were found in spleens of Treg-depleted and vaccinated mice than in vaccinated-only mice (P = .009).ConclusionsDepletion of Treg alone or in combination with a whole tumor cell vaccine promotes a tumor-specific immune response. Thus, strategies incorporating Treg depletion might improve the efficacy of cancer vaccines.

Selective axillary surgery in breast cancer patients based on positron emission tomography with 18F-fluoro-2-deoxy-D-glucose: not yet!

We prospectively evaluated 31 patients with invasive breast cancer. Preoperative positron emission tomography (PET) with 18F-fluoro-2-deoxy-D-glucose (18F-FDG) for detection of axillary lymph node metastases was compared with the histopathologic status of the sentinel lymph node (SLN). Sensitivity of PET imaging was 43%, specificity and negative predictive value were 94 and 67%, respectively. The smallest metastasis detected by PET measured 3 mm in diameter. The results of this study suggest that detection of small axillary lymph node metastases is limited by the currently achievable spatial resolution of PET imaging. Selective axillary surgery in breast cancer patients based on 18F-FDG PET is yet not possible.

Increasing rates of contralateral prophylactic mastectomy – A trend made in USA?

BACKGROUND Numerous recent studies conducted in the USA reported a considerable rise in the rates of contralateral prophylactic mastectomy (CPM) in early-stage breast cancer (BC). However, this aggressive surgical approach only showed an evidence-based improvement in prognosis for a small subgroup of high-risk BC patients. We present the first European study reporting CPM rates in an unselected cohort of patients with BC. PATIENTS & METHODS The data of 881 patients (≤ 80 years) who underwent surgery for stage I-III BC from 1995 to 2009 at the University of Basel Breast Center was analyzed. RESULTS CPM was performed in 23 of 881 patients (2.6%). Of the entire patient population, 37.5% underwent ipsilateral mastectomy and of those, only 7.0% chose to undergo CPM. Importantly, there was no trend over time in the rate of CPM. Women who chose CPM were significantly younger (54 vs. 60 years, p < 0.001), had more often a positive family history (39.1% vs. 24.4%, p = 0.032) and tumors of lobular histology (30.5% vs. 13.9%, p = 0.035). CONCLUSIONS Our analysis of CPM rates in BC patients, conducted at a European University breast center, does not show the considerably rising CPM rates observed in the USA. We hypothesize that different medico-social and cultural factors, which are highlighted by a different public perception of BC and a different attitude toward plastic surgery, determine the varying CPM rates between the USA and Europe.

High Myeloperoxidase Positive Cell Infiltration in Colorectal Cancer Is an Independent Favorable Prognostic Factor

Background Colorectal cancer (CRC) infiltration by adaptive immune system cells correlates with favorable prognosis. The role of the innate immune system is still debated. Here we addressed the prognostic impact of CRC infiltration by neutrophil granulocytes (NG). Methods A TMA including healthy mucosa and clinically annotated CRC specimens (n = 1491) was stained with MPO and CD15 specific antibodies. MPO+ and CD15+ positive immune cells were counted by three independent observers. Phenotypic profiles of CRC infiltrating MPO+ and CD15+ cells were validated by flow cytometry on cell suspensions derived from enzymatically digested surgical specimens. Survival analysis was performed by splitting randomized data in training and validation subsets. Results MPO+ and CD15+ cell infiltration were significantly correlated (p<0.0001; r = 0.76). However, only high density of MPO+ cell infiltration was associated with significantly improved survival in training (P = 0.038) and validation (P = 0.002) sets. In multivariate analysis including T and N stage, vascular invasion, tumor border configuration and microsatellite instability status, MPO+ cell infiltration proved an independent prognostic marker overall (P = 0.004; HR = 0.65; CI:±0.15) and in both training (P = 0.048) and validation (P = 0.036) sets. Flow-cytometry analysis of CRC cell suspensions derived from clinical specimens showed that while MPO+ cells were largely CD15+/CD66b+, sizeable percentages of CD15+ and CD66b+ cells were MPO−. Conclusions High density MPO+ cell infiltration is a novel independent favorable prognostic factor in CRC.

EpCAM expression varies significantly and is differentially associated with prognosis in the luminal B HER2 + , basal-like, and HER2 intrinsic subtypes of breast cancer

Background:Epithelial cell adhesion molecule (EpCAM) is frequently expressed in breast cancer, and its expression has been associated with poor prognosis. Breast cancer can be subdivided into intrinsic subtypes, differing in prognosis and response to therapy.Methods:To investigate the association between EpCAM expression and prognosis in the intrinsic subtypes of breast cancer, we performed immunohistochemical studies on a tissue microarray encompassing a total of 1365 breast cancers with detailed clinicopathological annotation and outcomes data.Results:We observed EpCAM expression in 660 out of 1365 (48%) cases. EpCAM expression varied significantly in the different intrinsic subtypes. In univariate analyses of all cases, EpCAM expression was associated with a significantly worse overall survival. In the intrinsic subtypes, EpCAM expression was associated with an unfavourable prognosis in the basal-like and luminal B HER2+ subtypes but associated with a favourable prognosis in the HER2 subtype. Consistently, specific ablation of EpCAM resulted in increased cell viability in the breast cancer cell line SKBR3 (ER−, PR−, and HER2+) but decreased viability in the breast cancer cell line MDA-MB-231 (ER−, PR−, and HER2− ).Conclusion:The differential association of EpCAM expression with prognosis in intrinsic subtypes has important implications for the development of EpCAM-targeted therapies in breast cancer.

Molecular investigation of lymph nodes in colon cancer patients using one-step nucleic acid amplification (OSNA): a new road to better staging?

A new diagnostic system, called one‐step nucleic acid amplification (OSNA), has recently been designed to detect cytokeratin 19 mRNA as a surrogate for lymph node metastases. The objective of this prospective investigation was to compare the performance of OSNA with both standard hematoxylin and eosin (H&E) analysis and intensive histopathology in the detection of colon cancer lymph node metastases.

Sentinel lymph node biopsy is associated with improved survival compared to level I & II axillary lymph node dissection in node negative breast cancer patients.

OBJECTIVE The few long-term follow-up data for sentinel lymph node (SLN) negative breast cancer patients demonstrate a 5-year disease-free survival of 96-98%. It remains to be elucidated whether the more accurate SLN staging defines a more selective node negative patient group and whether this is associated with better overall and disease-free survival compared with level I & II axillary lymph node dissection (ALND). METHODS Three-hundred and fifty-five consecutive node negative patients with early stage breast cancer (pT1 and pT2< or =3 cm, pN0/pN(SN)0) were assessed from our prospective database. Patients underwent either ALND (n=178) in 1990-1997 or SLN biopsy (n=177) in 1998-2004. All SLN were examined by step sectioning, stained with H&E and immunohistochemistry. Lymph nodes from ALND specimens were examined by standard H&E only. Neither immunohistochemistry nor step sections were performed in the analysis of ALND specimen. RESULTS The median follow-up was 49 months in the SLN and 133 months in the ALND group. Patients in the SLN group had a significantly better disease-free (p=0.008) and overall survival (p=0.034). After adjusting for other prognostic factors in Cox proportional hazard regression analysis, SLN procedure was an independent predictor for improved disease-free (HR: 0.28, 95% CI: 0.10-0.73, p=0.009) and overall survival (HR: 0.34, 95% CI: 0.14-0.84, p=0.019). CONCLUSIONS This is the first prospective analysis providing evidence that early stage breast cancer patients with a negative SLN have an improved disease-free and overall survival compared with node negative ALND patients. This is most likely due to a more accurate axillary staging in the SLN group.