The PD‐1/PD‐L1 axis and human papilloma virus in patients with head and neck cancer after adjuvant chemoradiotherapy: A multicentre study of the German Cancer Consortium Radiation Oncology Group (DKTK‐ROG)

We examined the prognostic role of PD‐1+ and CD8+ tumor infiltrating lymphocytes (TILs), and PD‐L1+ cells in patients with squamous cell carcinoma of the head and neck (SCCHN) treated with surgery and postoperative chemoradiotherapy (CRT). FFPE samples from 161 patients were immunohistochemically stained for PD‐1, CD8 and PD‐L1. The immune marker expression was correlated with clinicopathologic characteristics, and overall survival (OS), local progression‐free survival (LPFS) and distant metastases free‐survival (DMFS), also in the context of HPV16 DNA/p16 status. The median follow‐up was 48 months (range: 4–100). The 2‐year‐OS was 84.1% for the entire cohort. High PD‐1 and PD‐L1 expression were more common in patients with positive HPV16 DNA (p < 0.001 and p = 0.008, respectively) and high infiltration by CD8+ TILs (p < 0.001 for both markers). High PD‐L1 expression correlated with superior OS (p = 0.025), LPFS (p = 0.047) and DMFS (p = 0.048) in multivariable analysis, whereas no significance could be demonstrated for PD‐1. Patients with CD8^high/PD‐L1^high expression had favorable outcome (p < 0.001 for all endpoints) compared to other groups. We validated the superior OS data on CD8^high/PD‐L1^high using the Cancer Genome Atlas TCGA dataset (n = 518; p = 0.032). High PD‐L1 expression was a favorable prognostic marker in HPV16‐negative but not HPV16‐positive patients. In conclusion, HPV‐positive tumors showed higher expression of immune markers. PD‐L1 expression constitutes an independent prognostic marker in SCCHN patients post‐adjuvant CRT. In conjunction with CD8 status, these data provide an important insight on the immune contexture of SCCHN and are directly relevant for future treatment stratification with PD‐1/PD‐L1 immune checkpoint inhibitors to complement CRT.     

Heat shock protein 70 and tumor‐infiltrating NK cells as prognostic indicators for patients with squamous cell carcinoma of the head and neck after radiochemotherapy: A multicentre retrospective study of the German Cancer Consortium Radiation Oncology Group (DKTK‐ROG)

Tumor cells frequently overexpress heat shock protein 70 (Hsp70) and present it on their cell surface, where it can be recognized by pre‐activated NK cells. In our retrospective study the expression of Hsp70 was determined in relation to tumor‐infiltrating CD56⁺ NK cells in formalin‐fixed paraffin embedded (FFPE) tumor specimens of patients with SCCHN (N = 145) as potential indicators for survival and disease recurrence. All patients received radical surgery and postoperative cisplatin‐based radiochemotherapy (RCT). In general, Hsp70 expression was stronger, but with variable intensities, in tumor compared to normal tissues. Patients with high Hsp70 expressing tumors (scores 3–4) showed significantly decreased overall survival (OS; p = 0.008), local progression‐free survival (LPFS; p = 0.034) and distant metastases‐free survival (DMFS; p = 0.044), compared to those with low Hsp70 expression (scores 0–2), which remained significant after adjustment for relevant prognostic variables. The adverse prognostic value of a high Hsp70 expression for OS was also observed in patient cohorts with p16‐ (p = 0.001), p53‐ (p = 0.0003) and HPV16 DNA‐negative (p = 0.001) tumors. The absence or low numbers of tumor‐infiltrating CD56⁺ NK cells also correlated with significantly decreased OS (p = 0.0001), LPFS (p = 0.0009) and DMFS (p = 0.0001). A high Hsp70 expression and low numbers of tumor‐infiltrating NK cells have the highest negative predictive value (p = 0.00004). In summary, a strong Hsp70 expression and low numbers of tumor‐infiltrating NK cells correlate with unfavorable outcome following surgery and RCT in patients with SCCHN, and thus serve as negative prognostic markers.     

PD‐L1 expression of the residual tumor serves as a prognostic marker in local advanced breast cancer after neoadjuvant chemotherapy

This study sought to investigate the prevalence of programmed death ligand 1 (PD‐L1) and its prognostic value in patients with residual tumors after neoadjuvant chemotherapy (NCT) for locally advanced breast cancer. A total of 309 patients considered as non‐pathological complete responders (non‐pCR) after NCT followed by mastectomy were selected. The expression of PD‐L1 and tumor‐infiltrating lymphocytes (TILs) in residual breast cancer cells was assessed by immunohistochemistry in surgical specimens. The median density was used to classify PD‐L1 expression from low to high. The prognostic value of various clinicopathological factors was evaluated. The expression of PD‐L1 was more commonly observed in patients with low levels of total TILs (p < 0.001), high levels of FOXP3+ TILs (p < 0.001) and low levels of CD8+ TILs (p < 0.001). This served as an independent prognostic factor for both relapse‐free survival (Hazard ratio = 1.824, p = 0.013) and overall survival (OS) (Hazard ratio = 2.585, p = 0.001). High expression of PD‐L1 was correlated to worse survival, which is most significantly observed in triple‐negative patients. Patients classified as PD‐L1‐high/CD8‐low exhibited relatively unfavorable survival, whereas patients with either low expression of PD‐L1 or high expression of CD8 had similar outcomes. PD‐L1 expression in residual tumor can be used as a prognostic marker in non‐pCR patients after receiving NCT for breast cancer, which highlights the importance of immune evasion in the therapeutic vulnerability of chemoresistant cancer cells as well as the potential of anti‐PD‐L1 treatments in non‐pCR responders.     

Different outcome of invasive cervical cancer associated with high‐risk versus intermediate‐risk HPV genotype

Human papillomavirus (HPV) DNA sequences are associated with the large majority of invasive cervical carcinoma but the role of specific genotype(s) in the outcome of the disease is still debated. To determine the viral epidemiology in the French population of patients and the prognostic value of HPV genotypes in cervical cancer, we performed a retrospective study in 515 patients treated in our Institution from 1985 to 2005. Ninety‐six percent of the cases were found associated with HPV DNA whereas 4% remained HPV negative. High‐risk HPV 16/18 genotypes were found in 70% of the cases. HPV 18 was more frequently associated with adenocarcinoma (40.6%) than HPV 16 (10.4%) and found in tumours developed in younger women (mean age, 45.8 years) than HPV 16 (48.3 years) or other HPV types (53.6 years). In multivariate analysis, node involvement (p < 0.0001), parametria invasion (p = 0.009), tumour size (p = 0.01) and HPV status (p = 0.02) were associated with disease‐free survival (median follow‐up 95 months). Disease outcome was better in tumours associated with intermediate risk HPV types (HPV 31, 33, 35, 39, 52, 53, 58, 59, 73) than in tumours with high oncogenic types (HPV 16, 18, 45) (p = 0.03). Node status and tumour size remained prognostic factor for overall survival. Our data show that HPV genotype is one of the biological factors associated with the outcome of cervical cancer. One third of invasive carcinoma were not associated with HPV 16/18, indicating that the screening for cervical neoplasia should be maintained after prophylactic vaccination against these HPV genotypes. © 2008 Wiley‐Liss, Inc.     

Human papillomavirus DNA load and p16INK4a expression predict for local control in patients with anal squamous cell carcinoma treated with chemoradiotherapy

As the detection rate of HPV‐DNA in anal carcinoma commonly exceeds 90%, a comparison between sole HPV‐positive and HPV‐negative cancers with respect to treatment response following chemoradiotherapy (CRT) and long‐term oncological outcome is challenging. Against this background, we aimed to assess HPV types and HPV DNA load in formalin‐fixed paraffin‐embedded tissue (FFPE) of 95 patients treated with standard CRT for anal cancer to correlate viral load (≤/> median) with local failure, distant metastases, cancer‐specific (CSS) and overall survival (OS) rates. Various clinicopathologic parameters and the immunohistochemical marker p16^INK4a were evaluated for any correlation with HPV16 DNA load and were included in uni‐ and multivariate analyses. The overall prevalence of HPV DNA was 95.8% with HPV16 monoinfection being the most commonly encountered HPV type (78.9%), followed by HPV16 and 31, 35, 39, 44, 58, 66 and 81 dual infection in 9 patients (9.5%). HPV16 DNA load was significantly associated with p16^INK4a expression (p = 0.001). Patients with HPV16 DNA load ≤ median and low p16^INK4a expression showed significantly worse local control (HPV16 DNA load: univariate p = 0.023, multivariate p = 0.042; p16^INK4a: univariate p = 0.021), and OS (HPV16 DNA load: univariate p = 0.02, multivariate p = 0.03). Moreover, a combined HPV16 DNA load and p16^INK4a variable revealed a significant correlation to decreased local failure, and increased CSS and OS (p = 0.019, p = 0.04 and p = 0.031). In conclusion, these data indicate that HPV16 DNA load and p16^INK4a expression are significant prognostic factors for local tumor control and overall survival of patients with anal SCC following CRT.     

Predictive factors of the tumor immunological microenvironment for long‐term follow‐up in early stage breast cancer

The aim of this research was to investigate the correlation of immunologic factors in the tumor environment of breast cancer, using immunohistological staining to evaluate the expression of programmed death 1/programmed death ligand 1 (PD‐1/PD‐L1), phosphatase and tensin homolog (PTEN), tumor infiltrating lymphocytes (TILs), and macrophages, and to analyze the association between the immunologic factors and clinical outcome for patients with early stage breast cancer (EBC). A total of 97 EBC patients who underwent standard surgery were investigated. Expression of PD‐1/PD‐L1 and PTEN and the density of CD3⁺ TILs, CD8⁺ TILs, and CD163⁺ macrophages were evaluated by immunohistochemical analysis. The association between the immunologic factors and clinical outcome was statistically analyzed. The density of CD3⁺ TILs, CD8⁺ TILs, and CD163⁺ macrophages and non‐expression of PTEN was significantly higher in cases of triple negative breast cancer. CD8⁺ TIL density and CD8⁺/PD‐L1⁺ expression were predictive factors for disease‐free survival and overall survival (OS). Human epidermal growth factor 2 (HER2)‐positive patients with PTEN expression and luminal/HER2‐negative patients without PD‐L1 expression had significantly longer OS compared to patients without PTEN expression (P = 0.049) and with PD‐L1 expression (P = 0.036), respectively. Furthermore, patients with PD‐L1⁺/CD8⁺ expression had worse median progression‐free survival (P = 0.022) and median OS (P = 0.037) compared with patients without PD‐L1⁺/CD8⁺ expression. The CD3⁺ TILs, CD8⁺ TILs, and CD163⁺ macrophages were shown to infiltrate the tumor area of EBC. In particular, triple negative breast cancer had a higher rate of TIL infiltration within the tumor environment. Expression of PTEN and lack of PD‐L1 expression were associated with favorable survival in HER2‐positive and luminal/HER2‐negative EBC patients, respectively. The PD‐L1 expression combined with CD8⁺ density was significantly associated with an aggressive clinical outcome.     

Prognostic significance of tumor‐infiltrating lymphocytes in nondisseminated nasopharyngeal carcinoma: A large‐scale cohort study

The American Joint Committee on Cancer (AJCC) staging system is inadequate for an accurate prognosis in nasopharyngeal carcinoma (NPC). Thus, new biomarkers are under intense investigation. Here, we investigated whether the density of TILs could predict prognosis in NPC. First, we used 1490 cases of nasopharyngeal carcinoma samples from two independent cohorts to evaluate the density and distribution of tumor‐infiltrating lymphocytes (TILs). Second, in one cohort, we assessed associations between TILs and clinical outcomes in 593 randomly selected samples (defined as the training set) and validated findings in the remaining 593 samples (defined as the validation set). Furthermore, we confirmed the prognostic value of TILs in a second independent cohort of 304 cases (defined as the independent set). Based on multivariable Cox regression analysis, we also established an effective prognostic nomogram including TILs to improve accuracy in predicting disease‐free survival (DFS) for patients with nondisseminated NPC. We found that high TILs in the training set were significantly associated with favorable DFS [hazard ratio (HR) 0.41, 95% confidence interval (CI) 0.28–0.58, p < 0.001], overall survival (OS, HR 0.42, 95% CI 0.27–0.64, p < 0.001), distant metastasis‐free survival (DMFS, HR 0.37, 95% CI 0.23–0.58, p < 0.001) and local‐regional recurrent free survival (LRRFS, HR 0.43, 95% CI 0.25–0.73, p = 0.002). Multivariate analysis showed that TILs are an independent prognostic indicator for DFS in all cohorts. In summary, this study indicated that TILs may reflect the immunological heterogeneity of NPC and could represent a new prognostic biomarker.     

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.     

Prognostic value of baseline seric Syndecan‐1 in initially unresectable metastatic colorectal cancer patients: a simple biological score

In first‐line metastatic colorectal cancer (mCRC), baseline prognostic factors allowing death risk and treatment strategy stratification are lacking. Syndecan‐1 (CD138) soluble form was never described as a prognostic biomarker in mCRC. We investigated its additional prognostic value for overall survival (OS). mCRC patients with unresectable disease at diagnosis were treated with bevacizumab‐based chemotherapy in two independent prospective clinical trials (development set: n = 126, validation set: n = 51, study NCT00489697 and study NCT00544011, respectively). Serums were collected at baseline for CD138 measurement. OS determinants were assessed and, based on the final multivariate model, a prognostic score was proposed. Two independent OS prognostic factors were identified: Lactate Dehydrogenase (LDH) high level (p = 0.0066) and log‐CD138 high level (p = 0.0190). The determination of CD138 binary information (cutoff: 75 ng/mL) allowed the assessment of a biological prognostic score with CD138 and LDH values, identifying three risk groups for death (median OS= 38.9, 30.1 and 19.8 months for the low, intermediate and high risk groups, respectively; p < 0.0001). This score had a good discrimination ability (C‐index = 0.63). These results were externally confirmed in the validation set. Our study provides robust evidence in favor of the additional baseline soluble CD138 prognostic value for OS, in mCRC patients. A simple biological scoring system is proposed including LDH and CD138 binary status values.     

CD8‐alpha T‐cell infiltration in human papillomavirus‐related oropharyngeal carcinoma correlates with improved patient prognosis

Patients with human papillomavirus (HPV)‐related oropharyngeal tumors display improved prognosis. The biological basis of this tumor phenotype is poorly understood. We investigated whether increased lymphocyte infiltrate in HPV‐positive oropharyngeal squamous cell carcinomas could account for better prognosis. We previously identified, in an Affymetrix GeneChip analysis of 83 HPV‐unrelated and 11 HPV‐related squamous cell carcinoma of the oropharynx, several candidate genes, including CD8α and CD3ζ. Their expression was validated in this study by qRT–PCR on an independent clinical series of 144 oropharyngeal tumors. Immunohistochemical staining of tumor specimens was performed to evaluate infiltration of tumor stroma by CD8+ and CD4+ lymphocytes. The prognostic value of CD8α and CD3ζ expression levels was measured by Kaplan–Meier and Cox regression model analyses. Immune response‐related signaling pathways were found to be deregulated in HPV‐positive oropharyngeal tumors. Expression of CD8α, CD3ζ, granzyme K, CD28 and integrin αL RNAs was upregulated in HPV‐positive lesions when compared with HPV‐unrelated tumors (p < 0.05). Stroma of HPV‐positive tumors was frequently and strongly infiltrated by CD8α‐ and CD3ζ‐positive T cells. CD8α RNA expression correlated with both improved global (Kaplan–Meier; p = 0.005; Cox regression: p = 0.003) and disease‐free (Cox regression: p = 0.04) survival. CD3ζ RNA expression correlated with improved overall survival (Cox regression: p = 0.024). These results suggest that an increased cytotoxic T‐cell‐based antitumor immune response is involved in improved prognosis of patients with HPV‐positive tumors.     

The detection of circulating human papillomavirus‐specific T cells is associated with improved survival of patients with deeply infiltrating tumors

A detailed analyses of HPV‐specific immunity was performed in a large group of patients with HPV‐induced cervical cancer (CxCa) in relation to HLA‐types and prognostic factors. Patients were HLA‐typed and HPV16/18‐specific T‐cell immunity was assessed by proliferation assay and cytometric bead array using freshly isolated PBMC and by phenotypic analysis of HPV‐specific T cells. The results were analyzed in relation to known disease‐related HLA‐types (DR7, DR13, DR15/DQ06), invasion‐depth and size of tumor, lymph node (LN) status and disease free survival. In total 119 HLA‐typed patients with CxCa were analyzed. Patients expressing the HLA‐DR13 haplotype were underrepresented as compared to the Dutch population (p = 0.014), whereas HLA‐DR7 was overrepresented in patients with HPV16+ CxCa (p = 0.006). In 29 of 94 patients (31%) from whom blood could be tested, a proliferative response to HPV16/18 was detected, which was associated with increased numbers of HPV‐specific CD4+CD25+ (activated) T cells (p = 0.03) and HPV‐specific CD4+CD25+FoxP3‐positive T cells (p = 0.04). The presence of both FoxP3‐positive and negative HPV‐specific CD4+CD25+ T cells was significantly correlated (p = 0.01). Interestingly, the detection of HPV‐specific proliferation was associated with invasion depth (p = 0.020) but not with HLA type, tumor size nor LN status. Moreover, the detection of HPV‐specific immunity was associated with an improved disease free survival (p = 0.04) in patients with deeply infiltrating tumors. In conclusion, HPV‐specific proliferative T‐cell response, comprising higher percentages of HPV‐specific CD25+ and CD25+FoxP3‐positive CD4+T cells, are more frequently detected in patients with deep infiltrating CxCa tumors and associated with an improved survival.     

Double positivity for HPV DNA/p16 in tonsillar and base of tongue cancer improves prognostication: Insights from a large population‐based study

The aim was to explore the overall survival (OS) for palatine tonsillar squamous cell carcinoma (TSCC), subdivided, according to certainty of tonsillar tumour origin, into specified tonsillar squamous cell carcinomas (STSCCs) and nonspecified tonsillar squamous cell carcinomas (NSTSCCs), and base of tongue squamous cell carcinoma (BSCC) when stratifying for HPV DNA status, p16 expression and combined HPV/p16 status. We included all patients (n = 797) diagnosed with TSCCs and BSCCs in Eastern Denmark as registered in the Danish Head and Neck Cancer Group (DAHANCA) database and the Danish Pathology Databank, 2000–2010. Patients were treated according to national guidelines (radiotherapy +/? concomitant cisplatin). All specimens were analysed using HPV DNA PCR and p16 immunohistochemistry. Clinical information was retrieved from the DAHANCA database and the Danish National Patient Registry. Information on vital status was obtained from the Danish Civil Registration System. We observed improved OS for HPV+/p16+ BSCCs compared to HPV?/p16? (hazard ratio for death [HR], 0.15; 95% CI, 0.09–0.24). Among STSCCs, HPV+/p16+ showed the lowest HR (0.19, 95% CI, 0.13–0.29); whereas, HPV?/p16+ showed an intermediate HR (0.39; 95% CI, 0.22–0.70). For NSTSCCs, HPV+/p16+ and HPV?/p16+ showed similar OS (HRs, 0.39; 95% CI, 0.26–0.59; and 0.48; 95% CI, 0.24–0.95, respectively). Combined HPV+/p16+ was a significantly better prognostic marker in BSCCs and STSCCs than HPV DNA and p16, alone (all p‐values < 0.05). Whereas, combined testing in NSTSCC was not better than p16 (p = 0.53), alone. In conclusion, double positivity for HPV/p16 in conjunction with the certainty of tumour site improved prognosis.     

CD8+ and CD4+ tumour infiltrating lymphocytes in relation to human papillomavirus status and clinical outcome in tonsillar and base of tongue squamous cell carcinoma

Patients with human papillomavirus (HPV) positive tonsillar and base of tongue squamous cell carcinoma (TSCC and BOTSCC, respectively) have a better clinical outcome than those with HPV negative tumours, irrespective of treatment. However, to better individualise treatment, additional biomarkers are needed together with HPV status. In a pilot study, we showed that high numbers of CD8⁺ tumour infiltrating lymphocytes (TILs) in HPV_DNA+ p16^INK4a+ TSCC indicated a better outcome. Here this study was extended.Totally 203 TSCC and 77 BOTSCC formalin fixed paraffin embedded tumour biopsies, earlier tested for HPV DNA (79% HPV_DNA+) and p16^INK4a from patients treated with curative intention, were analysed for CD8⁺ and CD4⁺ TILs by immunohistochemistry. Data obtained for 275 patients were correlated to HPV_DNA and p16^INK4a status, overall survival (OS) and disease free survival (DFS).In both HPV_DNA+ and HPV_DNA+ p16^INK4a+ tumours higher CD8⁺ TIL counts correlated to a better 3-year OS (logrank test, both p < 0.001) and 3-year DFS (logrank test, p = 0.003 and p = 0.004 respectively) as compared to the lowest quartile in the groups. A similar pattern was observed when analysing TSCC alone, while for BOTSCC significance was obtained only for 3-year OS. In HPV_DNA? tumours the trend was similar, but significance was obtained again only for 3-year OS. The number of CD4⁺ TILs did not generally correlate to survival.In conclusion, in HPV_DNA+ and/or HPV_DNA+ p16^INK4a+ tumours high CD8⁺ TIL counts indicated a better 3-year OS. This suggests that high CD8⁺ TIL counts together with HPV_DNA+ or HPV_DNA+ p16^INK4a+ could be used when selecting patients for more individualised treatment.     

O6‐methylguanine DNA methyltransferase repairs platinum‐DNA adducts following cisplatin treatment and predicts prognoses of nasopharyngeal carcinoma

Cisplatin (CDDP) is an important anti‐cancer drug commonly used in various human cancers, including nasopharyngeal carcinoma (NPC). How to overcome the drug resistance of CDDP provides opportunities to improve clinical outcomes of NPC. O⁶‐methylguanine–DNA methyltransferase (MGMT) has been well‐characterized to be a therapeutic determinant of O⁶‐alkylguanine alkylating drugs. However, the underlying mechanism and clinical relevance between MGMT and CDDP remain poorly defined in NPC. In this study, we showed that MGMT‐proficient cells were highly resistant to the cytotoxic effects of CDDP as compared to MGMT‐deficient cells. Further studies showed that the platinum level of DNA after CDDP exposure was significantly lower in MGMT‐proficient cells than in MGMT‐deficient cells. Host cell reactivation assay revealed that MGMT protected NPC cells from CDDP‐induced DNA damage by enhancing DNA repair capacity. Importantly, we demonstrated for the first time that MGMT protein directly bound to CDDP‐induced DNA damages. Subsequently, CDDP‐bound MGMT protein became ubiquitinated and was degraded through ubiquitin‐mediated proteasome system. We further analyzed the relationship between MGMT expression and clinical survivals in a cohort of 83 NPC patients. NPC patients who received CDDP‐based concurrent chemoradiotherapy (CCRT), with high MGMT expression level, exhibited shorter progression‐free survival (PFS; p = 0.022) and overall survival (OS; p = 0.015), than patients with low MGMT expression level. Furthermore, high MGMT expression level remained to be an independent prognostic factor for worse PFS (p = 0.01, hazard ratio 2.23) and OS (p = 0.018, hazard ratio 2.14). Our findings suggest that MGMT protein is important to determine the efficacy of CDDP in NPC.     

CD56‐positive lymphocyte infiltration in relation to human papillomavirus association and prognostic significance in oropharyngeal squamous cell carcinoma

Human papillomavirus (HPV)‐related squamous cell carcinoma of the oropharynx (OSCC) are clinical and biological distinct from their HPV‐unrelated counterparts. Patients with HPV‐related OSCC display improved prognosis and therefore we investigated possible immune cell infiltrations associated with this tumor phenotype. We retrospectively analyzed a randomly selected cohort of 140 OSCC for presence of immune cells and HPV by immunohistochemistry and PCR followed by bead‐based hybridization (Luminex technology). HPV prevalence was 24.3% as determined by positive staining of p16INK4a and detection of high risk HPV‐DNA. We found significantly higher numbers of CD56 positive (CD56+) cells in tumor and surrounding microenvironment in HPV‐associated compared to HPV‐negative OSCC (t‐test: p = 0.004 and p = 0.002). For the entire cohort presence of CD56+ cells was associated with increased overall survival independent from HPV (Kaplan–Meier: p = 0.002; Cox regression: p = 0.042). Presence of CD56+ cells also correlated with a better outcome in HPV‐negative and especially in HPV‐negative OSCC with alcohol consumption ≤2 standard drinks per day (Kaplan–Meier: p = 0.05 and p = 0.003). Immunofluorescence localization of granular Granzyme B (GZMB) within CD56+ cells and coexpression of CD16 and CD56 suggests that detected CD56+ cells mainly represent cytotoxic Natural Killer (NK) cells. The fraction of potentially cytotoxic NK cells was significantly higher in HPV‐associated compared to HPV‐negative OSCC (Mann‐Whitney‐U‐Test: p = 0.011). The elevated abundance and activity of cytotoxic NK cells in OSCC with HPV driven carcinogenesis might contribute to favorable outcome in HPV‐related OSCC.     

Neuropilin‐2 and its ligand VEGF‐C predict treatment response after transurethral resection and radiochemotherapy in bladder cancer patients

The standard treatment for invasive bladder cancer is radical cystectomy. In selected patients, bladder‐sparing therapy can be performed by transurethral resection (TURBT) and radio‐chemotherapy (RCT) or radiotherapy (RT). Our published in vitro data suggest that the Neuropilin‐2 (NRP2)/VEGF‐C axis plays a role in therapy resistance. Therefore, we studied the prognostic impact of NRP2 and VEGF‐C in 247 bladder cancer patients (cN0M0) treated with TURBT and RCT (n = 198) or RT (n = 49) and a follow‐up time up to 15 years. A tissue microarray was analyzed by immunohistochemistry. NRP2 expression emerged as a prognostic factor in overall survival (OS; HR: 3.42; 95% CI: 1.48 – 7.86; p = 0.004) and was associated with a 3.85‐fold increased risk of an early cancer specific death (95% CI: 0.91 – 16.24; p = 0.066) in multivariate analyses. Cancer specific survival (CSS) dropped from 166 months to 85 months when NRP2 was highly expressed (p = 0.037). Patients with high VEGF‐C expression have a 2.29‐fold increased risk of shorter CSS (95% CI: 1.03–5.35; p = 0.043) in univariate analysis. CSS dropped from 170 months to 88 months in the case of high VEGF‐C expression (p = 0.041). Additionally, NRP2 and VEGF‐C coexpression is a prognostic marker for OS in multivariate models (HR: 7.54; 95% CI: 1.57–36.23; p = 0.012). Stratification for muscle invasiveness (T1 vs. T2‐T4) confirmed the prognostic role of NRP2 and NRP2/VEGF‐C co‐expression in patients with T2‐T4 but also with high risk T1 disease. In conclusion, immunohistochemistry for NRP2 and VEGF‐C has been determined to predict therapy outcome in bladder cancer patients prior to TURBT and RCT.     

CD8+tumor‐infiltrating lymphocytes at primary sites as a possible prognostic factor of cutaneous angiosarcoma

Tumor‐infiltrating lymphocytes (TILs) have been reported as a prognostic factor in various cancers and are a promising target for immunotherapy. To investigate whether TILs have any impact on the prognosis of angiosarcoma patients, 55 non‐treated patients (40 patients at stage 1 with cutaneous localized tumors, 4 patients at stage 2 with lymph node metastases and 11 patients at stage 3 with distant metastases) with angiosarcoma were evaluated retrospectively by immunohistochemistry stained CD4, CD8, FOXP3 and Ki67. The Kaplan–Meier method was used to estimate overall survival with patients at stage 1. Survival differences were analyzed by the log‐rank test. Patients with higher numbers of CD8⁺ TILs in their primary tumors survived significantly longer compared with patients with lower values. Moreover, the number of CD8 in TILs was positively correlated with a distant metastasis‐free period. The total number of primary TILs (CD4 plus CD8) and CD8⁺ primary TILs of stage 3 patients with distant metastases was positively correlated with their overall survival. To evaluate whether CD8⁺ effector T cells are activated or differentiated, flow cytometric analysis of peripheral blood mononuclear cells (PBMC) was performed. The percentages of CD8⁺ T cells producing IFN‐γ in PBMC were significantly higher in patients with angiosarcoma (n = 10) compared not only with that of healthy controls (n = 20) but also patients with advanced melanoma (n = 11). These results suggest that anti‐tumor immunity is clinically relevant in angiosarcoma.     

Significance of tumor‐infiltrating lymphocytes before and after neoadjuvant therapy for rectal cancer

Neoadjuvant therapy for locally advanced rectal cancer is becoming increasingly common. However, biomarkers predicting the response to neoadjuvant therapy have not been established. Tumor‐infiltrating lymphocytes (TILs) have a crucial effect on tumor progression and survival outcome as the primary host immune response, and an antitumor immune effect has been reported to contribute to the response to radiotherapy and chemotherapy. We investigated the significance of TILs before and after neoadjuvant treatment and the change in the density of those TILs. Sixty‐four patients who underwent radical resection after neoadjuvant treatment for locally advanced rectal cancer were enrolled. The number of TIL subsets was examined using immunohistochemical staining of pretreatment biopsy samples and post‐treatment resected specimens. In both the neoadjuvant chemotherapy cohort and the neoadjuvant chemoradiotherapy cohort, a low density of CD8⁺ TILs in pretreatment biopsy samples was associated with a poor response, and a low density of CD8⁺ TILs in post‐treatment resected specimens was similarly associated with a poor response. In the neoadjuvant chemoradiotherapy cohort, the density of CD8⁺ TILs in post‐treatment resected specimens was significantly increased compared with that in pretreatment biopsy samples. We concluded that T lymphocyte‐mediated immune reactions play an important role in tumor response to neoadjuvant treatment for rectal cancer, and the evaluation of TILs in pretreatment biopsy samples might be a predictor of the clinical effectiveness of neoadjuvant treatment. Furthermore, neoadjuvant therapy, especially chemoradiotherapy, could induce the activation of the local immune status.     

Combined P16 and human papillomavirus testing predicts head and neck cancer survival

While its prognostic significance remains unclear, p16^INK4a protein expression is increasingly being used as a surrogate marker for oncogenic human papillomavirus (HPV) infection in head and neck squamous cell carcinomas (HNSCC). To evaluate the prognostic utility of p16 expression in HNSCC, we prospectively collected 163 primary tumor specimens from histologically confirmed HNSCC patients who were followed for up to 9.4 years. Formalin fixed tumor specimens were tested for p16 protein expression by immunohistochemistry (IHC). HPV type‐16 DNA and RNA was detected by MY09/11‐PCR and E6/E7 RT‐PCR on matched frozen tissue, respectively. P16 protein expression was detected more often in oropharyngeal tumors (53%) as compared with laryngeal (24%), hypopharyngeal (8%) or oral cavity tumors (4%; p < 0.0001). With respect to prognosis, p16‐positive oropharyngeal tumors exhibited significantly better overall survival than p16‐negative tumors (log‐rank test p = 0.04), whereas no survival benefit was observed for nonoropharyngeal tumors. However, when both p16 and HPV DNA test results were considered, concordantly positive nonoropharyngeal tumors had significantly better disease‐specific survival than concordantly negative nonoropharyngeal tumors after controlling for sex, nodal stage, tumor size, tumor subsite, primary tumor site number, smoking and drinking [adjusted hazard ratio (HR) = 0.04, 0.01–0.54]. Compared with concordantly negative nonoropharyngeal HNSCC, p16(+)/HPV16(?) nonoropharyngeal HNSCC (n = 13, 7%) demonstrated no significant improvement in disease‐specific survival when HPV16 was detected by RNA (adjusted HR = 0.83, 0.22–3.17). Our findings show that p16 IHC alone has potential as a prognostic test for oropharyngeal cancer survival, but combined p16/HPV testing is necessary to identify HPV‐associated nonoropharyngeal HNSCC with better prognosis.