Oropharyngeal squamous cell carcinomas differentially express granzyme inhibitors.

OBJECTIVES: Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinomas (OPSCCs) have an improved prognosis compared to HPV-negative OPSCCs. Several theories have been proposed to explain this relatively good prognosis. One hypothesis is a difference in immune response. In this study, we compared tumor-infiltrating CD3+, CD4+, CD8+ T-cells, and granzyme inhibitors (SERPINB1, SERPINB4, and SERPINB9) between HPV-positive and HPV-negative tumors and the relation with survival. METHODS: Protein expression of tumor-infiltrating lymphocytes (TILs) (CD3, CD4, and CD8) and granzyme inhibitors was analyzed in 262 OPSCCs by immunohistochemistry (IHC). Most patients (67%) received primary radiotherapy with or without chemotherapy. Cox regression analysis was carried out to compare overall survival (OS) of patients with low and high TIL infiltration and expression of granzyme inhibitors. RESULTS: HPV-positive OPSCCs were significantly more heavily infiltrated by TILs (p < 0.001) compared to HPV-negative OPSCCs. A high level of CD3+ TILs was correlated with a favorable outcome in the total cohort and in HPV-positive OPSCCs, while it reached no significance in HPV-negative OPSCCs. There was expression of all three granzyme inhibitors in OPSCCs. No differences in expression were found between HPV-positive and HPV-negative OPSCCs. Within the group of HPV-positive tumors, a high expression of SERPINB1 was associated with a significantly worse overall survival. CONCLUSION: HPV-positive OPSCCs with a low count of CD3+ TILs or high expression of SERPINB1 have a worse OS, comparable with HPV-negative OPSCCs. This suggests that the immune system plays an important role in the carcinogenesis of the virally induced oropharynx tumors.

FGFR4 Is a Potential Predictive Biomarker in Oral and Oropharyngeal Squamous Cell Carcinoma.

OBJECTIVE: The aim of this study was to investigate whether fibroblast growth factor receptor 4 (FGFR4) could serve as a potential therapeutic target, prognostic biomarker or biomarker predicting radiotherapy sensitivity in oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC). METHODS: FGFR4 immunohistochemistry and FGFR4/CEN5q FISH were performed on tissue microarrays from 212 OSCC and 238 OPSCC patients. FGFR4 genotypes were determined by PCR and DNA sequencing in 76 random OPSCC samples. The response to radiotherapy was evaluated 3 months after the last radiotherapy treatment session by a head and neck radiation oncologist and/or surgeon during clinic visits. The results were correlated to overall survival and response to radiotherapy. RESULTS: The FGFR4 protein was overexpressed in 64% (153/238) of OPSCCs and 41% (87/212) of OSCCs. The FGFR4 gene was amplified in 0.47% (1/212) of OSCCs and 0.42% (1/238) of OPSCCs, and the FGFR4 Gly388Arg polymorphism was detected in 62% (47/76) of OPSCCs. FGFR4 protein expression, FGFR4 gene copy numbers and FGFR4 genotypes were not related to overall survival or response to radiotherapy in OSCC or OPSCC. CONCLUSION: FGFR4 is frequently overexpressed in OSCC and OPSCC in the absence of gene amplification, and may serve as a potential predictive marker for FGFR4-directed targeted therapy in OSCC and OPSCC.

TP53 Y220C Is a Hotspot Mutation in Oropharyngeal Squamous Cell Carcinoma.

OBJECTIVES: Although TP53 mutations in head and neck squamous cell carcinoma (HNSCC) have been extensively studied, their association with the different subsites in the head and neck region has never been described. METHODS: Sanger sequence analysis evaluating exons 4-9 in the TP53 gene was performed on 116 HNSCC patients. The exon location, exact codon and corresponding substitution in relation to the anatomical site (subsite) of the HNSCC were evaluated. RESULTS: We found nonsynonymous TP53 mutations in 70% (81/116) of the patients. In oral cavity carcinomas, most mutations occurred in exon 7 (37%). In oropharyngeal and laryngeal tumors, mutations were mainly found in exons 6 and 7. The most common mutation was located in codon 220, and all of these were an Y220C mutation. Five out of nine (56%) Y220C mutations occurred in oropharyngeal tumors. Additionally, 22% of all mutations observed in oropharyngeal squamous cell carcinoma (OPSCC) consisted of Y220C mutations. CONCLUSION: In this study, the subsite-related distribution of TP53 mutations underlines the biological diversity between tumors arising from different anatomical regions in the head and neck region. Moreover, the Y220C mutation was by far the most prevalent TP53 mutation in HNSCC and a relative hotspot mutation in the oropharynx. © 2015 S. Karger AG, Basel.

[Acute sore throat, not always innocent]. / Acute keelpijn, niet altijd onschuldig.

Acute sore throat is one of the most common problems in general practice. It usually concerns a viral oropharyngeal infection, with good recovery within 10 days. In current guidelines, antibiotics are recommended only in exceptional situations. Although rare, potentially life-threatening complications can occur. Three case histories, on epiglottitis, peritonsillar abscess, and Lemierre syndrome, respectively, demonstrate that acute sore throat can result in severe illness. Early recognition of alarm symptoms, alertness on a complicated disease course, and clinical (re)evaluation (within 1-2 days), are essential. This contributes to the differentiation between a harmless and a serious course, given that serious conditions also have an innocent onset. We highly recommend to consult an ENT specialist when there is doubt about the seriousness of the disease, or correctness of therapy, so timely co-assessment, treatment or transfer can follow. ECMO can be a life-saving treatment when conventional therapy is insufficiently supportive.

HIF-1a expression and differential effects on survival in patients with oral cavity, larynx, and oropharynx squamous cell carcinomas.

BACKGROUND: Hypoxia is a negative prognostic factor in head and neck squamous cell carcinomas. Under hypoxia, the hypoxia-inducible factor (HIF)-1a transcription factor is overexpressed. We investigated whether there were site differences in HIF-1a expression and its effect on patient outcomes per subsite. DESIGN/METHOD: A total of 941 patients with HNSCC in the squamous cell carcinoma of the oropharynx (OPSCC, n = 302), oral cavity (OSCC, n = 391), or larynx (LSCC, n = 248) were included. Expression of HIF-1a in tissue samples was investigated using immunohistochemistry. Overall survival (OS), disease-free survival (DFS), and locoregional control (LRC) were analyzed. RESULTS: HIF-1a expression was higher in OSCC than in LSCC and OPSCC. High HIF-1a expression led to worse prognosis in OPSCC (OS P = .029, DFS P = .085) and LSCC (OS P = .041, DFS P = .011) and better in OSCC (OS P = .055, DFS P = .012). There was no association between HIF-1a and LRC. CONCLUSIONS: High HIF-1a expression is related to poor outcome in OPSCC and LSCC and better outcome in OSCC.

[A woman with a swelling in the mouth]. / Een vrouw met een zwelling in de mond.

A 35-year-old woman developed acute swallowing problems caused by a big oral blood blister after eating nuts. The blister ruptured 32 hours later and healed without scarring. 'Angina bullosa haemorrhagica' was diagnosed after ruling out bleeding disorders.

Influence of tumor and microenvironment characteristics on diffusion-weighted imaging in oropharyngeal carcinoma: A pilot study.

OBJECTIVES: Diffusion weighted imaging (DWI) is a frequently performed MRI sequence in cancer patients. While previous studies have shown the clinical value of the apparent diffusion coefficient (ADC) for response prediction and response monitoring, less is known about the biological background of ADC. In the tumor microenvironment, hypoxia and increased proliferation of tumor cells contribute to resistance to (radio-)therapy, while high T-cell influx is related to better prognosis. We investigated the correlation between these three tissue characteristics and ADC in 20 oropharyngeal squamous cell carcinoma patients. MATERIALS AND METHODS: 20 patients with oropharyngeal squamous cell carcinoma (OPSCC) who underwent 1.5 T MRI, including DWI were included in this pilot study. Corresponding formalin-fixed paraffin-embedded tumor tissues were immunohistochemically analyzed for protein expression of hypoxia-inducible factor 1a (HIF-1a), Ki-67 and CD3. Expression of these markers was correlated with ADC. RESULTS: ADC negatively correlated with Ki-67 expression (p = .024) in tumor cells. There was a significant negative correlation between ADC and CD3-positive cell count (p = .009). No correlation was observed between HIF-1a expression and ADC. CONCLUSION: This study suggests that ADC reflects characteristics of tumor cells as well as the surrounding microenvironment. Interestingly, high tumor proliferation (a negative prognostic factor) and high T-cell influx (a beneficial prognostic factor) are both associated with a lower ADC. Further studies should be performed to correlate ADC to these histological characteristics in relation to previously known factors that affect ADC, to gain further knowledge on the role of DW-MRI in diagnostics and personalized medicine.

FGFR1 Is a Potential Prognostic Biomarker and Therapeutic Target in Head and Neck Squamous Cell Carcinoma.

PURPOSE: FGFR1 is a promising therapeutic target in multiple types of solid tumors, including head and neck squamous cell carcinoma (HNSCC). FGFR inhibitors have shown great therapeutic value in preclinical models. However, resistance remains a major setback. In this study, we have investigated the prognostic value of FGFR1 expression in HNSCC, the therapeutic relevance of targeting FGFR with AZD4547, and potential resistant mechanisms. EXPERIMENTAL DESIGN: IHC and FISH were applied on tissue microarrays to investigate FGFR1 protein expression and FGFR1 gene copy numbers in 452 HNSCCs. The sensitivity of HNSCC cell lines to AZD4547, either as single or combination treatment with the EGFR inhibitor gefitinib, was assessed using long-term colony formation assays, short-term viability assays, and biochemical analysis. RESULTS: FGFR1 protein overexpression occurred in 82% (36/44) of human papillomavirus (HPV)-positive HNSCC and 75% (294/392) of HPV-negative HNSCC and relates with poor overall survival and disease-free survival in HPV-negative HNSCC [HR, 3.07; 95% confidence interval (CI), 1.74-6.90; P = 0.001 and HR, 1.53; 95% CI, 1.04-2.39; P = 0.033]. Moreover, the FGFR1 gene was amplified in 3% (3/110) of HPV-negative HNSCC. Treatment of the high FGFR1-expressing cell line CCL30 with AZD4547 reduced cell proliferation and FGFR signaling. Two FGFR-amplified cell lines, SCC147 and BICR16, were resistant to AZD4547 treatment due to EGFR signaling. Combined AZD4547 and gefitinib treatment synergistically inhibited the proliferation of resistant cell lines. CONCLUSIONS: Here, we identify high FGFR1 expression as a candidate prognostic biomarker in HPV-negative HNSCC. Furthermore, we provide a rationale for treating FGFR1-expressing HNSCC with the FGFR inhibitor AZD4547 and for combining AZD4547 and gefitinib in FGFR inhibitor-resistant HNSCC patients. Clin Cancer Res; 22(15); 3884-93. ©2016 AACR.

Fibroblast growth factor receptor 3 protein is overexpressed in oral and oropharyngeal squamous cell carcinoma.

Fibroblast growth factor receptor 3 (FGFR3) is a member of the fibroblast growth factor receptor tyrosine kinase family. It has been identified as a promising therapeutic target in multiple types of cancer. We have investigated FGFR3 protein expression and FGFR3 gene copy-numbers in a single well-documented cohort of oral and oropharyngeal squamous cell carcinoma. Tissue microarray sets containing 452 formalin-fixed paraffin-embedded tissues were immunohistochemically stained with an anti-FGFR3 antibody and hybridized with a FGFR3 fluorescence in situ hybridization probe. FGFR3 protein expression was correlated with clinicopathological and survival data, which were retrieved from electronic medical records. FGFR3 mRNA data of 522 head and neck squamous cell carcinoma (HNSCC) were retrieved from The Cancer Genome Atlas (TCGA). Fibroblast growth factor receptor 3 (FGFR3) protein was overexpressed in 48% (89/185) of oral and 59% (124/211) of oropharyngeal squamous cell carcinoma. Overexpression of FGFR3 protein was not related to overall survival or disease-free survival in oral (HR[hazard ratio]: 0.94; 95% CI: 0.64-1.39; P = 0.77, HR: 0.94; 95% CI: 0.65-1.36; P = 0.75) and oropharyngeal squamous cell carcinoma (HR: 1.21; 95% CI: 0.81-1.80; P = 0.36, HR: 0.42; 95% CI: 0.79-1.77; P = 0.42). FGFR3 mRNA was upregulated in 3% (18/522) of HNSCC from the TCGA. The FGFR3 gene was gained in 0.6% (1/179) of oral squamous cell carcinoma but no amplification was found in oral and oropharyngeal squamous cell carcinoma. In conclusion, FGFR3 protein is frequently overexpressed in oral and oropharyngeal squamous cell carcinoma. Therefore, it may serve as a potential therapeutic target for FGFR3-directed therapies in oral and oropharyngeal squamous cell carcinoma.

Fibroblast Growth Factor Receptor Family Members as Prognostic Biomarkers in Head and Neck Squamous Cell Carcinoma: A Systematic Review.

BACKGROUND: Since head and neck cancer is characterized by poor survival rates, there is a demand for novel therapeutic targets and prognostic biomarkers. An upcoming therapeutic target is the fibroblast growth factor receptor (FGFR) family. However, their prognostic role in head and neck cancer remains unclear. OBJECTIVE: To systematically review current evidence on the prognostic value of FGFR family members in head and neck squamous cell carcinoma (HNSCC). METHODS: A systematic search of PubMed, Embase, and the Cochrane Library was performed for publications up to 14 May 2014. Two reviewers screened all articles and included prognostic studies on the molecular biomarkers FGFR1-5 in any type of HNSCC. Relevant studies were assessed on risk of bias using the Quality in Prognostic Studies (QUIPS) tool. Data on FGFR aberrations and survival outcome were extracted from relevant studies. The prognostic value of FGFR aberrations was compared among studies. RESULTS: The initial search yielded 1568 publications of which 12 fulfilled the inclusion criteria. Four studies reported FGFR1 gene amplification (9.3-17.4 %) and FGFR1 protein overexpression (11.8 %) in HNSCC. FGFR1 protein expression by cancer-associated fibroblasts correlated with poor survival outcome in one study (p < 0.01). Eight studies reported high rates of FGFR4 Gly388Arg polymorphisms (32.5-54.2 %) and FGFR4 protein overexpression (16-35 %), with varying correlations with survival. So far, no studies assessed the prognostic role of FGFR2, FGFR3, or FGFR5 in HNSCC. LIMITATIONS: Significant risk of bias has been identified among included studies. Therefore, cautious interpretation of the results is recommended. CONCLUSION: In conclusion, evidence was found for prognostic value of FGFR1 expression in cancer-associated fibroblasts in HNSCC. Prognostic evidence on the other FGFR family members in HNSCC is limited and conflicting. This emphasizes the need for future well-conducted prognostic studies.

Correlation of human papillomavirus status with apparent diffusion coefficient of diffusion-weighted MRI in head and neck squamous cell carcinomas.

BACKGROUND: Identification of prognostic patient characteristics in head and neck squamous cell carcinoma (HNSCC) is of great importance. Human papillomavirus (HPV)-positive HNSCCs have favorable response to (chemo)radiotherapy. Apparent diffusion coefficient, derived from diffusion-weighted MRI, has also shown to predict treatment response. The purpose of this study was to evaluate the correlation between HPV status and apparent diffusion coefficient. METHODS: Seventy-three patients with histologically proven HNSCC were retrospectively analyzed. Mean pretreatment apparent diffusion coefficient was calculated by delineation of total tumor volume on diffusion-weighted MRI. HPV status was analyzed and correlated to apparent diffusion coefficient. RESULTS: Six HNSCCs were HPV-positive. HPV-positive HNSCC showed significantly lower apparent diffusion coefficient compared to HPV-negative. This correlation was independent of other patient characteristics. CONCLUSION: In HNSCC, positive HPV status correlates with low mean apparent diffusion coefficient. The favorable prognostic value of low pretreatment apparent diffusion coefficient might be partially attributed to patients with a positive HPV status. © 2015 Wiley Periodicals, Inc. Head Neck 38: E613-E618, 2016.

FGFR Family Members Protein Expression as Prognostic Markers in Oral Cavity and Oropharyngeal Squamous Cell Carcinoma.

INTRODUCTION: Fibroblast growth factor receptor family member proteins (FGFR1-4) have been identified as promising novel therapeutic targets and prognostic markers in a wide spectrum of solid tumors. The present study investigates the expression and prognostic value of four FGFR family member proteins in a large multicenter oral cavity squamous cell carcinoma (OCSCC) and oropharyngeal squamous cell carcinoma (OPSCC) cohort. METHODS: Protein expression of FGFR1-4 was determined by immunohistochemistry on tissue microarrays containing 951 formalin-fixed paraffin embedded OCSCC and OPSCC tissues from the University Medical Center Utrecht and University Medical Center Groningen. Protein expression was correlated to overall survival using Cox regression models, and bootstrapping was performed as internal validation. RESULTS: FGFR proteins were highly expressed in 39-64 % of OCSCC and 63-79 % of OPSCC. Seventy-three percent (299/412) of OCSCC and 85 % (305/357) of OPSCC highly co-expressed two or more FGFR family member proteins. FGFR1 protein was more frequently highly expressed in human papillomavirus (HPV)-negative OPSCC than HPV-positive OPSCC (82 vs. 65 %; p = 0.008). Furthermore, protein expression of FGFR family members was not related to overall survival in OCSCC or OPSCC (p > 0.05). CONCLUSION: FGFR family members are frequently highly expressed in OCSCC and OPSCC. These FGFR family member proteins are therefore potential targets for novel therapies that are urgently required to improve survival of OCSCC and OPSCC patients.

The diagnostic value of 11q13 amplification and protein expression in the detection of nodal metastasis from oral squamous cell carcinoma: a systematic review and meta-analysis.

Despite improvements in both diagnostic and therapeutic strategies, the prognosis of oral squamous cell carcinoma (OSCC) has not changed significantly over the last decades. Prognosis of OSCC particularly depends on the presence of nodal metastasis in the neck. Therefore, proper determination of the nodal status is pivotal for appropriate treatment. Unfortunately, current available imaging techniques (magnetic resonance imaging or ultrasound even with fine needle aspiration of suspected lymph nodes (LNs)) fail to detect occult nodal disease accurately. Clinicians in head and neck oncology urgently need new diagnostic tools to reliably determine the presence of nodal metastasis of the neck. Gain of the chromosomal region 11q13 is one of the most prominent genetic alterations in head and neck cancer and is associated with poor prognosis and metastasis. The aim of this systematic review and meta-analysis was to determine the diagnostic value of either 11q13 amplification or amplification/protein overexpression of individual genes located on 11q13 to detect nodal metastasis in OSCC. A search was conducted in Pubmed, EMBASE, and Cochrane, and 947 unique citations were retrieved. Two researchers independently screened all articles and only 18 were found to meet our inclusion criteria and were considered of sufficient quality for meta-analysis. Pooled results of those show that both amplification of CCND1 and protein overexpression of cyclin D1 significantly correlate with lymph node metastasis (LNM) in OSCC. In addition, amplification of CCND1 shows a negative predictive value of 80 % in the detection of LNM in early stage OSCCs which are clinically lymph node negative although this evidence is sparse and should be validated in a larger homogeneous cohort of T1-2 OSCC.

Clinical relevance of copy number profiling in oral and oropharyngeal squamous cell carcinoma.

Current conventional treatment modalities in head and neck squamous cell carcinoma (HNSCC) are nonselective and have shown to cause serious side effects. Unraveling the molecular profiles of head and neck cancer may enable promising clinical applications that pave the road for personalized cancer treatment. We examined copy number status in 36 common oncogenes and tumor suppressor genes in a cohort of 191 oropharyngeal squamous cell carcinomas (OPSCC) and 164 oral cavity squamous cell carcinomas (OSCC) using multiplex ligation probe amplification. Copy number status was correlated with human papillomavirus (HPV) status in OPSCC, with occult lymph node status in OSCC and with patient survival. The 11q13 region showed gain or amplifications in 59% of HPV-negative OPSCC, whereas this amplification was almost absent in HPV-positive OPSCC. Additionally, in clinically lymph node-negative OSCC (Stage I-II), gain of the 11q13 region was significantly correlated with occult lymph node metastases with a negative predictive value of 81%. Multivariate survival analysis revealed a significantly decreased disease-free survival in both HPV-negative and HPV-positive OPSCC with a gain of Wnt-induced secreted protein-1. Gain of CCND1 showed to be an independent predictor for worse survival in OSCC. These results show that copy number aberrations, mainly of the 11q13 region, may be important predictors and prognosticators which allow for stratifying patients for personalized treatment of HNSCC.

Differences in methylation profiles between HPV-positive and HPV-negative oropharynx squamous cell carcinoma: a systematic review.

Oropharyngeal squamous cell carcinoma (OPSCC) is associated with human papillomavirus (HPV). HPV-positive OPSCC is considered a distinct molecular entity with a better prognosis than HPV-negative cases of OPSCC. However, the exact pathogenic mechanisms underlying the differences in clinical and molecular behavior between HPV-positive and HPV-negative OPSCC remain poorly understood. Epigenetic events play an important role in the development of cancer. Hypermethylation of DNA in promoter regions and global hypomethylation are 2 epigenetic changes that have been frequently observed in human cancers. It is suggested that heterogeneous epigenetic changes play a role in the clinical and biological differences between HPV-positive and HPV-negative tumors. Unraveling the differences in methylation profiles of HPV-associated OPSCC may provide for promising clinical applications and may pave the road for personalized cancer treatment. This systematic review aims to assess the current state of knowledge regarding differences in promoter hypermethylation and global methylation between HPV-positive and HPV-negative OPSCC.

HPV-positive oropharyngeal squamous cell carcinoma is associated with TIMP3 and CADM1 promoter hypermethylation.

Oropharyngeal squamous cell carcinoma (OPSCC) is associated with human papillomavirus (HPV) in a proportion of tumors. HPV-positive OPSCC is considered a distinct molecular entity with a prognostic advantage compared to HPV-negative cases. Silencing of cancer-related genes by DNA promoter hypermethylation may play an important role in the development of OPSCC. Hence, we examined promoter methylation status in 24 common tumor suppressor genes in a group of 200 OPSCCs to determine differentially methylated genes in HPV-positive versus HPV-negative primary OPSCC. Methylation status was correlated with HPV status, clinical features, and patient survival using multivariate methods. Additionally, methylation status of 16 cervical squamous cell carcinomas (SCC) was compared with HPV-positive OPSCC. Using methylation-specific probe amplification, HPV-positive OPSCC showed a significantly higher cumulative methylation index (CMI) compared to HPV-negative OPSCC (P=0.008). For the genes CDH13, DAPK1, and RARB, both HPV-positive and HPV-negative OPSCC showed promoter hypermethylation in at least 20% of the tumors. HPV status was found to be an independent predictor of promoter hypermethylation of CADM1 (P < 0.001), CHFR (P = 0.027), and TIMP3 (P < 0.001). CADM1 and CHFR showed similar methylation patterns in OPSCC and cervical SCC, but TIMP3 showed no methylation in cervical SCC in contrast to OPSCC. Methylation status of neither individual gene nor CMI was associated with survival. These results suggest that HPV-positive tumors are to a greater extent driven by promotor hypermethylation in these tumor suppressor genes. Especially CADM1 and TIMP3 are significantly more frequently hypermethylated in HPV-positive OPSCC and CHFR in HPV-negative tumors.

Promoter hypermethylation using 24-gene array in early head and neck cancer: better outcome in oral than in oropharyngeal cancer.

Silencing of tumor suppressor genes (TSGs) by DNA promoter hypermethylation is an early event in carcinogenesis and a potential target for personalized cancer treatment. In head and neck cancer, little is known about the role of promoter hypermethylation in survival. Using methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) we investigated the role of promoter hypermethylation of 24 well-described genes (some of which are classic TSGs), which are frequently methylated in different cancer types, in 166 HPV-negative early oral squamous cell carcinomas (OSCC), and 51 HPV-negative early oropharyngeal squamous cell carcinomas (OPSCC) in relation to clinicopathological features and survival. Early OSCC showed frequent promoter hypermethylation in RARB (31% of cases), CHFR (20%), CDH13 (13%), DAPK1 (12%), and APC (10%). More hypermethylation (≥ 2 genes) independently correlated with improved disease specific survival (hazard ratio 0.17, P = 0.014) in early OSCC and could therefore be used as prognostic biomarker. Early OPSCCs showed more hypermethylation of CDH13 (58%), TP73 (14%), and total hypermethylated genes. Hypermethylation of two or more genes has a significantly different effect on survival in OPSCC compared with OSCC, with a trend toward worse instead of better survival. This could have a biological explanation, which deserves further investigation and could possibly lead to more stratified treatment in the future.