Tine Plato Hansen

Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis

BACKGROUND Following the discovery that mutant KRAS is associated with resistance to anti-epidermal growth factor receptor (EGFR) antibodies, the tumours of patients with metastatic colorectal cancer are now profiled for seven KRAS mutations before receiving cetuximab or panitumumab. However, most patients with KRAS wild-type tumours still do not respond. We studied the effect of other downstream mutations on the efficacy of cetuximab in, to our knowledge, the largest cohort to date of patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab plus chemotherapy in the pre-KRAS selection era. METHODS 1022 tumour DNA samples (73 from fresh-frozen and 949 from formalin-fixed, paraffin-embedded tissue) from patients treated with cetuximab between 2001 and 2008 were gathered from 11 centres in seven European countries. 773 primary tumour samples had sufficient quality DNA and were included in mutation frequency analyses; mass spectrometry genotyping of tumour samples for KRAS, BRAF, NRAS, and PIK3CA was done centrally. We analysed objective response, progression-free survival (PFS), and overall survival in molecularly defined subgroups of the 649 chemotherapy-refractory patients treated with cetuximab plus chemotherapy. FINDINGS 40.0% (299/747) of the tumours harboured a KRAS mutation, 14.5% (108/743) harboured a PIK3CA mutation (of which 68.5% [74/108] were located in exon 9 and 20.4% [22/108] in exon 20), 4.7% (36/761) harboured a BRAF mutation, and 2.6% (17/644) harboured an NRAS mutation. KRAS mutants did not derive benefit compared with wild types, with a response rate of 6.7% (17/253) versus 35.8% (126/352; odds ratio [OR] 0.13, 95% CI 0.07-0.22; p<0.0001), a median PFS of 12 weeks versus 24 weeks (hazard ratio [HR] 1.98, 1.66-2.36; p<0.0001), and a median overall survival of 32 weeks versus 50 weeks (1.75, 1.47-2.09; p<0.0001). In KRAS wild types, carriers of BRAF and NRAS mutations had a significantly lower response rate than did BRAF and NRAS wild types, with a response rate of 8.3% (2/24) in carriers of BRAF mutations versus 38.0% in BRAF wild types (124/326; OR 0.15, 95% CI 0.02-0.51; p=0.0012); and 7.7% (1/13) in carriers of NRAS mutations versus 38.1% in NRAS wild types (110/289; OR 0.14, 0.007-0.70; p=0.013). PIK3CA exon 9 mutations had no effect, whereas exon 20 mutations were associated with a worse outcome compared with wild types, with a response rate of 0.0% (0/9) versus 36.8% (121/329; OR 0.00, 0.00-0.89; p=0.029), a median PFS of 11.5 weeks versus 24 weeks (HR 2.52, 1.33-4.78; p=0.013), and a median overall survival of 34 weeks versus 51 weeks (3.29, 1.60-6.74; p=0.0057). Multivariate analysis and conditional inference trees confirmed that, if KRAS is not mutated, assessing BRAF, NRAS, and PIK3CA exon 20 mutations (in that order) gives additional information about outcome. Objective response rates in our series were 24.4% in the unselected population, 36.3% in the KRAS wild-type selected population, and 41.2% in the KRAS, BRAF, NRAS, and PIK3CA exon 20 wild-type population. INTERPRETATION While confirming the negative effect of KRAS mutations on outcome after cetuximab, we show that BRAF, NRAS, and PIK3CA exon 20 mutations are significantly associated with a low response rate. Objective response rates could be improved by additional genotyping of BRAF, NRAS, and PIK3CA exon 20 mutations in a KRAS wild-type population. FUNDING Belgian Federation against Cancer (Stichting tegen Kanker).

External Quality Assessment for KRAS Testing Is Needed: Setup of a European Program and Report of the First Joined Regional Quality Assessment Rounds

The use of epidermal growth factor receptor-targeting antibodies in metastatic colorectal cancer has been restricted to patients with wild-type KRAS tumors by the European Medicines Agency since 2008, based on data showing a lack of efficacy and potential harm in patients with mutant KRAS tumors. In an effort to ensure optimal, uniform, and reliable community-based KRAS testing throughout Europe, a KRAS external quality assessment (EQA) scheme was set up. The first large assessment round included 59 laboratories from eight different European countries. For each country, one regional scheme organizer prepared and distributed the samples for the participants of their own country. The samples included unstained sections of 10 invasive colorectal carcinomas with known KRAS mutation status. The samples were centrally validated by one of two reference laboratories. The laboratories were allowed to use their own preferred method for histological evaluation, DNA isolation, and mutation analysis. In this study, we analyze the setup of the KRAS scheme. We analyzed the advantages and disadvantages of the regional scheme organization by analyzing the outcome of genotyping results, analysis of tumor percentage, and written reports. We conclude that only 70% of laboratories correctly identified the KRAS mutational status in all samples. Both the false-positive and false-negative results observed negatively affect patient care. Reports of the KRAS test results often lacked essential information. We aim to further expand this program to more laboratories to provide a robust estimate of the quality of KRAS testing in Europe, and provide the basis for remedial measures and harmonization.

Recommendations for reporting tumor budding in colorectal cancer based on the International Tumor Budding Consensus Conference (ITBCC) 2016.

Tumor budding is a well-established independent prognostic factor in colorectal cancer but a standardized method for its assessment has been lacking. The primary aim of the International Tumor Budding Consensus Conference (ITBCC) was to reach agreement on an international, evidence-based standardized scoring system for tumor budding in colorectal cancer. The ITBCC included nine sessions with presentations, a pre-meeting survey and an e-book covering the key publications on tumor budding in colorectal cancer. The ‘Grading of Recommendation Assessment, Development and Evaluation’ method was used to determine the strength of recommendations and quality of evidence. The following 10 statements achieved consensus: tumor budding is defined as a single tumor cell or a cell cluster consisting of four tumor cells or less (22/22, 100%). Tumor budding is an independent predictor of lymph node metastases in pT1 colorectal cancer (23/23, 100%). Tumor budding is an independent predictor of survival in stage II colorectal cancer (23/23, 100%). Tumor budding should be taken into account along with other clinicopathological features in a multidisciplinary setting (23/23, 100%). Tumor budding is counted on H&E (19/22, 86%). Intratumoral budding exists in colorectal cancer and has been shown to be related to lymph node metastasis (22/22, 100%). Tumor budding is assessed in one hotspot (in a field measuring 0.785 mm2) at the invasive front (22/22, 100%). A three-tier system should be used along with the budding count in order to facilitate risk stratification in colorectal cancer (23/23, 100%). Tumor budding and tumor grade are not the same (23/23, 100%). Tumor budding should be included in guidelines/protocols for colorectal cancer reporting (23/23, 100%). Members of the ITBCC were able to reach strong consensus on a single international, evidence-based method for tumor budding assessment and reporting. It is proposed that this method be incorporated into colorectal cancer guidelines/protocols and staging systems.

Prognostic factors in papillary and follicular thyroid carcinomas : p53 expression is a significant indicator of prognosis

To identify clinical and histologic prognostic factors and to investigate whether immunohistochemical detection of p53 expression might contain prognostic information, a retrospective study of patient and tumor characteristics was performed in 225 cases of papillary and follicular thyroid carcinomas. The analyses were based on cause‐specific and crude survival. In univariate analysis, age at diagnosis, tumor size, presence of distant metastases, histology (papillary contra follicular type), extrathyroidal invasion, necrosis in primary tumor, and p53 expression were significant prognostic indicators. For 211 patients (96%) all information was available and Coxs proportional hazard model was applied. The authors found that age, distant metastases, necrosis in primary tumor, extrathyroidal invasion, and p53 expression were significant prognostic factors. Analyses of cause‐specific and crude survival gave similar results. The authors conclude that age at diagnosis, presence of distant metastases, necrosis in primary tumor, and extrathyroidal invasion are important prognostic factors, and that immunohistochemical detection of p53 protein in the primary tumor is a significant and independent prognostic indicator, which might be of value in the treatment planning in patients with papillary or follicular thyroid carcinomas.

High expression of microRNA-625-3p is associated with poor response to first-line oxaliplatin based treatment of metastatic colorectal cancer

The backbone of current cytotoxic treatment of metastatic colorectal cancer (mCRC) consists of a fluoropyrimidine together with either oxaliplatin (XELOX/FOLFOX) or irinotecan (XELIRI/FOLFIRI). With an overall objective response rate of approximately 50% for either treatment combination, a major unsolved problem is that no predictors of response to these treatments are available. To address this issue, we profiled 742 microRNAs in laser‐capture microdissected cancer cells from responding and non‐responding patients receiving XELOX/FOLFOX as first‐line treatment for mCRC, and identified, among others, high expression of miR‐625‐3p, miR‐181b and miR‐27b to be associated with poor clinical response. In a validation cohort of 94 mCRC patients treated first‐line with XELOX, high expression of miR‐625‐3p was confirmed to be associated with poor response (OR = 6.25, 95%CI [1.8; 21.0]). Independent analyses showed that miR‐625‐3p was not dysregulated between normal and cancer samples, nor was its expression associated with recurrence of stage II or III disease, indicating that miR‐625‐3p solely is a response marker. Finally, we also found that these miRNAs were up‐regulated in oxaliplatin resistant HCT116/oxPt (miR‐625‐3p, miR‐181b and miR‐27b) and LoVo/oxPt (miR‐181b) colon cancer cell lines as compared with their isogenic parental cells. Altogether, our results suggest an association between miR‐625‐3p and response to first‐line oxaliplatin based chemotherapy of mCRC.

Esophageal Atresia: Gastroesophageal functional follow-up in 5–15 year old children

PURPOSE Esophageal atresia (EA) is one of the most frequent congenital alimentary tract anomalies with a considerable morbidity throughout childhood. This study evaluates the gastroesophageal problems in 5-15 year old children with EA and aims to identify factors predisposing to esophagitis in EA. MATERIAL AND METHODS Fifty-nine patients primarily operated at Odense University Hospital, Denmark, during 1993-2005 were included in this follow-up study. The patients underwent the following examinations: Interview, upper endoscopy, endoscopic ultrasonography, high-resolution esophageal manometry (HREM), and pH- and multichannel intraluminal impedance (MII) measurements. Twenty-five patients with suspected gastro-esophageal reflux disease (GERD) underwent the same investigations and served as controls. RESULTS Median age was 10.2 years (7.1-13.3). Thirty-three (55.9%) presented with GERD symptoms, 41 (69.5%) with dysphagia, and 33 (55.9%) with respiratory symptoms. Twenty-nine (49.2%) had endoscopic esophagitis, and 26 (44.1%) histological esophagitis. Median reflux index (RI) was 8.3 (4.8-14.9). In 32 (55.2%) RI was above 7. Ten percent had eosinophilic inflammation. HREM showed dysmotility in the esophagus in all EA patients, 83.3% had no propagating swallows. No predictive factors predisposing the development of endoscopic esophagitis were identified. CONCLUSIONS Gastroesophageal problems in children born with EA are common. Routine follow-up with endoscopy and pH-metry in EA patients is warranted.

Hamartomatous polyposis syndromes: a review.

Hamartomatous Polyposis Syndromes (HPS) are genetic syndromes, which include Peutz-Jeghers syndrome, Juvenile polyposis syndrome, PTEN hamartoma tumour syndrome (Cowden Syndrom, Bannayan-Riley-Ruvalcaba and Proteus Syndrome) as well as hereditary mixed polyposis syndrome. Other syndromes such as Gorlin Syndrome and multiple endocrine neoplasia syndrome 2B are sometimes referred to as HPS. HPS is characterized by the development of hamartomatous polyps in the gastrointestinal tract as well as several extra-intestinal findings such as dermatological and dysmorphic features or extra-intestinal cancer. The syndromes are rare and inherited in an autosomal dominant manner.The diagnosis of HPS has traditionally been based on clinical criteria, but can sometimes be difficult as the severity of symptoms range considerably from only a few symptoms to very severe cases - even within the same family. De novo cases are also frequent. However, because of the discovery of several associated germline-mutations as well as the rapid development in genetics it is now possible to use genetic testing more often in the diagnostic process. Management of the syndromes is different for each syndrome as extra-intestinal symptoms and types of cancers differs.Clinical awareness and early diagnosis of HPS is important, as affected patients and at-risk family members should be offered genetic counselling and surveillance. Surveillance in children with HPS might prevent or detect intestinal or extra-intestinal complications, whereas in adulthood surveillance is recommended due to an increased risk of cancer e.g. intestinal cancer or breast cancer.

Endoscopic ultrasound, endoscopic sonoelastography, and strain ratio evaluation of lymph nodes with histology as gold standard

BACKGROUND AND STUDY AIMS Accurate lymph node staging is essential for the selection of an optimal treatment in patients with upper gastrointestinal cancer. Endoscopic ultrasound (EUS) and fine-needle aspiration (FNA) are considered to be the most accurate method for locoregional staging. Endoscopic sonoelastography (ESE) assesses the elasticity of lymph nodes and has been used to differentiate lymph nodes with promising results. The aim of this study was to evaluate the use of EUS, EUS - FNA, ESE, and ESE-strain ratio using histology as the gold standard. PATIENTS AND METHODS Patients with upper gastrointestinal cancer who were referred for EUS examination were enrolled if surgical treatment was planned and the patient had a lymph node that was accessible for EUS - FNA and EUS-guided fine-needle marking (FNM). The lymph node was classified using EUS, ESE, and ESE-strain ratio. Finally, EUS - FNA and EUS - FNM were performed. The marked lymph node was isolated during surgery for histological examination. RESULTS The marked lymph node was isolated for separate histological examination in 56 patients, of whom 22 (39 %) had malignant lymph nodes and 34 (61 %) had benign lymph nodes. There were no complications of EUS - FNM. The sensitivity of EUS for differentiation between malignant and benign lymph nodes was 86 % compared with 55 % - 59 % for the different ESE modalities. The specificity of EUS was 71 % compared with 82 % - 85 % using ESE modalities. CONCLUSION The use of the EUS - FNM technique enabled the identification of a specific lymph node and thereby the use of histology as gold standard. ESE and ESE-strain ratio were no better than standard EUS in differentiating between malignant and benign lymph nodes in patients with resectable upper gastrointestinal cancer.

Establishment and characterization of models of chemotherapy resistance in colorectal cancer: Towards a predictive signature of chemoresistance

Current standard treatments for metastatic colorectal cancer (CRC) are based on combination regimens with one of the two chemotherapeutic drugs, irinotecan or oxaliplatin. However, drug resistance frequently limits the clinical efficacy of these therapies. In order to gain new insights into mechanisms associated with chemoresistance, and departing from three distinct CRC cell models, we generated a panel of human colorectal cancer cell lines with acquired resistance to either oxaliplatin or irinotecan. We characterized the resistant cell line variants with regards to their drug resistance profile and transcriptome, and matched our results with datasets generated from relevant clinical material to derive putative resistance biomarkers. We found that the chemoresistant cell line variants had distinctive irinotecan‐ or oxaliplatin‐specific resistance profiles, with non‐reciprocal cross‐resistance. Furthermore, we could identify several new, as well as some previously described, drug resistance‐associated genes for each resistant cell line variant. Each chemoresistant cell line variant acquired a unique set of changes that may represent distinct functional subtypes of chemotherapy resistance. In addition, and given the potential implications for selection of subsequent treatment, we also performed an exploratory analysis, in relevant patient cohorts, of the predictive value of each of the specific genes identified in our cellular models.

Expression of T-cell KV1.3 potassium channel correlates with pro-inflammatory cytokines and disease activity in ulcerative colitis

BACKGROUND AND AIMS Potassium channels, KV1.3 and KCa3.1, have been suggested to control T-cell activation, proliferation, and cytokine production and may thus constitute targets for anti-inflammatory therapy. Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by excessive T-cell infiltration and cytokine production. It is unknown if KV1.3 and KCa3.1 in the inflamed mucosa are markers of active UC. We hypothesized that KV1.3 and KCa3.1 correlate with disease activity and cytokine production in patients with UC. METHODS Mucosal biopsies were collected from patients with active UC (n=33) and controls (n=15). Protein and mRNA expression of KV1.3 and KCa3.1, immune cell markers, and pro-inflammatory cytokines were determined by quantitative-real-time-polymerase-chain-reaction (qPCR) and immunofluorescence, and correlated with clinical parameters of inflammation. In-vitro cytokine production was measured in human CD3(+) T-cells after pharmacological blockade of KV1.3 and KCa3.1. RESULTS Active UC KV1.3 mRNA expression was increased 5-fold compared to controls. Immunofluorescence analyses revealed that KV1.3 protein was present in inflamed mucosa in 57% of CD4(+) and 23% of CD8(+) T-cells. KV1.3 was virtually absent on infiltrating macrophages. KV1.3 mRNA expression correlated significantly with mRNA expression of pro-inflammatory cytokines TNF-α (R(2)=0.61) and IL-17A (R(2)=0.51), the mayo endoscopic subscore (R(2)=0.13), and histological inflammation (R(2)=0.23). In-vitro blockade of T-cell KV1.3 and KCa3.1 decreased production of IFN-γ, TNF-α, and IL-17A. CONCLUSIONS High levels of KV1.3 in CD4 and CD8 positive T-cells infiltrates are associated with production of pro-inflammatory IL-17A and TNF-α in active UC. KV1.3 may serve as a marker of disease activity and pharmacological blockade might constitute a novel immunosuppressive strategy.