Han van Krieken

Deficiency of interleukin-18 in mice leads to hyperphagia, obesity and insulin resistance

Here we report the presence of hyperphagia, obesity and insulin resistance in knockout mice deficient in IL-18 or IL-18 receptor, and in mice transgenic for expression of IL-18 binding protein. Obesity of Il18−/− mice resulted from accumulation of fat tissue based on increased food intake. Il18−/− mice also had hyperinsulinemia, consistent with insulin resistance and hyperglycemia. Insulin resistance was secondary to obesity induced by increased food intake and occurred at the liver level as well as at the muscle and fat-tissue level. The molecular mechanisms responsible for the hepatic insulin resistance in the Il18−/− mice involved an enhanced expression of genes associated with gluconeogenesis in the liver of Il18−/− mice, resulting from defective phosphorylation of STAT3. Recombinant IL-18 (rIL-18) administered intracerebrally inhibited food intake. In addition, rIL-18 reversed hyperglycemia in Il18−/− mice through activation of STAT3 phosphorylation. These findings indicate a new role of IL-18 in the homeostasis of energy intake and insulin sensitivity.

Histopathology, cell proliferation indices and clinical outcome in 304 patients with mantle cell lymphoma (MCL): a clinicopathological study from the European MCL Network

Mantle cell lymphoma (MCL) is a distinct lymphoma subtype with a particularly poor clinical outcome. The clinical relevance of the morphological characteristics of these tumours remains uncertain. The European MCL Network reviewed 304 cases of MCL to determine the prognostic significance of histopathological characteristics. Cytomorphological subtypes, growth pattern and markers of proliferation (mitotic and Ki‐67 indices) were analysed. In addition to the known cytological subtypes, classical (87·5%), small cell (3·6%), pleomorphic (5·9%) and blastic (2·6%), we identified new pleomorphic subgroups with mixtures of cells (classical + pleomorphic type; 1·6%) or transitions (classical/pleomorphic type; 1·6%), which, however, did not differ significantly in overall survival time. Exactly 80·5% of cases displayed a diffuse growth pattern, whereas 19·5% of cases had a nodular growth pattern, which was associated with a slightly more favourable prognosis. A high proliferation rate (mitotic or Ki‐67 indices) was associated with shorter overall survival. Cut‐off levels were defined that allowed three subgroups with different proliferation rates to be discriminated, which showed significantly different clinical outcomes (P < 0·0001). Based on this large clinicopathological study of prospective clinical trials, multivariate analysis confirmed the central prognostic role of cell proliferation and its superiority to all other histomorphological and clinical criteria.

Evolution of acquired severe aplastic anaemia to myelodysplasia and subsequent leukaemia in adults

Myelodysplasia (MDS) and leukaemia following acquired aplastic anaemia has been reported as a rare event occurring in about 5% of patients. Improved results in survival of patients with severe aplastic anaemia (SAA) and subsequent prolonged follow‐up created the possibility of evaluating the occurrence of MDS and leukaemia in 38 adult patients with acquired SAA surviving two or more years without bone marrow transplantation. Five patients, age 22, 35, 47, 56, 72 years, two females, three males, all with idiopathic SAA and normal cytogenetic analysis developed a refractory anaemia (RA) 7, 30, 48, 56, 142 months after diagnosis of SAA. In 3/5 RA evolved into an acute myeloid leukaemia (AML) either via a chronic myelomonocytic leukaemia (CMML) (2/3) or via RA with excess of blasts (RAEB) (1/3). Three patients revealed a monosomy 7 during MDS and/or leukaemic phase. One patient died during RA phase without cytogenetic abnormalities. A pattern of evolution could be identified in these patients revealing well‐documented SAA ‐ improvement of bone marrow haematopoiesis ‐ dyshaematopoietic features of one or more cell lines with predominance of dyserythropoiesis ‐ RA ‐ RAEB or CMML ‐ AML. These five patients represent more than 10% of all patients surviving at least 2 years. This implies that the risk of developing MDS and leukaemia in SAA patients surviving with autologous marrow, might increase with longer follow‐up.

Intragenic deletion of CDH1 as the inactivating mechanism of the wild-type allele in an HDGC tumour

Mutations in CDH1, encoding E-cadherin, are the underlying genetic defect in approximately one-third of the hereditary diffuse gastric cancer (HDGC) families described so far. Tumours arising in these families show abnormal or absence of E-cadherin expression, following the model of tumour suppressor gene inactivation. A single study has been reported showing inactivation of the CDH1 wild-type allele in tumour cells from HDGC families either by promoter methylation or by somatic mutation. In order to find the genetic alteration responsible for the presence of diffuse gastric cancers in four members of a Caucasian family, we have screened the coding sequence of CDH1 for germline mutations and searched for the second inactivating hit in the tumour samples. In this family, we have found a germline splice-site mutation in all members affected by gastric cancer and, in one tumour, a somatic deletion affecting at least exon 8 of CDH1. Our results show that a CDH1 intragenic deletion is the second hit inactivating the wild-type allele, in one of the tumours in this family.

Preoperative Chemoradiotherapy with Capecitabine and Oxaliplatin in Locally Advanced Rectal Cancer. A Phase I–II Multicenter Study of the Dutch Colorectal Cancer Group

BackgroundWe studied the maximum tolerated dose (MTD) and efficacy of oxaliplatin added to capecitabine and radiotherapy (Capox-RT) as neoadjuvant therapy for rectal cancer.MethodsT3-4 rectal cancer patients received escalating doses of oxaliplatin (day 1 and 29) with a fixed dose of capecitabine of 1000 mg/m2 twice daily (days 1–14, 25–38) added to RT with 50.4 Gy and surgery after 6–8 weeks. The MTD, determined during phase I, was used in the subsequent phase II, in which R0 resection rate (a negative circumferential resection margin) was the primary end point.ResultsTwenty-one patients were evaluable. In the phase I part, oxaliplatin at 85 mg/m2 was established as MTD. In phase II, the main toxicity was grade III diarrhea (18%). All patients underwent surgery, and 20 patients had a resectable tumor. An R0 was achieved in 17/21 patients, downstaging to T0-2 in 7/21 and a pCR in 2/21.ConclusionCombination of Capox-RT has an acceptable acute toxicity profile and a high R0 resection rate of 81% in locally advanced rectal cancer. However the pCR rate was low.

Morphological changes in tumour type after radiotherapy are accompanied by changes in gene expression profile but not in clinical behaviour.

The morphological features of neoplastic cells, combined with a stromal reaction, determine the presence of cancer at the microscopic level. Malignant tumours arise through a series of genetic alterations, but these do not entirely explain invasive and metastatic behaviour and correlate only weakly with morphological changes. In order to understand the relationship between the morphology of cancer tissue, gene expression, and clinical behaviour, a study of radiation‐induced mucinous rectal carcinoma was performed. Short‐term radiotherapy of rectal carcinoma results in an increased incidence of mucinous carcinoma. A cohort of rectal carcinomas (n = 1304), from patients who participated in a randomized radiotherapy trial, was evaluated for the presence and amount of a mucinous component. The results were compared with data from the pre‐irradiation biopsies and revealed the presence of two distinct classes of mucinous carcinoma in the radiotherapy group, namely pre‐existing (un‐induced; n = 24) and induced mucinous carcinoma (n = 29). Clinical data, pathological parameters, and immunohistochemical data from these patients and their tumours showed that induced mucinous carcinomas were more comparable to typical adenocarcinomas than to pre‐existing mucinous carcinomas. The prognosis of patients with induced mucinous carcinoma was significantly better than that of patients with pre‐existing mucinous carcinomas (91.2% versus 39.3% recurrence‐free interval at 2 years, p = 0.02). Gene expression profiles of the different groups of mucinous carcinomas and adenocarcinomas were analysed using Affymetrix Human Cancer Chips. Surprisingly, despite the difference in prognosis, the expression profile of radiation‐induced mucinous carcinomas was very closely related to that of their un‐induced counterparts. It is shown in the present study that radiation therapy of rectal cancer leads within a few days to substantial changes in both morphology and expression profile. However, the morphology of the pre‐therapy biopsy predicts patient survival far better than post‐therapy expression profiles. It is concluded that tumour morphology equates to expression profile, but that external factors might influence both, leading to sub‐optimal prognostication. Copyright

Next generation diagnostic molecular pathology: Critical appraisal of quality assurance in Europe

Tumor evaluation in pathology is more and more based on a combination of traditional histopathology and molecular analysis. Due to the rapid development of new cancer treatments that specifically target aberrant proteins present in tumor cells, treatment decisions are increasingly based on the molecular features of the tumor. Not only the number of patients eligible for targeted precision medicine, but also the number of molecular targets per patient and tumor type is rising. Diagnostic molecular pathology, the discipline that determines the molecular aberrations present in tumors for diagnostic, prognostic or predictive purposes, is faced with true challenges. The laboratories have to meet the need of comprehensive molecular testing using only limited amount of tumor tissue, mostly fixed in formalin and embedded in paraffin (FFPE), in short turnaround time. Choices must be made for analytical methods that provide accurate, reliable and cost‐effective results. Validation of the test procedures and results is essential. In addition, participation and good performance in internal (IQA) and external quality assurance (EQA) schemes is mandatory. In this review, we critically evaluate the validation procedure for comprehensive molecular tests as well as the organization of quality assurance and assessment of competence of diagnostic molecular pathology laboratories within Europe.

T-cell Landscape in a Primary Melanoma Predicts the Survival of Patients with Metastatic Disease after Their Treatment with Dendritic Cell Vaccines

Tumor-infiltrating lymphocytes appear to be a predictor of survival in many cancers, including cutaneous melanoma. We applied automated multispectral imaging to determine whether density and distribution of T cells within primary cutaneous melanoma tissue correlate with survival of metastatic melanoma patients after dendritic cell (DC) vaccination. CD3(+) T cell infiltration in primary tumors from 77 metastatic melanoma patients was quantified using the ratio of intratumoral versus peritumoral T-cell densities (I/P ratio). Patients with longer survival after DC vaccination had stronger T-cell infiltration than patients with shorter survival in a discovery cohort of 19 patients (P = 0.000026) and a validation cohort of 39 patients (P = 0.000016). I/P ratio was the strongest predictor of survival in a multivariate analysis including M substage and serum lactate dehydrogenase level. To evaluate I/P ratio as a predictive biomarker, we analyzed 19 chemotherapy-treated patients. Longer survival times of DC-vaccinated compared with chemotherapy-treated patients was observed for high (P = 0.000566), but not low (P = 0.154) I/P ratios. In conclusion, T-cell infiltration into primary melanoma is a strong predictor of survival after DC vaccination in metastatic melanoma patients who, on average, started this therapy several years after primary tumor resection. The infiltration remains predictive even after adjustment for late-stage prognostic markers. Our findings suggest that the I/P ratio is a potential predictive biomarker for treatment selection. Cancer Res; 76(12); 3496-506. ©2016 AACR.

Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL

The clinical demand for mutation detection within multiple genes from a single tumour sample requires molecular diagnostic laboratories to develop rapid, high-throughput, highly sensitive, accurate and parallel testing within tight budget constraints. To meet this demand, many laboratories employ next-generation sequencing (NGS) based on small amplicons. Building on existing publications and general guidance for the clinical use of NGS and learnings from germline testing, the following guidelines establish consensus standards for somatic diagnostic testing, specifically for identifying and reporting mutations in solid tumours. These guidelines cover the testing strategy, implementation of testing within clinical service, sample requirements, data analysis and reporting of results. In conjunction with appropriate staff training and international standards for laboratory testing, these consensus standards for the use of NGS in molecular pathology of solid tumours will assist laboratories in implementing NGS in clinical services.

Next generation sequencing in synovial sarcoma reveals novel gene mutations

Over 95% of all synovial sarcomas (SS) share a unique translocation, t(X;18), however, they show heterogeneous clinical behavior. We analyzed multiple SS to reveal additional genetic alterations besides the translocation. Twenty-six SS from 22 patients were sequenced for 409 cancer-related genes using the Comprehensive Cancer Panel (Life Technologies, USA) on an Ion Torrent platform. The detected variants were verified by Sanger sequencing and compared to matched normal DNAs. Copy number variation was assessed in six tumors using the Oncoscan array (Affymetrix, USA). In total, eight somatic mutations were detected in eight samples. These mutations have not been reported previously in SS. Two of these, in KRAS and CCND1, represent known oncogenic mutations in other malignancies. Additional mutations were detected in RNF213, SEPT9, KDR, CSMD3, MLH1 and ERBB4. DNA alterations occurred more often in adult tumors. A distinctive loss of 6q was found in a metastatic lesion progressing under pazopanib, but not in the responding lesion. Our results emphasize t(X;18) as a single initiating event in SS and as the main oncogenic driver. Our results also show the occurrence of additional genetic events, mutations or chromosomal aberrations, occurring more frequently in SS with an onset in adults.