Florian Grabellus

Resection of residual disease in patients with metastatic gastrointestinal stromal tumors responding to treatment with imatinib

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract. Long‐term survival of patients with metastatic disease has only been observed in patients with completely resected disease. Recently, the tyrosine kinase inhibitor imatinib has been found to yield responses in the majority of patients with metastatic GIST suggesting improved resectability in responding patients. Combined treatment approaches including resective surgery after imatinib treatment in patients with advanced metastatic disease have rarely been explored. We report a series of 90 patients with metastatic GIST in whom treatment with imatinib enabled 12 patients with mostly recurrent and extensive disease to be considered for resection of residual disease. In 11 of these patients, complete resection could be achieved. Viable tumor cells were found in all but one resected specimens suggesting that despite favorable radiological or clinical responses, imatinib is unlikely to induce pathological complete responses. Until more mature data from prospective trials are available, these data suggest that an early aggressive surgical approach should be considered for all patients with metastatic GIST. Further trials investigating a combined surgical and pre/postoperative treatment with imatinib in patients with advanced metastatic GIST are warranted.

Differential miRNA expression profiles in variants of papillary thyroid carcinoma and encapsulated follicular thyroid tumours

Background:Recent studies showed a significant upregulation of distinct microRNAs (miRNAs) in papillary thyroid carcinoma (PTC). The objective of this study was to explore whether this upregulation could also be assigned to distinct histomorphological variants of PTC, especially the follicular variant and other encapsulated follicular thyroid tumours.Methods:We used total RNA of 113 formalin-fixed paraffin-embedded tissues of 50 PTCs ((10 conventional type (PTC-CT), 10 tall cell variants (PTC-TCVs), 30 follicular variants (PTC-FVs)), 10 follicular adenomas (FAs), 10 multinodular goitres (MNGs), 21 follicular thyroid carcinomas and 22 well-differentiated tumours of unknown malignant potential (WDT-UMP) to analyse the miRNA expression pattern of five selected miRNAs (146b, 181b, 21, 221 and 222) using RT–PCR TaqMan miRNA assay to explore the diagnostic utility of this method.Results:The mean values of the expression pattern of all miRNAS in PTCs show a statistically significant difference from those in MNG and FA with fold changes up to 90 for miRNA 146b, P<0.001. No differences in expression pattern could be showed between MNG and FA. The PTC-FVs differ significantly from FA in all five miRNAS, from MNG in three and from WDT-UMP in one miRNA with fold changes between 1.7 and 21.2, but failed to be of diagnostic value regarding individual cases with substantial overlaps.Conclusion:We conclude that analysis of a set of five selected miRNAS distinguish common variants of PTC from FA/MNG but failed to be a useful diagnostic method in individual and doubtful cases, especially in the differential diagnosis of encapsulated follicular thyroid tumours.

Conjunctival Melanomas Harbor BRAF and NRAS Mutations and Copy Number Changes Similar to Cutaneous and Mucosal Melanomas

Purpose: Conjunctival melanoma is a rare but potentially deadly tumor of the eye. Despite effective local therapies, recurrence and metastasis remain frequent. Once the tumor has metastasized, treatment options are limited and the prognosis is poor. To date, little is known of the genetic alterations in conjunctival melanomas. Experimental Design: We conducted genetic analysis of 78 conjunctival melanomas, to our knowledge the largest cohort reported to date. An oncogene hotspot array was run on 38 samples, screening for a panel of known cancer-relevant mutations. Thirty tumors were analyzed for genome-wide copy number alterations (CNA) using array-based comparative genomic hybridization. Sanger sequencing of selected target mutations was conducted in all samples. Results: BRAF mutations were identified in 23 of 78 (29%) tumors. NRAS mutations, previously not recognized as relevant in conjunctival melanoma, were detected in 14 of 78 (18%) tumors. We found CNAs affecting various chromosomes distributed across the genome in a pattern reminiscent of cutaneous and mucosal melanoma but differing markedly from uveal melanoma. Conclusions: The presence of NRAS or BRAF mutations in a mutually exclusive pattern in roughly half (47%) of conjunctival melanomas and the pattern of CNAs argue for conjunctival melanoma being closely related to cutaneous and mucosal melanoma but entirely distinct from uveal melanoma. Patients with metastatic conjunctival melanoma should be considered for therapeutic modalities available for metastatic cutaneous and mucosal melanoma including clinical trials of novel agents. Clin Cancer Res; 19(12); 3143–52. ©2013 AACR.

A mouse model of osteochondromagenesis from clonal inactivation of Ext1 in chondrocytes

We report a mouse model of multiple osteochondromas (MO), an autosomal dominant disease in humans, also known as multiple hereditary exostoses (MHE or HME) and characterized by the formation of cartilage-capped osseous growths projecting from the metaphyses of endochondral bones. The pathogenesis of these osteochondromas has remained unclear. Mice heterozygous for Ext1 or Ext2, modeling the human genotypes that cause MO, occasionally develop solitary osteochondroma-like structures on ribs [Lin et al. (2000) Dev Biol 224(2):299–311; Stickens et al. (2005) Development 132(22):5055–5068]. Rather than model the germ-line genotype, we modeled the chimeric tissue genotype of somatic loss of heterozygosity (LOH), by conditionally inactivating Ext1 via head-to-head loxP sites and temporally controlled Cre-recombinase in chondrocytes. These mice faithfully recapitulate the human phenotype of multiple metaphyseal osteochondromas. We also confirm homozygous disruption of Ext1 in osteochondroma chondrocytes and their origin in proliferating physeal chondrocytes. These results explain prior modeling failures with the necessity for somatic LOH in a developmentally regulated cell type.

Prognostic relevance of activated Akt kinase in node-negative breast cancer: a clinicopathological study of 99 cases

Patients with lymphnode-negative breast cancer show a 10-year tumor recurrence rate of approximately 30%. Therefore, it is important to identify high-risk patients who would benefit from further adjuvant therapy. For this purpose, we examined the activation state of two kinases important in the regulation of cell proliferation and apoptosis in a series of 99 node-negative breast cancer cases with a mean follow-up of 10 years: Akt and extracellular regulated kinase (ERK1/2). The activation of Akt and ERK1/2 was investigated by immunohistochemistry using phospho-specific antibodies. The results were correlated with HER-2/neu expression, histological grading, receptor status, overall survival (OS) as well as with cell proliferation (Ki67 immunoreactivity, mitotic count) and tumor apoptosis assessed by TUNEL staining. Activation of Akt (pAkt) but not activation of ERK1/2 (pERK1/2) correlated with HER-2/neu overexpression (P<0.05) and was related to reduced tumor apoptosis (P<0.05). No association was found between pAkt or pERK1/2 with cell proliferation assessed by Ki67 and mitotic count (MC). Survival analysis of receptor status, HER2/neu expression, histological grading, MC and pAkt immunoexpression showed a significant correlation with decreased OS, but only pAkt reached statistical significance in the multivariate Cox regression analysis (P=0.015). Activation of Akt in node-negative breast cancer may indicate aggressive tumor behavior and may constitute an independent prognostic factor of OS. The determination of pAkt status may be of value in identifying high-risk patients, who would benefit from adjuvant therapy, and gives a rationale to investigate new therapy strategies by specific inhibition of the Akt signaling pathway in breast cancer.

Targeting Bcl-2 family proteins modulates the sensitivity of B-cell lymphoma to rituximab-induced apoptosis

The chimeric monoclonal antibody rituximab is the standard of care for patients with B-cell non-Hodgkin lymphoma (B-NHL). Rituximab mediates complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity of CD20-positive human B cells. In addition, rituximab sensitizes B-NHL cells to cytotoxic chemotherapy and has direct apoptotic and antiproliferative effects. Whereas expression of the CD20 antigen is a natural prerequisite for rituximab sensitivity, cell-autonomous factors determining the response of B-NHL to rituximab are less defined. To this end, we have studied rituximab-induced apoptosis in human B-NHL models. We find that rituximab directly triggers apoptosis via the mitochondrial pathway of caspase activation. Expression of antiapoptotic Bcl-xL confers resistance against rituximab-induced apoptosis in vitro and rituximab treatment of xenografted B-NHL in vivo. B-NHL cells insensitive to rituximab-induced apoptosis exhibit increased endogenous expression of multiple antiapoptotic Bcl-2 family proteins, or activation of phosphatidylinositol-3-kinase signaling resulting in up-regulation of Mcl-1. The former resistance pattern is overcome by treatment with the BH3-mimetic ABT-737, the latter by combining rituximab with pharmacologic phosphatidylinositol-3-kinase inhibitors. In conclusion, sensitivity of B-NHL cells to rituximab-induced apoptosis is determined at the level of mitochondria. Pharmacologic modulation of Bcl-2 family proteins or their upstream regulators is a promising strategy to overcome rituximab resistance.

Inhibitors of Deacetylases Suppress Oncogenic KIT Signaling, Acetylate HSP90, and Induce Apoptosis in Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT or platelet-derived growth factor receptor A (PDGFRA), and treatment with the tyrosine kinase inhibitor imatinib yields responses in the majority of patients. However, most patients develop secondary resistance, which is associated with a dismal prognosis. Histone deacetylase inhibitors (HDACI) have been shown to enhance imatinib activity in imatinib-resistant chronic myelogenous leukemia. Against this background, we explored whether HDACI might provide an alternative therapeutic strategy to KIT/PDGFRA kinase inhibitors in GIST. Inhibition of cell proliferation by HDACI was seen in KIT-positive but not in KIT-negative GIST cell lines, suggesting that HDACI activity is mainly conferred by targeting oncogenic KIT. KIT activity, expression, and activation of downstream pathways were strongly inhibited by several HDACI (SAHA, LBH589, VPA, trichostatin A, and NaButyrate). SAHA and LBH589 induced apoptosis in KIT-positive GIST, and strong synergism with imatinib was observed at low concentrations of SAHA and LBH589. Mechanistically, treatment with HDACI reduced KIT mRNA transcript levels and led to strong acetylation of HSP90, interfering with its activity as KIT chaperone. These results provide preclinical evidence for a disease-specific effect of HDACI in KIT-positive GIST, which could translate into therapeutic activity.

High expression of focal adhesion kinase (p125FAK) in node-negative breast cancer is related to overexpression of HER-2/neu and activated Akt kinase but does not predict outcome

IntroductionFocal adhesion kinase (FAK) regulates multiple cellular processes including growth, differentiation, adhesion, motility and apoptosis. In breast carcinoma, FAK overexpression has been linked to cancer progression but the prognostic relevance remains unknown. In particular, with regard to lymph node-negative breast cancer it is important to identify high-risk patients who would benefit from further adjuvant therapy.MethodsWe analyzed 162 node-negative breast cancer cases to determine the prognostic relevance of FAK expression, and we investigated the relationship of FAK with major associated signaling pathways (HER2, Src, Akt and extracellular regulated kinases) by immunohistochemistry and western blot analysis.ResultsElevated FAK expression did not predict patient outcome, in contrast to tumor grading (P = 0.005), Akt activation (P = 0.0383) and estrogen receptor status (P = 0.0033). Significant positive correlations were observed between elevated FAK expression and HER2 overexpression (P = 0.001), as well as phospho-Src Tyr-215 (P = 0.021) and phospho-Akt (P < 0.001), but not with phospho-ERK1/2 (P = 0.108). Western blot analysis showed a significant correlation of FAK Tyr-861 activation and HER2 overexpression (P = 0.01).ConclusionsImmunohistochemical detection of FAK expression is of no prognostic significance in node-negative breast cancer but provides evidence that HER2 is involved in tumor malignancy and metastatic ability of breast cancer through a novel signaling pathway participating FAK and Src.

TERT promoter mutations in ocular melanoma distinguish between conjunctival and uveal tumours

Background:Recently, activating mutations in the TERT promoter were identified in cutaneous melanoma. We tested a cohort of ocular melanoma samples for similar mutations.Methods:The TERT promoter region was analysed by Sanger sequencing in 47 uveal (ciliary body or choroidal) melanomas and 38 conjunctival melanomas.Results:Mutations of the TERT promoter were not identified in uveal melanomas, but were detected in 12 (32%) conjunctival melanomas. Mutations had a UV signature and were identical to those found in cutaneous melanoma.Conclusion:Mutations of TERT promoter with UV signatures are frequent in conjunctival melanomas and favour a pathogenetic kinship with cutaneous melanomas. Absence of these mutations in uveal melanomas emphasises their genetic distinction from cutaneous and conjunctival melanomas.

Magnetic resonance colonography without bowel cleansing: a prospective cross sectional study in a screening population

Background and aim: To evaluate the diagnostic accuracy of magnetic resonance colonography (MRC) without bowel cleansing in a screening population and compare the results to colonoscopy as a standard of reference. Methods: 315 screening patients, older than 50 years with a normal risk profile for colorectal cancer, were included in this study. For MRC, a tagging agent (5.0% Gastrografin, 1.0% barium sulphate, 0.2% locust bean gum) was ingested with each main meal within 2 days prior to MRC. No bowel cleansing was applied. For the magnetic resonance examination, a rectal water enema was administered. Data collection was based on contrast enhanced T1 weighted images and TrueFISP images. Magnetic resonance data were analysed for image quality and the presence of colorectal lesions. Conventional colonoscopy and histopathological samples served as reference. Results: In 4% of all colonic segments, magnetic resonance image quality was insufficient because of untagged faecal material. Adenomatous polyps >5 mm were detected by means of MRC, with a sensitivity of 83.0%. Overall specificity was 90.2% (false positive findings in 19 patients). However, only 16 of 153 lesions <5 mm and 9 of 127 hyperplastic polyps could be visualised on magnetic resonance images. Conclusions: Faecal tagging MRC is applicable for screening purposes. It provides good accuracy for the detection of relevant (ie, adenomatous) colorectal lesions >5 mm in a screening population. However, refinements to optimise image quality of faecal tagging are needed.