Florian Haller

Surgical Management After Neoadjuvant Imatinib Therapy in Gastrointestinal Stromal Tumours (GISTs) with Respect to Imatinib Resistance Caused by Secondary KIT Mutations

BackgroundIn metastasized GISTs, resistance to imatinib after initial tumour response has been associated with observation of secondary mutations in the activation loop of KIT. The aim of the current study was to evaluate the tumour response and observance of secondary KIT mutations in a case of GIST undergoing neoadjuvant imatinib therapy.MethodsWe report on a case of an initially unresectable gastric GIST with curative resection after 10 months of neoadjuvant imatinib therapy. Mutation analysis of KIT was performed on a pretherapeutic biopsy specimen, as well as on the resected tumour specimen.ResultsThe pretherapeutic biopsy revealed cKit positive tumour cells with mutation of KIT exon 11 Del 560–576. The remaining tumour mass after neoadjuvant imatinib therapy almost exclusively consisted of hypocellular myxohyalinale stroma with rare microfoci of cKit positive tumour cells. Laser microdissection of several tumour microfoci revealed two additional point mutations located in the activation loop of KIT exon 17, C809G and N822Y, each observed separately in a distinct microfocus. Neither of these two point mutations has been reported in a GIST so far.ConclusionsNeoadjuvant imatinib therapy successfully reduces tumour size in GISTs. Since resistance relevant secondary mutations of the activation loop of KIT may be observed after neoadjuvant imatinib therapy, the time elapse with preoperative imatinib therapy should be chosen as short as curative tumour resection or function sparing surgery can be carried out. The determination of the optimal time point for surgery is therefore a critical event and will be discussed.

Solitary Fibrous Tumors/Hemangiopericytomas with Different Variants of the NAB2-STAT6 Gene Fusion Are Characterized by Specific Histomorphology and Distinct Clinicopathological Features

Recurrent somatic fusions of the two genes, NGFI-A-binding protein 2 (NAB2) and STAT6, located at chromosomal region 12q13, have been recently identified to be presumable tumor-initiating events in solitary fibrous tumors (SFT). Herein, we evaluated a cohort of 52 SFTs/hemangiopericytomas (HPCs) by whole-exome sequencing (one case) and multiplex RT-PCR (all 52 cases), and identified 12 different NAB2-STAT6 fusion variants in 48 cases (92%). All 52 cases showed strong and diffuse nuclear positivity for STAT6 by IHC. We categorized the fusion variants according to their potential functional effects within the predicted fusion protein and found strong correlations with relevant clinicopathological features. Tumors with the most common fusion variant, NAB2ex4-STAT6ex2/3, corresponded to classic pleuropulmonary SFTs with diffuse fibrosis and mostly benign behavior and occurred in older patients (median age, 69 years). In contrast, tumors with the second most common fusion variant, NAB2ex6-STAT6ex16/17, were found in much younger patients (median age, 47 years) and represented typical HPCs from deep soft tissue with a more aggressive phenotype and clinical behavior. In summary, these molecular genetic findings support the concept that classic pleuropulmonary SFT and deep-seated HPC are separate entities that share common features but correlate to different clinical outcome.

An oncogenetic tree model in gastrointestinal stromal tumours (GISTs) identifies different pathways of cytogenetic evolution with prognostic implications

To model the cytogenetic evolution in gastrointestinal stromal tumour (GIST), an oncogenetic tree model was reconstructed using comparative genomic hybridization data from 203 primary GISTs (116 gastric and 87 intestinal GISTs, including 151 newly analysed cases), with follow‐up available in 173 cases (mean 40 months; maximum 133 months). The oncogenetic tree model identified three major cytogenetic pathways: one initiated by − 14q, one by − 1p, and another by − 22q. The − 14q pathway mainly characterized gastric tumours with predominantly stable karyotypes and more favourable clinical course. On the other hand, the − 1p pathway was more characteristic of intestinal GISTs, with an increased capacity for cytogenetic complexity and more aggressive clinical course. Loss of 22q, more closely associated with − 1p than − 14q, appeared to initiate the critical transition to an unfavourable cytogenetic subpathway. This − 22q pathway included accumulation of + 8q, − 9p, and − 9q, which could all predict disease‐free survival in addition to tumour site. Thus, insights into the cytogenetic evolution obtained from oncogenetic tree models may eventually help to gain a better understanding of the heterogeneous site‐dependent biological behaviour of GISTs. Copyright

Localization- and mutation-dependent microRNA (miRNA) expression signatures in gastrointestinal stromal tumours (GISTs), with a cluster of co-expressed miRNAs located at 14q32.31.

The molecular biology and clinical behaviour of gastrointestinal stromal tumours (GISTs) are associated with their anatomical localization (stomach or intestine), and also with the mutation status of the receptor tyrosine kinases KIT and PDGFRA. Twelve GISTs were evaluated for differential miRNA expression signatures by use of microarrays representing 734 human miRNAs. Thirty‐two miRNAs were found to be differentially expressed according to localization and mutation status. Differential expression was further analysed and confirmed for four miRNAs (miR‐132, miR‐221, miR‐222, and miR‐504) by qRT‐PCR in 49 additional GISTs. Differentially expressed miRNAs were functionally mapped to KIT/PDGFRA signalling and G1/S‐phase transition of the cell cycle, revealing 22 predicted miRNA/mRNA interactions for ten gene targets from KIT/PDGFRA signalling, and 12 interactions for 12 gene targets of G1/S‐phase transition. Moreover, the expression of 44 miRNAs clustered in a genetically imprinted region at 14q32.31 was found to be strongly correlated in the microarray analysis. This was confirmed for two selected miRNAs (miR‐134 and miR‐370) from the 14q32.31 cluster by qRT‐PCR in 49 additional GISTs, and the expression of these two miRNAs was significantly lower in GISTs with 14q loss, and also in GISTs with tumour progress. miRNA profiling may prove to be a key determinant of the biology and clinical features of GISTs Copyright

Prognostic role of E2F1 and members of the CDKN2A network in gastrointestinal stromal tumors.

Purpose: The aim of the current study was to examine the prognostic relevance of the CDKN2A tumor suppressor pathway in gastrointestinal stromal tumors (GIST). Experimental Design: We determined the mRNA expression of p1INK4A, p14ARF, CDK4, RB1, MDM2, TP53, and E2F1 by quantitative reverse transcription-PCR in 38 cases of GISTs and correlated the findings with clinicopathologic factors, including mutation analysis of KIT and PDGFRA. Results: The k-means cluster analysis yielded three prognostic subgroups of GISTs with distinct mRNA expression patterns of the CDKN2A pathway. GISTs with low mRNA expression of the CDKN2A transcripts p16INK4A and p14ARF but high mRNA expression of CDK4, RB1, MDM2, TP53, and E2F1 were associated with aggressive clinical behavior and unfavorable prognosis, whereas GISTs with a low mRNA expression of CDK4, RB1, MDM2, TP53, and E2F1 were not. GISTs with a moderate to high mRNA expression of all examined genes also seemed to be associated with unfavorable prognosis. Regarding mutation analysis, we found significant differences in the KIT/PDGFRA genotype among the three clusters. Univariate analysis revealed high expression of E2F1 to be associated with mitotic count, proliferation rate, KIT mutation, and aggressive clinical behavior. These findings on mRNA level could be confirmed by immunohistochemistry. Conclusion: Our findings implicate differential regulation schemes of the CDKN2A tumor suppressor pathway converging to up-regulation of E2F1 as the critical link to increased cell proliferation and adverse prognosis of GISTs.

Aberrant DNA hypermethylation of SDHC: a novel mechanism of tumor development in Carney triad

Carney triad (CT) is a rare condition with synchronous or metachronous occurrence of gastrointestinal stromal tumors (GISTs), paragangliomas (PGLs), and pulmonary chondromas in a patient. In contrast to Carney-Stratakis syndrome (CSS) and familial PGL syndromes, no germline or somatic mutations in the succinate dehydrogenase (SDH) complex subunits A, B, C, or D have been found in most tumors and/or patients with CT. Nonetheless, the tumors arising among patients with CT, CSS, or familial PGL share a similar morphology with loss of the SDHB subunit on the protein level. For the current study, we employed massive parallel bisulfite sequencing to evaluate DNA methylation patterns in CpG islands in proximity to the gene loci of all four SDH subunits. For the first time, we report on a recurrent aberrant dense DNA methylation at the gene locus of SDHC in tumors of patients with CT, which was not present in tumors of patients with CSS or PGL, or in sporadic GISTs with KIT mutations. This DNA methylation pattern was correlated to a reduced mRNA expression of SDHC, and concurrent loss of the SDHC subunit on the protein level. Collectively, these data suggest epigenetic inactivation of the SDHC gene locus with functional impairment of the SDH complex as a plausible alternate mechanism of tumorigenesis in CT.

SMARCB1(INI1)-deficient Sinonasal Basaloid Carcinoma A Novel Member of the Expanding Family of SMARCB1-deficient Neoplasms

Poorly differentiated sinonasal carcinomas are a heterogenous group of aggressive neoplasms that encompasses squamous cell carcinoma including basaloid variant, lymphoepithelial carcinoma, sinonasal undifferentiated carcinoma, and neuroendocrine-type small cell carcinoma. We herein describe 3 cases of a hitherto unreported variant combining features of basaloid carcinoma with variable intermingled rhabdoid cells. Patients were 2 women (aged 28 and 35) and a man (52 y) who presented with sinonasal masses. All had advanced local disease with bone involvement (pT4). None had a history of irradiation or a family history of rhabdoid tumors. Treatment was surgery and adjuvant chemoradiation. One patient developed liver, lung, pleural, and pericardial metastases (63 mo) and is currently (70 mo) alive under palliative treatment. Another developed recurrent cervical lymph node metastases and died of disease 8.5 years later. The youngest patient was disease-free at last follow-up 7 years later. Histologic features were very similar in all 3 cases and showed intimate admixture of compact basaloid cell nests with peripheral palisading, perivascular pseudorosettes, and a few scattered rhabdoid cells. Rhabdoid cells were more extensive in the metastasis in 1 case but formed a minor inconspicuous component in the primary tumors in all cases. Striking features common to all cases were (1) basaloid “blue” appearance at low power, (2) papilloma-like exophytic component, (3) extensive pagetoid surface growth with prominent denuding features, and (4) replacement of underlying mucous glands mimicking an inverted papilloma. Clear-cut origin from benign papilloma and overt squamous differentiation were lacking. Diffuse (2) or partial (1) p16 expression was noted, but all cases lacked human papillomavirus DNA by molecular tests. In situ hybridization was negative for Epstein-Barr virus. Immunohistochemistry showed diffuse expression of pancytokeratin. CK5 and vimentin showed intermingling of CK5+/vimentin− basaloid and CK5−/vimentin+ rhabdoid cells. Complete loss of nuclear SMARCB1 expression was seen in all cases including also the denuding carcinoma in situ–like surface lesions. To our knowledge, this variant of sinonasal carcinoma has not been reported before. The identical features in all 3 cases suggest a specific disease rather than a nonspecific dedifferentiated phenotype. Awareness of this rare variant and thus reporting of additional cases is necessary for defining its full morphologic and biological spectrum.

Loss of 9p leads to p16INK4A down-regulation and enables RB/E2F1-dependent cell cycle promotion in gastrointestinal stromal tumours (GISTs)†

Loss of chromosome 9p is a reliable predictor of malignant behaviour in gastrointestinal stromal tumours (GISTs). p16INK4A located at 9p21 inhibits the CDK4/6/cyclin D complex from phosphorylating RB. Phosphorylation of RB through CDK4/6/cyclin D in early G1 phase frees the transcription factor E2F1 from RB and enables mRNA transcription of genes essential for G1/S phase transition. This study aims to determine the impact of 9p loss on mRNA and protein expression of p16INK4A and further key cell cycle regulators in the different phases of the cell cycle. Sixty primary GISTs previously characterized for 9p loss by comparative genomic hybridization were analysed for mRNA expression of p16INK4A, p15INK4B, CDK4, CDK6, cyclin D, p21CIP1p27KIP1, CDK2, cyclin E, cyclin B, RB and E2F1, using quantitative RT–PCR. The protein expression of CDK6, CDK2, p21CIP1, p27KIP1 and phosphorylated RB (S807/S811) was evaluated using protein arrays as a novel and highly sensitive platform for profiling of protein abundance and protein phosphorylation. In parallel, the nuclear percentages of immunohistochemical staining for p16INK4A, cyclin D, E2F1 and RB were quantified on a tissue microarray. GISTs with 9p loss had significantly higher proliferation rates, higher metastatic behaviour and shorter disease‐free survival. On the molecular level, GISTs with 9p loss had a significantly reduced mRNA as well as nuclear protein expression of p16INK4A. RB was significantly more phosphorylated in these tumours, together with increased mRNA expression and nuclear staining for E2F1. Furthermore, GISTs with 9p loss had up‐regulation of the late G1/S phase promoters CDK2 and cyclin E. We conclude that loss of 9p accompanied by early G1 phase inhibitor p16INK4A down‐regulation in GISTs facilitates phosphorylation of RB, enabling E2F1‐dependent transcription of genes essential for late G1/S phase transition. This study provides a possible basis for the accelerated proliferation and particularly malignant behaviour in GISTs with 9p loss. Copyright

Altered regulation of Prox1-gene-expression in liver tumors

BackgroundProspero-related homeobox 1 (Prox1) transcription factor was described as a tumor-suppressor gene in liver tumors. In contrast, Prox1 knock out in murine embryos drastically reduces proliferation of hepatoblasts.MethodsWe have studied the expression of Prox1 in normal liver, liver cirrhosis and peritumoral liver samples in comparison to hepatocellular (HCC) and cholangiocellular carcinoma (CCC) at mRNA, protein and functional levels.ResultsProx1 was found in hepatocytes of normal liver, while normal bile duct epithelial cells were negative. However, Prox1+ cells, which co-expressed biliary epithelial makers and showed ductular morphology, could be detected within fibrotic septa of cirrhotic livers, and in both HCC and CCC. Two Prox1 mRNA isoforms (2.9 kb and 7.9 kb) were identified with a prevalence of the longer isoform in several HCC samples and the shorter in most CCC samples. Evidence was provided that Myc-associated zinc finger protein (MAZ) might significantly contribute to the gene expression of Prox1 in HCC, while neo-expression of Prox1 in CCC remains to be resolved. A point mutation in the prospero domain of Prox1 was found in one HCC sample.ConclusionOur study shows dysregulation of Prox1 in liver cirrhosis, HCC and CCC, such as neo-expression in cells with biliary epithelial phenotype in liver cirrhosis, and in CCC. Altered Prox1 mRNA expression is partly regulated by MAZ, and mutation of the prospero domain in HCC indicates an involvement for Prox1 during tumor progression.

Multicentric sporadic gastrointestinal stromal tumors (GISTs) of the stomach with distinct clonal origin: differential diagnosis to familial and syndromal GIST variants and peritoneal metastasis.

Most sporadic gastrointestinal stromal tumors (GISTs) occur solitary, whereas a multicentric appearance is suspicious for a familial or syndromal setting such as with germline mutations of proto-oncogene tyrosine protein kinase Kit (KIT) or platelet derived growth factor receptor alpha (PDGFRA), or even for metastases. The aim of this study was to evaluate whether multicentric sporadic GISTs are of clonal origin. Four patients with 1 clinically apparent tumor (mean size 5.6 cm) and 1 to 3 further small incidental tumors (mean size 0.7 cm) were analysed by mutation analysis and comparative genomic hybridization for mutations of KIT and PDGFRA and chromosomal imbalances in their tumors. No clinicopathologic features have been found being indicative of one of the established familial or syndromal GIST variants. Each of the small GISTs were localized in the muscularis propria, and were visible from the serosal but not from the mucosal side. Different mutations of KIT and PDGFRA were present among individual tumors of each patient, and germline mutation of KIT and PDGFRA could be excluded. Comparative genomic hybridization revealed a mean count of 7 chromosomal imbalances in the clinically apparent tumors compared with a mean count of 0.3 in the small incidental counterparts. Sporadic GISTs can appear multicentric by coincidence. They are an important differential diagnosis to familial and syndromal GIST variants, or even to peritoneal metastases. Different mutations of KIT and PDGFRA among individual tumors in 1 patient refer to different clonal origin of multicentric sporadic GISTs. The type of mutation of KIT and PDGFRA was independent of tumor size, whereas small GISTs <1 cm rarely had genomic imbalances.