Michel P. Bihl

Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas

Somatic STAT3 mutations present in a subset of inflammatory hepatocellular adenomas result in the generation of constitutively active STAT3 proteins that homodimerize independently of IL-6 stimulation.

V600E BRAF mutations are alternative early molecular events in a subset of KIT/PDGFRA wild-type gastrointestinal stromal tumours

Background: A small subset (10–15%) of gastrointestinal stromal tumours (GISTs) lack mutations in KIT and PDGFRA (wild-type GIST). Recently, a novel BRAF exon 15 mutation (V600E) was detected in imatinib-naive wild-type high-risk intestinal GISTs (4%). However, the frequency and distribution of BRAF mutations within the spectrum of GISTs, and whether they might represent secondary events acquired during tumour progression, remain unknown. Methods: 69 GISTs (39 KIT mutants, 2 PDGFRA mutants and 28 wild-type) were analysed for mutations in BRAF exon 15 and KRAS exon 2. To assess the stage at which these mutations might occur in GIST, a considerable number of incidental gastric (n = 23) and intestinal (n = 2) tumours were included. Results: BRAF mutations (V600E) were detected in 2 of 28 wild-type GISTs (7%), but in none of the 41 KIT/PDGFRA mutants. No KRAS mutation was detected. The two BRAF-mutated GISTs measured 4 mm in diameter and originated in the gastric body and the jejunum in two men (mean age, 76 years). Both tumours were mitotically inactive KIT-positive spindle-cell GISTs that were indistinguishable histologically from their more common KIT-mutated counterparts. Conclusion: BRAF mutations represent an alternative molecular pathway in the early tumorigenesis of a subset of KIT/PDGFRA wild-type GISTs and are per se not associated with a high risk of malignancy. Mutations in KIT, PDGFRA and BRAF were mutually exclusive in this study. Results from this and a previous study indicate that BRAF-mutated GISTs show a predilection for the small bowel (four of five tumours), but this needs further evaluation in larger studies.

Clinicopathological and protein characterization of BRAF- and K-RAS-mutated colorectal cancer and implications for prognosis.

Recent evidence highlights the potential prognostic and predictive value of BRAF and K‐RAS gene alterations in patients with colorectal cancer. However, a comprehensive evaluation of BRAF and K‐RAS mutations and their specific clinicopathological features, histomorphological presentation and effect on protein expression have not been systematically analyzed. The aim of this study was to characterize the clinicopathological, histomorphological and protein expression profiles of BRAF‐ and K‐RAS‐mutated colorectal cancers and determine their impact on patient survival. Molecular analysis for microsatellite instability (MSI), K‐RAS and BRAF was carried out on paraffin‐embedded samples from 404 patients with primary colorectal cancer. Using tissue microarrays, 36 tumor‐associated and 14 lymphocyte/inflammatory‐associated markers were evaluated by immunohistochemistry. BRAF mutation was associated with right‐sided tumor location (p < 0.001), higher tumor grade (p = 0.029), absence of peritumoral lymphocytic inflammation (p = 0.026) and MSI‐H (p < 0.001). In right‐sided tumors, loss of CDX2 expression was observed in 23 of 24 cases (95.8%). BRAF mutation was a poor prognostic indicator in patients with right‐sided disease (p = 0.01). This result was maintained in multivariable analysis (p < 0.001; HR = 2.82; 95% CI: 1.5–5.5) with pT, pN and vascular invasion and independent of CDX2 expression. K‐RAS mutation, in contrast, was not associated with any of the features analyzed. BRAF gene mutation is an adverse prognostic factor in right‐sided colon cancer patients independent of MSI status and, moreover, in patients with lymph node‐negative disease. These results indicate that molecular analysis for BRAF may be a useful biomarker for identifying patients with right‐sided colon cancer with poor outcome who may benefit from a more individualized course of therapy.

Comprehensive epidermal growth factor receptor gene analysis from cytological specimens of non-small-cell lung cancers

Epidermal growth factor receptor (EGFR) gene mutations and increased copy numbers are considered as predictors of response to EGFR tyrosine kinase inhibitors (EGFR-TKI) in non-small-cell lung cancer (NSCLC). Lung cancer diagnosis is often based on cytology alone. However, almost all published data on EGFR gene analyses were obtained from biopsies. This study tested the feasibility of EGFR gene analyses on cytological specimens. Eighty-four cytological specimens from NSCLCs were prospectively analysed for EGFR gene mutation in exons 18–21 and EGFR gene copy numbers were evaluated by fluorescence in situ hybridisation (FISH). A FISH-positive result was defined according to the criteria by Cappuzzo et al established for biopsies of NSCLCs. Fluorescence in situ hybridisation results of cytological specimens were compared to the FISH results on matching biopsies (n=33). Initial diagnosis of NSCLC was solely based on cytology in 37 out of 84 (44.0%) patients. Out of 80 NSCLCs, 6 (7.5%) showed EGFR gene mutations. Out of 67 cancers, 45 (67.2%) were FISH positive on cytological specimens. Comparison of FISH showed a FISH-positive result in 21 out of 33 (63.6%) cytological specimens but in only 8 out of 33 (24.2%) matched biopsies. Epidermal growth factor receptor gene analyses are well applicable to cytological specimens. The high FISH-positive rate of NSCLC on cytological specimens contrasts with the low rate on biopsies when previously suggested criteria are used. New criteria for a positive EGFR FISH status to predict response to therapy with EGFR-TKI need to be defined for cytological specimens.

Combined analysis of specific KRAS mutation, BRAF and microsatellite instability identifies prognostic subgroups of sporadic and hereditary colorectal cancer

Confounding effects of specific KRAS gene alterations on colorectal cancer (CRC) prognosis stratified by microsatellite instability (MSI) and BRAFV600E have not yet been investigated. The aim of our study was to evaluate the combined effects of MSI, BRAFV600E and specific KRAS mutation (Gly → Asp; G12D, Gly → Asp, G13D; Gly → Val; G12V) on prognosis in 404 sporadic and 94 hereditary CRC patients. MSI status was determined according to the Bethesda guidelines. Mutational status of KRAS and BRAFV600E was assessed by direct DNA sequencing. In sporadic CRC, KRAS G12D mutations had a negative prognostic effect compared to G13D and wild‐type cancers (p = 0.038). With MSI, specific KRAS and BRAFV600E mutations, 3 distinct prognostic subgroups were observed in univariate (p = 0.006) and multivariable (p = 0.051) analysis: patients with (i) KRAS mutation G12D, G12V or BRAFV600E mutation, (ii) KRAS/BRAFV600E wild‐type or KRAS G13D mutations in MSS/MSI‐L and (iii) MSI‐H and KRAS G13D mutations. Moreover, none of the sporadic MSI‐H or hereditary patients with KRAS G13 mutations had a fatal outcome. Specific KRAS mutation is an informative prognostic factor in both sporadic and hereditary CRC and applied in an algorithm with BRAFV600E and MSI may identify sporadic CRC patients with poor clinical outcome.

Interaction of C/EBPα and the glucocorticoid receptor in vivo and in nontransformed human cells

Belonging to the family of steroid hormones, glucocorticoids are essential for development and survival of vertebrates. The cellular response to glucocorticoids is attributed to the glucocorticoid receptor, which functions as a transcription factor. However, the majority of glucocorticoid‐modulated genes lack a DNA binding site for the glucocorticoid receptor, raising the question of which mechanism mediates the responses to glucocorticoids. It has been suggested that besides direct DNA binding of the glucocorticoid receptor, interaction with members of other transcription factor families modulates the effect of the glucocorti‐coid receptor. However, the significance of such transcription factor interaction is not clear. In cultured human mesenchymal cells and peripheral blood leukocytes of human volunteers treated with glucocorticoids, we detected the formation of a complex between the GR and the CCAAT/enhancer binding protein a.Inin vitro experiments, this interaction turned out to be responsible for the inhibitory action of glucocorticoids on lymphocytic and mesenchymal cell proliferation. Our results suggest that complex formation of the GR with C/EBPα accounts for a novel pathway of glucocorticoid action.—Rüdiger, J. J., Roth, M., Bihl, M. P., Cornelius, B. C., Johnson, M., Ziesche, R., Block, L.‐H. Interaction of C/EBPα and the glucocorticoid receptor in vivo and in nontransformed human cells. FASEB J. 16, 177–184 (2002)

Microscopic gastrointestinal stromal tumors in esophageal and intestinal surgical resection specimens: a clinicopathologic, immunohistochemical, and molecular study of 19 lesions.

Microscopic gastrointestinal stromal tumors (GISTs) (synonyms: sporadic interstitial cell of Cajal hyperplasia, seedling GISTs, minimal GISTs) are common incidental findings in gastroesophageal resections (9% to 35%). To our knowledge, their frequency, clinicopathologic features, and molecular pathogenesis from nongastroesophageal sites have so far not been sufficiently analyzed. We studied 19 lesions from distal esophagus (n=8), gastroesophageal junction (n=2), sigmoid colon (n=5), and vermiform appendix, cecum, rectum, and small intestine (1 each). Microscopic GISTs were detected in 0.2%, 0.1%, and 0.01% of routinely processed resection specimens from sigmoid colon, vermiform appendix, and rectum, respectively. Patients were 11 men and 8 women with a mean age of 66 years (range, 57 to 86 y). Thirteen patients had GI cancers and 5 had diverticular disease. None has a family history of GIST or features of neurofibromatosis 1. Lesions were 0.5 to 4 mm in size (mean, 1.12 mm), were all spindled and had noncircumscribed infiltrating borders. All arose in the muscularis propria and 2 were predominantly subserosal. Immunohistochemistry revealed a CD117+/CD34+/smooth muscle actin-negative phenotype in 18/19 lesions. Three KIT exon 11 mutations (2 point mutations and 1 deletion, all involving W557) were detected in 3/12 lesions with successful molecular analysis. In conclusion, incidental microscopic GISTs are uncommon in intestinal resections (≤0.1%), contrasting with their gastroesophageal counterparts (≥9%). Somatic KIT mutations are early initiating molecular events in a subset of them. The remarkable variation in the incidence of microscopic GISTs at different GI sites suggests an origin from heterogeneous subsets of interstitial cells of Cajal with varying potentials for neoplastic transformation.

Comprehensive analysis of CpG island methylator phenotype (CIMP)-high, -low, and -negative colorectal cancers based on protein marker expression and molecular features†‡

CpG island methylator phenotype (CIMP) is being investigated for its role in the molecular and prognostic classification of colorectal cancer patients but is also emerging as a factor with the potential to influence clinical decision‐making. We report a comprehensive analysis of clinico‐pathological and molecular features (KRAS, BRAF and microsatellite instability, MSI) as well as of selected tumour‐ and host‐related protein markers characterizing CIMP‐high (CIMP‐H), ‐low, and ‐negative colorectal cancers. Immunohistochemical analysis for 48 protein markers and molecular analysis of CIMP (CIMP‐H: ≥ 4/5 methylated genes), MSI (MSI‐H: ≥ 2 instable genes), KRAS, and BRAF were performed on 337 colorectal cancers. Simple and multiple regression analysis and receiver operating characteristic (ROC) curve analysis were performed. CIMP‐H was found in 24 cases (7.1%) and linked (p < 0.0001) to more proximal tumour location, BRAF mutation, MSI‐H, MGMT methylation (p = 0.022), advanced pT classification (p = 0.03), mucinous histology (p = 0.069), and less frequent KRAS mutation (p = 0.067) compared to CIMP‐low or ‐negative cases. Of the 48 protein markers, decreased levels of RKIP (p = 0.0056), EphB2 (p = 0.0045), CK20 (p = 0.002), and Cdx2 (p < 0.0001) and increased numbers of CD8+ intra‐epithelial lymphocytes (p < 0.0001) were related to CIMP‐H, independently of MSI status. In addition to the expected clinico‐pathological and molecular associations, CIMP‐H colorectal cancers are characterized by a loss of protein markers associated with differentiation, and metastasis suppression, and have increased CD8+ T‐lymphocytes regardless of MSI status. In particular, Cdx2 loss seems to strongly predict CIMP‐H in both microsatellite‐stable (MSS) and MSI‐H colorectal cancers. Cdx2 is proposed as a surrogate marker for CIMP‐H. Copyright

Calcifying fibrous tumor of the stomach: clinicopathologic and molecular study of seven cases with literature review and reappraisal of histogenesis.

Calcifying fibrous tumor (CFT) is a rare benign mesenchymal tumor composed of hyalinized fibrous tissue with interspersed bland fibroblastic spindled cells, scattered psammomatous, and/or dystrophic calcifications and variably prominent mononuclear inflammatory infiltrate. CFTs show a predilection for the abdominal cavity and soft tissue. To date, 6 gastric and 3 intestinal CFTs have been reported. We analyzed 7 gastric CFTs including 6 new cases. Patients were 4 men and 3 women with a mean age of 53 years (range, 40 to 77). Mean tumor size was 2.2 cm. Most tumors originated in the gastric body (6/7). Six were incidental findings at autopsy or during surgery for other diseases. One ulcerated tumor caused iron deficiency anemia and ulcer symptoms. Six tumors involved the muscularis propria with variable submucosal and subserosal extension and 1 arose within thickened muscularis mucosae adjacent to a mucosal invagination. Histology was typical with uniformly hypocellular vaguely storiform collagen, lymphoplasmacytic infiltrates, lymphoid aggregates and psammomatous, and dystrophic calcifications. Peritumoral lymphoid aggregates were seen in 3 cases. Adjacent muscle coat contained lymphoid aggregates with fiber degeneration (2), minute CFT-like foci (1), and calcifications (1). In none of the cases were there remnants of burnt-out GIST, inflammatory fibroid polyp, inflammatory myofibroblastic tumor, leiomyoma, schwannoma, or other specific lesion. All tumors were negative for CD117, S100, smooth muscle actin, desmin, ALK1, h-caldesmon, and PDGFRA. Two stained focally with CD34. Scattered IgG4-positive plasma cells were seen in 4 of 6 cases stained with this marker. All 5 tumors with available tissue for molecular analysis were wild-type for KIT and PDGFRA. Three patients had follow-up (range, 12 to 24 mo); none developed recurrence. Gastric CFTs are distinct from sclerosing GIST and other mesenchymal gut lesions and may represent a localized inflammatory fibrosclerosis in response to immune-mediated or other-type tissue injury affecting the muscularis propria. They differ from soft tissue CFTs by smaller size, older age at presentation and lack of recurrence, and from peritoneal CFTs by equal gender distribution, older age, and absent multifocal occurrence.

Multiple Sporadic Gastrointestinal Stromal Tumors (GISTs) of the Proximal Stomach are Caused by Different Somatic KIT Mutations Suggesting a Field Effect

Multifocal gastrointestinal stromal tumors (GISTs) are observed in patients with germline KIT or PDGFRA mutations, and in those with neurofibromatosis 1. However, the pathogenesis of apparently sporadic multifocal gastric GISTs in adults is poorly understood. We analyzed 27 GISTs from 11 patients (mean age, 75 y) with 2 to 4 tumors each. All tumors represented incidental findings in surgical (n=8) and autopsy (n=3) specimens and were located in the gastric body or fundus within ≤4 cm distance from each other. The 8 surgical cases represented 10% of GISTs involving the proximal stomach in our case material. Tumor size ranged from 1.5 mm to 45 mm (mean, 9 mm). Histology revealed a uniform spindle cell morphology with a variable sclerosis/calcification and a low mitotic activity (<5 mitoses/50 high-power fields). All tumors were KIT+/CD34+. Nineteen of 22 tumors (79%) revealed mutations in KIT exon 11 (13 deletions and 6 point mutations). Individual lesions from the same patient displayed different mutations in all, but 1 case, thus ruling out germline mutations and neurofibromatosis 1. Our findings indicate that multifocal gastric GISTs in elderly patients are unrelated to hereditary GIST syndromes. Clustering of these lesions in the proximal stomach, their close proximity, and the demonstration of different KIT mutations in individual lesions from the same patient point to the existence of distinct subsets of interstitial cells of Cajal with a higher propensity for different somatic KIT exon 11 mutations, possibly as a result of a field effect involving premutational epigenetic alterations or yet unidentified etiologic factors.