Evolutionary patterns of chromosomal instability and mismatch repair deficiency in proximal and distal colorectal cancer.

AIM: Colorectal carcinomas (CRCs) progress through heterogeneous pathways. The aim of this study was to analyse whether or not the cytogenetic evolution of CRC is linked to tumour site, level of chromosomal imbalance and metastasis. METHOD: A set of therapy-naïve pT3 CRCs comprising 26 proximal and 49 distal pT3 CRCs was studied by combining immunohistochemistry of mismatch repair (MMR) proteins, microsatellite analyses and molecular karyotyping as well as clinical parameters. RESULTS: A MMR deficient/microsatellite-unstable (dMMR/MSI-H) status was associated with location of the primary tumour proximal to the splenic flexure, and dMMR/MSI-H tumours presented with significantly lower levels of chromosomal imbalances compared with MMR proficient/microsatellite-stable (pMMR/MSS) tumours. Oncogenetic tree modelling suggested two evolutionary clusters characterized by dMMR/MSI-H and chromosomal instability (CIN), respectively, for both proximal and distal CRCs. In CIN cases, +13q, -18q and +20q were predicted as preferentially early events, and -1p, -4 -and -5q as late events. Separate oncogenetic tree models of proximal and distal cases indicated similar early events independent of tumour site. However, in cases with high CIN defined by more than 10 copy number aberrations, loss of 17p occurred earlier in cytogenetic evolution than in cases showing low to moderate CIN. Differences in the oncogenetic trees were observed for CRCs with lymph node and distant metastasis. Loss of 8p was modelled as an early event in node-positive CRC, while +7p and +8q comprised early events in CRC with distant metastasis. CONCLUSION: CRCs characterized by CIN follow multiple, interconnected genetic pathways in line with the basic 'Vogelgram' concept proposed for the progression of CRC that places the accumulation of genetic changes at centre of tumour evolution. However, the timing of specific genetic events may favour metastatic potential.

The clinicopathologic spectrum and genomic landscape of de-/trans-differentiated melanoma.

Dedifferentiation and transdifferentiation are rare and only poorly understood phenomena in cutaneous melanoma. To study this disease more comprehensively we have retrieved 11 primary cutaneous melanomas from our pathology archives showing biphasic features characterized by a conventional melanoma and additional areas of de-/trans-differentiation as defined by a lack of immunohistochemical expression of all conventional melanocytic markers (S-100 protein, SOX10, Melan-A, and HMB-45). The clinical, histologic, and immunohistochemical findings were recorded and follow-up was obtained. The patients were mostly elderly (median: 81 years; range: 42-86 years) without significant gender predilection, and the sun-exposed skin of the head and neck area was most commonly affected. The tumors were deeply invasive with a mean depth of 7 mm (range: 4-80 mm). The dedifferentiated component showed atypical fibroxanthoma-like features in the majority of cases (7), while additional rhabdomyosarcomatous and epithelial transdifferentiation was noted histologically and/or immunohistochemically in two tumors each. The background conventional melanoma component was of desmoplastic (4), superficial spreading (3), nodular (2), lentigo maligna (1), or spindle cell (1) types. For the seven patients with available follow-up data (median follow-up period of 25 months; range: 8-36 months), two died from their disease, and three developed metastases. Next-generation sequencing of the cohort revealed somatic mutations of established melanoma drivers including mainly NF1 mutations (5) in the conventional component, which was also detected in the corresponding de-/trans-differentiated component. In summary, the diagnosis of primary cutaneous de-/trans-differentiated melanoma is challenging and depends on the morphologic identification of conventional melanoma. Molecular analysis is diagnostically helpful as the mutated gene profile is shared between the conventional and de-/trans-differentiated components. Importantly, de-/trans-differentiation does not appear to confer a more aggressive behavior.

Goal-Directed Perfusion Methodology for Determining Oxygenator Performance during Clinical Cardiopulmonary Bypass.

New generation oxygenators incorporate arterial line filtration either sequential to, or directly in, the gas exchange module. This unique design may affect gas exchange performance by altering the operational characteristics of the device. The present study was designed to evaluate three oxygenators in a clinical setting using a goal-directed perfusion algorithm during cardiopulmonary bypass (CPB). After institutional review board approval, 60 adult patients undergoing cardiac surgery for acquired heart disease were matched for disease state and body size into three groups based on oxygenator type: Terumo SX18™ (n = 20), Terumo FX15™ (n = 20), and LivaNova Inspire6F™ 6 Dual (n = 20). An external arterial line filter was used with the FX15, whereas the SX18 and Inspire6F had integrated arterial filters. All perfusion, anesthetic and postoperative care management was standardized using institutional goal-directed patient management processes. Data were collected and stored according to quality improvement guidelines. There were no differences in demographics or type of surgical procedure performed among groups. The Inspire6F patients required lower fraction of inspired oxygen values as compared to the SX18 (67.9% ± 6.2% vs. 75.4% ± 6.5%, p < .005) and FX15 (79.1% ± 8.4%, p < .0001) groups. Arterial oxygen content and oxygen delivery were slightly higher in the FX15 group as compared to SX18 (13.1 ± 1.4 mL O2/dL vs. 12.4 ± 1.1 mL O2/dL, 611.1 ± 150.4 mL O2 vs. 528.2 ± 102.3 mL O2, p < .05). The FX15 patients had significantly higher CPB hematocrits compared to SX18 patients (30.3% ± 3.9% vs. 27.7% ± 2.6%, p < .05), but were not different when compared to the Inspire6F group (28.8% ± 3.5%, p < .50). There were no differences in intraoperative transfusion rates, but a higher percent of patients received postoperative transfusions in the SX18 group as compared to either FX15 or Inspire6F groups (p < .039). There were no differences in postoperative morbidity or complications in any group. In conclusion, the use of the SX18 was comparable to newer generation oxygenators in regard to gas exchange performance and the degree of hemodilution.

Duodenal GIST: a single center experience.

PURPOSE: The duodenum as primary site for gastrointestinal stromal tumors (GISTs) is rare and mitotic rate, tumor size, type of mutation and number of chromosomal aberrations have prognostic implications. METHODS: We analyzed the outcome of 13 patients with duodenal GISTs who underwent surgical tumor resection. Either segmental duodenectomy or pylorus-preserving duodenopancreatectomy was performed. The tumors were histopathologically examined and the risk of progression was assessed based on tumor size and mitotic count. Additionally, mutation analysis of the KIT and PDGFRA receptor tyrosine kinase genes and comparative genomic hybridization (CGH) were performed in all cases. RESULTS: Eight patients underwent segmental duodenectomy and five patients were treated with pylorus-preserving duodenopancreatectomy. None of the five GISTs with low or no risk for malignancy according to the Miettinen classification developed tumor progress. In contrast, five of eight cases (62.5%) with high-risk tumors revealed tumor progress, and four of these patients died (50%). The median overall survival for all patients was 66 months, and the median disease-free survival 41 months. The operative procedure and type of mutation did not correlate with long-term survival. CGH analysis displayed -15q in 12/13 tumors, and -1p in 11/13 cases as characteristic chromosomal aberrations for intestinal origin. Notably, -22q was present in three of four cases with tumor progress. CONCLUSIONS: Both segmental duodenectomy and pylorus-preserving duodenopancreatectomy are appropriate options to treat duodenal GIST and should be implemented depending on resectability and the patient's performing state. The Miettinen classification and CGH findings correlate with the clinical course.

Genomic Complexity as a Biomarker to De-Escalate Adjuvant Imatinib Treatment in High-Risk Gastrointestinal Stromal Tumor.

PURPOSE: Adjuvant imatinib treatment is recommended for patients with localized gastrointestinal stromal tumor (GIST) at high risk of recurrence. Almost half of high-risk patients are cured by surgery alone, indicating a need for improved selection of patients for adjuvant therapy. The aim of this study was to investigate if genomic tumor complexity could be used as a prognostic biomarker. METHODS: The discovery cohort consisted of patients who underwent resection of primary GIST at Oslo University Hospital between 1998 and 2020. Karyotypes were categorized as simple if they had ≤ 5 chromosomal changes and complex if there were > 5 chromosomal aberrations. Validation was performed in an independent patient cohort where chromosomal imbalances were mapped using comparative genomic hybridization. RESULTS: Chromosomal aberrations were detected in 206 tumors, of which 76 had a complex karyotype. The most frequently observed changes were losses at 14q, 22q, 1p, and 15q. The 5-year recurrence-free survival (RFS) in patients classified as very low, low, or intermediate risk was 99%. High-risk patients with a simple tumor karyotype had an estimated 5-year RFS of 94%, and patients with a complex karyotype had an estimated 5-year RFS of 51%. A complex karyotype was associated with poor RFS in patients with and without adjuvant imatinib treatment and in multivariable analysis adjusted for tumor site, size, mitotic count, and rupture. The prognostic impact of genomic complexity was confirmed in the validation cohort. In both cohorts, the 5-year disease-specific survival was > 90% for high-risk patients with genomically simple tumors. CONCLUSION: Genomic tumor complexity is an independent prognostic biomarker in localized, high-risk GIST. Recurrences were infrequent for tumors with simple karyotypes. De-escalation of adjuvant imatinib treatment should be explored in patients with cytogenetically simple GISTs.

Unexpected long-term survival of Stage IV pancreatic cancer patient with synchronic liver metastases after multimodal therapy including upfront surgery.

We report the case of a 56-year-old male with pancreatic cancer and 25 liver metastases. The patient underwent a distal pancreatectomy with 11 metastasectomies in the left liver lobe. Histological examination demonstrated a moderately differentiated ductal adenocarcinoma with pT3N0M1, Stage IVb. Three weeks later, we performed transarterial chemoembolization for the right lobe of the liver, and after 6 weeks we started systemic chemotherapy with FOLFIRINOX. After 31 months, computer tomography examination showed increases in size of the remaining lesions at segment VII/VIII of the right lobe. All liver metastases were surgically removed and a new chemotherapy was initiated. Nevertheless, after 40 months the patient developed two brain metastases. One was surgically resected and the smaller lesion was treated by gamma knife. Unfortunately, the patient died 42 months after the first presentation. Conclusively, in very selected patients with synchronic liver metastasis, multimodal treatment including repeated surgery, TACE and chemotherapy may prolong survival.

Role of biliary complications in chronic graft rejection after living donor liver transplantation.

Postoperative biliary complications remain a substantial challenge after living donor liver transplantation, especially due to its heterogeneous clinical presentation.

Prevention of severe injuries of child passengers in motor vehicle accidents: is re-boarding sufficient?

PURPOSE: The purpose of this study was to evaluate whether prolonged re-boarding of restraint children in motor vehicle accidents is sufficient to prevent severe injury. METHODS: Data acquisition was performed using the Trauma Register DGU® (TR-DGU) in the time period from 2010 to 2019 of seriously injured children (AIS 2 +) aged 0-5 years as motor vehicle passengers (MVP). Primarily treated and transferred patients where included. RESULTS: The study group included 727 of 2030 (35.8%) children, who were severely injured (AIS 2 +) in road traffic accidents, among them 268 (13.2%) as MVPs in the age groups: 0-1 years (42.5%), 2-3 years (26.1%) and 4-5 years (31.3%). The pattern of severe injury was head/brain (56.0%), thoracic (42.2%), abdominal (13.1%), fractures (extremities and pelvis, 52.6%) and spine/severe whiplash (19.8%). The 0-1-year-old MVPs showed the significantly highest proportion of brain injuries with Glasgow Coma Score (GCS) < 8 and severe injury to the spine. The 2-3-year-olds showed the significantly highest proportion of fractures especially the lower extremity and highest proportion of cervical spine injuries of all spine injuries, while the 4-5-year-olds, the significantly highest proportion of abdominal injury and second highest proportion of cervical spine injury of all spine injuries. MVPs of the 0-1-year-old and 2-3-year-old groups showed a higher median Injury Severity Score (ISS) of 21.5 and 22.1 points than the older children (17.0 points). They also suffered an AIS-6-injury significantly more often (9 of 21) of spine (p = 0.001). Especially the cervical spine was significantly more often involved. Passengers at the age of 0-1 years were treated with cardiopulmonary resuscitation (CPR) three times as often as older children in the prehospital setting and twice as often at admission in the Trauma Resuscitation Unit (TRU). Their survival rate was 7 out of 8 (0-1 years), 1 out of 6 (2-3 years) and 1 out of 4 (4-5 years). CONCLUSION: Although the younger MVPs are restraint in a re-boarding position, severe injury to the spine and head occurred more often, while older children as front-faced positioned MVPs suffered from significantly higher rates of abdominal and more often severe facial injury. Our data show, that it is more important to properly restrain children in their adequate car seats (i-size-Norm) and additionally consider the age-related physiological and anatomical specific risks of injury as well as co-factors in road traffic accidents, than only prolonging the re-boarding position over the age of 15 months as a single method.

IgG4-related pseudotumours: a series of 12 cases and a review of the literature.

IgG4-related pseudotumours (IgG4-RPT) represent a distinctive manifestation in the broad spectrum of IgG4-related diseases (IgG4-RD). Due to their wide morphology and rarity, IgG4-RPTs represent a diagnostic challenge in the differential between reactive lesions and a fibrous soft tissue tumours. Thus, our aim was to characterise our cases and review the literature, focusing on the macroscopic and microscopic features of the lesions. In this paper, we summarise the possible presentations and histomorphological features of IgG4-RPT based on data collected from the literature and from cases at our institute and provide an overview of the pathogenesis and histological characteristics based on the knowledge accumulated in recent years. We collected surgical cases with a diagnosis of IgG4-RPT over the period 2013-2020 at two centres and analysed their macroscopic, histological, and immunohistochemical profiles. Furthermore, we performed a literature research in the MEDLINE and EBSCO databases regarding case reports and studies with the explicit diagnosis of IgG4-RPT. Our cases consist of nine men and three women, with an average age of 60±14 years, representing about 0.05% of the lesions evaluated at the two departments. The involved sites include the kidney, lung, gallbladder, pterygopalatine fossa, spleen, tongue, mediastinum, and submandibular gland. Grossly, nine lesions showed sharp margins. On histological examination, all the lesions showed an abundant inflammatory infiltrate with lymphocytes and IgG4-positive plasma cells as well as characteristic fibroblastic storiform proliferation. The literature search revealed 266 cases and similar histomorphological features in 23 locations. In 30 of these cases (11%), IgG4-RPTs were multifocal. IgG4-RPT are exceedingly rare lesions, which makes them challenging to diagnose. They can affect different sites, and the histomorphological presentation may differ.

Diagnostic value of chest radiography in the early management of severely injured patients with mediastinal vascular injury.

INTRODUCTION: Time is of the essence in the management of severely injured patients. This is especially true in patients with mediastinal vascular injury (MVI). This rare, yet life threatening injury needs early detection and immediate decision making. According to the ATLS guidelines [American College of Surgeon Committee on Trauma in Advanced Trauma Life Support (ATLS®), 10th edn, 2018], chest radiography (CXR) is one of the first-line imaging examinations in the Trauma Resuscitation Unit (TRU), especially in patients with MVI. Yet thorough interpretation and the competence of identifying pathological findings are essential for accurate diagnosis and drawing appropriate conclusion for further management. The present study evaluates the role of CXR in detecting MVI in the early management of severely injured patients. METHOD: We addressed the question in two ways. (1) We performed a retrospective, observational, single-center study and included all primary blunt trauma patients over a period of 2 years that had been admitted to the TRU of a Level-I Trauma Center. Mediastinal/chest (M/C) ratio measurements were calculated from CXRs at three different levels of the mediastinum to identify MVI. Two groups were built: with MVI (VThx) and without MVI (control). The accuracy of the CXR findings were compared with the results of whole-body computed tomography scans (WBCT). (2) We performed another retrospective study and evaluated the usage of sonography, CXR and WBCT over 15 years (2005-2019) in level-I-III Trauma Centers in Germany as documented in the TraumaRegister DGU® (TR-DGU). RESULTS: Study I showed that in 2 years 267 patients suffered from a significant blunt thoracic trauma (AIS ≥ 3) and met the inclusion criteria. 27 (10%) of them suffered MVI (VThx). Through the initial CXR in a supine position, MVI was detected in 56-92.6% at aortic arch level and in 44.4-100% at valve level, depending on different M/C-ratios (2.0-3.0). The specificity at different thresholds of M/C ratio was 63.3-2.9% at aortic arch level and 52.9-0.4% at valve level. The ROC curve showed a statistically random process. No significant differences of the cardiac silhouette were observed between VThx and Control (mean cardiac width was 136.5 mm, p = 0.44). Study II included 251,095 patients from the TR-DGU. A continuous reduction of the usage of CXR in the TRU could be observed from 75% in 2005 to 25% in 2019. WBCT usage increased from 35% in 2005 to 80% in 2019. This development was observed in all trauma centers independently from their designated level of care. CONCLUSION: According to the TRU management guidelines (American College of Surgeon Committee on Trauma in Advanced Trauma Life Support (ATLS®), 10th edn, 2018; Reissig and Kroegel in Eur J Radiol 53:463-470, 2005) CXR in supine position is performed to detect pneumothorax, hemothorax and MVI. Our study showed that sensitivity and specificity of CXR in detecting MVI was statistically and clinically not reliable. Previous studies have already shown that CXR is inferior to sonography in detecting pneumothorax and hemothorax. Therefore, we challenge the guidelines and suggest that the use of CXR in the early management of severely injured patients should be individualized. If sonography and WBCT are available and reasonable, CXR is unnecessary and time consuming. The clinical reality reflected in the usage of CXR and WBCT over time, as documented in the TR-DGU, seems to support our statement.

Epithelioid/mixed phenotype in gastrointestinal stromal tumors with KIT mutation from the stomach is associated with accelerated passage of late phases of the cell cycle and shorter disease-free survival.

In gastrointestinal stromal tumors (GISTs), the occurrence of an epithelioid/mixed phenotype has been correlated to PDGFRA mutations, gastric localization and favorable outcome. On the other hand, the prognostic significance of an epithelioid/mixed growth pattern occasionally observed in GISTs with KIT mutation is unclear. The aim of this study was to evaluate the prognostic significance of an epithelioid/mixed phenotype in correlation to anatomical localization, genotype, and expression of cell-cycle markers in a series of 116 primary GISTs with KIT mutation on a tissue microarray. Independent of their anatomical localization, the majority of KIT-mutated GISTs displayed a pure spindled phenotype (72%), with the remaining tumors showing an epithelioid/mixed growth pattern. In KIT-mutated GISTs from the stomach, the occurrence of an epithelioid/mixed growth pattern was significantly correlated with larger tumor diameters (P=0.005), higher mitotic counts (P=0.0001), high-risk category (P=0.001), higher expression of the G2-phase cell-cycle marker cyclin B1 (P=0.04), higher expression of the G1 to M-phase proliferation marker Ki67 (P=0.02) and a significantly shorter disease-free survival (P=0.003) compared with tumors with pure spindled morphology. In contrast, there were no significant differences between pure spindled and epithelioid/mixed GISTs from the small/large bowel. Our findings indicate that the epithelioid/mixed phenotype in KIT-mutant gastric GISTs represents a secondary tumor growth pattern associated with tumor progression and adverse outcome, probably through accelerated G1/S-phase restriction point passage.

Number of pathologists in Germany: comparison with European countries, USA, and Canada.

The rapid development of pathology is in contrast to a shortage of qualified staff. The aims of the present study are to compile basic information on the numbers of German physicians in pathology and to compare it with the situation in Europe and overseas. In addition, model calculations will shed light on the effects of part-time working models. Various publicly accessible databases (EuroStat) as well as publications of medical associations and professional associations of European countries and the USA/Canada were examined. In addition, a survey was carried out among the institutes of German universities. Figures from 24 European countries and the USA/Canada were evaluated. With one pathologist per 47,989 inhabitants, the density of pathologists in Germany in relation to the population is the second-lowest in Europe (average: 32,018). Moreover, the proportion of pathologists among the physicians working in Germany is the lowest in Europe and at the same time lower than in the USA and Canada (Germany: 1:200, USA: 1:70, Canada: 1:49). The ratio of pathologists to medical specialists is shifted in the same direction. The survey among university pathologists revealed a relevant increase in the workload over the last 10 years. The majority of institutes can manage this workload only with considerable difficulties. With a ratio between specialists and residents of 1:1, the university institutes show a high commitment in the area of training. The results of this study indicate a shortage of pathologists in Germany that could lead to a bottleneck in large parts of the health system.

Sequential osseointegration from osseohealing to osseoremodeling - Histomorphological comparison of novel 3D porous and solid Ti-6Al-4V titanium implants.

In the present study, we analyzed the histological characteristics of osseointegration of an open-porous Ti-6Al-4V material that was produced in a space holder method creating a 3-D through-pores trabecular design that mimics the inhomogeneity and size relationships of trabecular bone in macro- as well as microstructure. Pairs of cylindrical implants with a porosity of 49 % and an average pore diameter of 400 µm (PI) or equal sized solid, corundum blasted devices (SI) as reference were bilaterally implanted press fit in the lateral condyles of 16 rabbits. Histological examination was performed after 4 weeks of short-term osseohealing and 12 weeks of mid-term osseoremodeling and we summarized the criteria for sequential osseointegration. After 4 weeks, osteoid had already been largely replaced by mineralized woven bone in both types of implants but was only represented to a greater extent in the deeper pores of PI. The cortical as well as trabecular region showed regular osseohealing with excessive and spatially undirected formation of immature woven bone. A dense bone mass was found in the cortical area, while in the trabecular region the bone mass was reduced distinctly, presenting large lacuna-like recesses and a demarcating trabecular structure. The pores near the implant surface contained more mineralized woven bone than the deeper pores. After 12 weeks, the osseoremodeling was largely completed with a physiological maturation to lamellar bone. The newly formed bone mass increased for PI and SI compared to the 4-week group and osteoid was only detectable in the deeper pores. The inhomogeneous trabecular design of the pores enables an excellent ingrowth of mineralized lamellar bone after remodeling to a pore depth of 1800 µm, which proves a functional load transfer from the surrounding bone into the implant. According to the concept of osseointegration by Branemark and Albrektsson, the histological evaluation confirms a successful, superior osseointegration of the presented porous properties improving long-term implant stability. The presented study protocol allows an excellent evaluation and comparison of the sequential osseointegration from short-term osseohealing to mid-term osseoremodeling.

Osseointegration of a novel 3D porous Ti-6Al-4V implant material - Histomorphometric analysis in rabbits.

Porous structure properties are known to conduct initial and long-term stability of titanium alloy implants. This study aims to assess the histomorphometric effect of a 3-D porosity in Ti-6Al-4V implants (PI) on osseointegration in comparison to solid Ti-6Al-4V implants (SI). The PI was produced in a spaceholder method and sintering and has a pore size of mean 400 µm (50 µm to 500 µm) and mimics human trabecular bone. Pairs of PI and equal sized SI as reference were bilaterally implanted at random in the lateral femoral condyle of 16 Chinchilla-Bastard rabbits. The animals were sacrificed after 4 and 12 weeks for histomorphometric analysis. The histomorphometric evaluation confirmed a successful short-term osseohealing (4 weeks) and mid-term osseoremodeling (12 weeks) for both types of implants. The total newly formed bone area was larger for PI than for SI after 4 and 12 weeks, with the intraporous bone area being accountable for the significant difference (p<0.05). A more detailed observation of bone area distribution revealed a bony accumulation in a radius of +/- 500 µm around the implant surface after remodeling. The bone-to-implant contact (BIC) increased significantly (p<0.05) from 4 to 12 weeks (PI 26.23 % to 42.68 %; SI 28.44 % to 47.47 %) for both types of implants. Due to different surface properties, however, PI had a significant (p<0.05) larger absolute osseous contact (mm) to the implant circumference compared to the SI (4 weeks: 7.46 mm vs 5.72 mm; 12 weeks: 11.57 mm vs 9.52 mm [PI vs. SI]). The regional influences (trabecular vs. cortical) on bone formation and the intraporous distribution were also presented. Conclusively, the porous structure and surface properties of PI enable a successful and regular osseointegration and enhance the bony fixation compared to solid implants under experimental conditions.

Lmcd1/Dyxin, a novel Z-disc associated LIM protein, mediates cardiac hypertrophy in vitro and in vivo.

To identify new mediators of cardiac hypertrophy, we performed a genome-wide mRNA screen of stretched neonatal rat cardiomyocytes (NRCMs). In addition to known members of the hypertrophic gene program, we found the novel sarcomeric Z-disc LIM protein Lmcd1/Dyxin markedly upregulated. Consistently, Lmcd1 was also induced in several mouse models of myocardial hypertrophy suggesting a causal role in cardiac hypertrophy. We overexpressed Lmcd1 in NRCM, which led to cardiomyocyte hypertrophy and induction of the hypertrophic gene program. Likewise, the calcineurin-responsive gene RCAN1-4 was found significantly upregulated. Conversely, knockdown of Lmcd1 blunted the response to hypertrophic stimuli such as stretch and phenylephrine (PE), suggesting that Lmcd1 is required for the hypertrophic response. Furthermore, PE-mediated activation of calcineurin was completely blocked by knockdown of Lmcd1. To confirm these results in vivo, we generated transgenic mice with cardiac-restricted overexpression of Lmcd1. Despite normal cardiac function, adult transgenic mice displayed significant cardiac hypertrophy, again accompanied by induction of hypertrophic marker genes such as ANF and alpha-skeletal actin. Likewise, Rcan1-4 was found upregulated. Moreover, when crossed with transgenic mice overexpressing constitutionally active calcineurin, Lmcd1 transgenic mice revealed an exacerbated cardiomyopathic phenotype with depressed contractile function and further increased cardiomyocyte hypertrophy. We show that the novel z-disc protein Lmcd1/Dyxin is significantly upregulated in several models of cardiac hypertrophy. Lmcd1/Dyxin potently induces cardiomyocyte hypertrophy both in vitro and in vivo, while knockdown of this molecule prevents hypertrophy. Mechanistically, Lmcd1/Dyxin appears to signal through the calcineurin pathway. Lmcd1/Dyxin may thus represent an attractive target for novel antihypertrophic strategies.

Pitfalls in mutational testing and reporting of common KIT and PDGFRA mutations in gastrointestinal stromal tumors.

BACKGROUND: Mutation analysis of KIT and PDGFRA genes in gastrointestinal stromal tumors is gaining increasing importance for prognosis of GISTs and for prediction of treatment response. Several groups have identified specific mutational subtypes in KIT exon 11 associated with an increased risk of metastatic disease whereas GISTs with PDGFRA mutations often behave less aggressive. Furthermore, in advanced GIST disease with proven KIT exon 9 mutation the doubled daily dose of 800 mg imatinib increases the progression free survival and is now recommended both in the European and the American Guidelines. In Germany, there are still no general rules how to perform mutational analysis. METHODS: When comparing results from six different molecular laboratories we recognized the need of standardisation. Six German university laboratories with experience in mutation analysis in GISTs joined together to develop recommendations for the mutation analysis of the most common and clinically relevant hot spots, i. e. KIT exons 9 and 11 and PDGFRA exon 18. We performed a three-phased interlaboratory trial to identify pitfalls in performing molecular analysis in GISTs. RESULTS: We developed a design for a continuous external laboratory trial. In 2009 this external trial was conducted by 19 laboratories via the initiative for quality assurance in pathology (QuiP) of the German Society of Pathology and the Professional Association of German Pathologists. CONCLUSIONS: By performing a three-phased internal interlaboratory trial and conducting an external trial in Germany we were able to identify potential pitfalls when performing KIT and PDGFRA mutational analysis in gastrointestinal stromal tumors. We developed standard operation procedures which are provided with the manuscript to allow other laboratories to prevent these pitfalls.

Expression of p16INK4A in gastrointestinal stromal tumours (GISTs): two different forms exist that independently correlate with poor prognosis.

AIMS: To determine the prognostic impact of p16INK4A expression in gastrointestinal stromal tumours (GISTs), which is currently being questioned, with both loss and overexpression said to be correlated with poor prognosis. METHODS AND RESULTS: Two different forms of p16INK4A were identified, presenting with predominantly nuclear and cytoplasmic expression pattern, respectively. The immunohistochemical expression of the two forms and their correlation with E2F1 and prognosis were analysed in a series of 120 GISTs with clinical follow-up. Low nuclear p16INK4A expression correlated with E2F1 up-regulation, higher mitotic counts, and tumour progression. The prognostic value of nuclear p16INK4A expression was only marginally significant (P=0.05). Strong expression of the cytoplasmic p16INK4A form was significantly associated with shorter disease-free survival (P=2x10(-5)). The prognostic impact of strong expression of the cytoplasmic p16INK4A form was independent of anatomical localization, tumour size and mitotic counts, and significant even among the cohort of tumours with high malignant potential. CONCLUSIONS: Low expression of the nuclear p16INK4A form and strong expression of the cytoplasmic p16INK4A form both represent two independent parameters each associated with tumour progression in GISTs. Low nuclear p16INK4A expression enables E2F1 up-regulation and consecutive accelerated cell proliferation. In contrast, strong cytoplasmic p16INK4A expression probably reflects a negative feedback loop as a result of (as yet unknown) oncogenic events.

Optimal thresholds of risk parameters for gastrointestinal stromal tumors.

BACKGROUND: Gastrointestinal stromal tumors (GIST) are the most frequent mesenchymal neoplasms of the gastrointestinal tract with highly variable potential for relapse. Tumor size and mitotic index (MI) are major risk factors that predict the outcome of GIST patients. Recent risk stratification schemes include some or all of the empirical size thresholds of 2 cm, 5 cm, and 10 cm and MI thresholds of 5 per 50 high-power fields (hpf) and 10 per 50 hpf. However, data that verify these numbers are sparse. METHODS: By exhaustive regression tree analysis, maximally selected rank statistics and survival difference analysis with bootstrap sampling on a naive GIST population of 161 patients with a mean follow-up of 44 months, current stratification schemes using tumor size and MI were analyzed herein. RESULTS: /Conclusions: Thresholds that optimally stratify the risk of recurrence are observed at tumor sizes of 4-5 cm and 10-11 cm and at mitotic indices of about 5 per 50 hpf and 10 per 50 hpf, respectively. While these data validate the canonical thresholds for size and MI used in risk stratification of GIST, transition regions as well as differences in the implementation of these thresholds between the different classification schemes proposed in the recent years should be considered when classifying GIST.

Expression of stem cell factor and its receptor c-Kit during the development of intrahepatic cholangiocarcinoma.

Stem cell factor (SCF) and its receptor, c-Kit, constitute an important signal transduction system with proliferative and anti-apoptotic functions. Besides regulating hemopoietic stem cell proliferation and liver regeneration, it has been implicated in the regulation of human malignancies. However, the cellular expression of the SCF-c-Kit gene system in the liver during cholangiocarcinogenesis has not been studied to date. The protein- and mRNA-expression levels of SCF and c-Kit genes were examined in normal rat liver, in isolated normal rat liver cells and in a thioacetamide-induced rat model of intrahepatic cholangiocarcinoma (CC). Immunohistochemical analysis of the normal liver showed that SCF is expressed in the wall of the hepatic artery and in some cells, which were located along the sinusoids, although it was absent from hepatocytes and biliary epithelial cells. The mRNA analysis of isolated normal liver cell populations revealed a co-expression of SCF- and c-Kit-mRNA in sinusoidal endothelial cells and in Kupffer cells, whereas passaged and cultured liver myofibroblasts (MFs) expressed only SCF. Low levels of the SCF- and c-Kit-mRNA expression could be detected in isolated hepatocytes of the normal liver. Immunohistochemical analysis of the CC tissue showed SCF positivity in proliferating biliary cells (CK-19(+)), in macrophages (ED-1(+)) and in MFs (alpha-smooth-muscle-actin, alpha-SMA(+)) of the tumoral microenvironment. c-Kit-positivity could be detected on hepatocytes of the regenerating nodules and on the proliferating bile ducts of CC. Compared with the normal liver tissue, SCF-mRNA from the CC tissue was upregulated up to 20-fold, whereas c-Kit-mRNA was upregulated up to fivefold. These data indicate that several cell populations may become able to express SCF and/or c-Kit during cholangiocarcinogenesis. Therefore, the SCF-c-Kit system may contribute to tumor development, for instance, by inducing proliferation of hepatocytes and of biliary cells and by acting as a surviving factor for CC cells.

Distinct biphasic histomorphological pattern in gastrointestinal stromal tumours (GISTs) with common primary mutations but divergent molecular cytogenetic progression.

AIMS: The morphological diversity of gastrointestinal stromal tumours (GISTs) is well appreciated. The aim of this study was to shed light on the molecular pathogenesis of GISTs displaying a distinct biphasic histomorphological pattern, which is poorly understood. METHODS AND RESULTS: Six biphasic gastric GISTs (four high, one intermediate and one low risk for aggressive behaviour) were studied by histological, immunohistochemical, molecular and comparative genomic hybridization methods. The different tumour components were designated as primary and secondary compartments, based on cellularity and mitotic index. In addition, metastases from two patients were analysed separately. According to the classification of Miettinen et al., four biphasic histomorphological patterns were seen: (i) sclerosing spindle cell/dyscohesive or paraganglioma-like epithelioid (n = 2); (ii) sarcomatous spindle cell/pleomorphic sarcomatous spindle cell (n = 1); (iii) sarcomatous spindle cell/sarcomatous epithelioid (n = 2); and (iv) sclerosing epithelioid/hypercellular epithelioid/sarcomatous epithelioid (n = 1) morphology. In each case, both tumour compartments revealed the same KIT (n = 5) or platelet-derived growth factor receptor-alpha (n = 1) mutation, as well as common chromosomal imbalances reflecting their common clonal origin. Additional chromosomal imbalances were detected in the secondary tumour compartments and their respective metastases. CONCLUSIONS: Our results indicate that the intratumoral phenotypic diversity in GIST reflects histomorphological progression, which is associated with higher chromosomal instability, irrespective of the primary mutation.