Combined Hepatocellular-Cholangiocarcinoma: Biology, Diagnosis, and Management.

Background: Combined hepatocellular-cholangiocarcinoma (cHCC-iCCA) is a rare type of primary liver cancer displaying characteristics of both hepatocytic and cholangiocytic differentiation. Summary: Because of its aggressive nature, patients with cHCC-iCCA exhibit a poorer prognosis than those with HCC. Surgical resection and liver transplantation may be considered curative treatment approaches; however, only a minority of patients are eligible at the time of diagnosis, and postoperative recurrence rates are high. For cases that are not eligible for surgery, locoregional and systemic therapy are often administered based on treatment protocols applied for HCC or iCCA. Owing to the rarity of this cancer, there are still no established standard treatment protocols; therefore, the choice of therapy is often personalized and guided by the suspected predominant component. Further, the genomic and molecular heterogeneity of cHCC-iCCA can severely compromise the efficacy of the available therapies. Key Messages: In the present review, we summarize the latest advances in cHCC-iCCA and attempt to clarify its terminology and molecular biology. We provide an overview of the etiology of cHCC-iCCA and present new insights into the molecular pathology of this disease that could contribute to further studies aiming to improve the patient outcomes through new systemic therapies.

Multi-location trials and population-based genotyping reveal high diversity and adaptation to breeding environments in a large collection of red clover.

Red clover (Trifolium pratense L.) is an outcrossing forage legume that has adapted to a wide range of climatic and growing conditions across Europe. Red clover is valued for its high yield potential and its forage quality. The high amount of genetic diversity present in red clover provides an invaluable, but often poorly characterized resource to improve key traits such as yield, quality, and resistance to biotic and abiotic stresses. In this study, we examined the genetic and phenotypic diversity within a diverse set of 395 diploid red clover accessions via genome wide allele frequency fingerprinting and multi-location field trials across Europe. We found that the genetic structure of accessions mostly reflected their geographic origin and only few cases were detected, where breeders integrated foreign genetic resources into their local breeding pools. The mean dry matter yield of the first main harvesting season ranged from 0.74 kg m-2 in Serbia and Norway to 1.34 kg m-2 in Switzerland. Phenotypic performance of accessions in the multi-location field trials revealed a very strong accession x location interaction. Local adaptation was especially prominent in Nordic red clover accessions that showed a distinct adaptation to the growing conditions and cutting regime of the North. The traits vigor, dry matter yield and plant density were negatively correlated between the trial location in Norway and the locations Great Britain, Switzerland, Czech Republic and Serbia. Notably, breeding material and cultivars generally performed well at the location where they were developed. Our results confirmed that red clover cultivars were bred from regional ecotypes and show a narrow adaptation to regional conditions. Our study can serve as a valuable basis for identifying interesting materials that express the desired characteristics and contribute to the adaptation of red clover to future climatic conditions.

Phenotypic variation and quantitative trait loci for resistance to southern anthracnose and clover rot in red clover.

KEY MESSAGE: High variability for and candidate loci associated with resistance to southern anthracnose and clover rot in a worldwide collection of red clover provide a first basis for genomics-assisted breeding. Red clover (Trifolium pratense L.) is an important forage legume of temperate regions, particularly valued for its high yield potential and its high forage quality. Despite substantial breeding progress during the last decades, continuous improvement of cultivars is crucial to ensure yield stability in view of newly emerging diseases or changing climatic conditions. The high amount of genetic diversity present in red clover ecotypes, landraces, and cultivars provides an invaluable, but often unexploited resource for the improvement of key traits such as yield, quality, and resistance to biotic and abiotic stresses. A collection of 397 red clover accessions was genotyped using a pooled genotyping-by-sequencing approach with 200 plants per accession. Resistance to the two most pertinent diseases in red clover production, southern anthracnose caused by Colletotrichum trifolii, and clover rot caused by Sclerotinia trifoliorum, was assessed using spray inoculation. The mean survival rate for southern anthracnose was 22.9% and the mean resistance index for clover rot was 34.0%. Genome-wide association analysis revealed several loci significantly associated with resistance to southern anthracnose and clover rot. Most of these loci are in coding regions. One quantitative trait locus (QTL) on chromosome 1 explained 16.8% of the variation in resistance to southern anthracnose. For clover rot resistance we found eight QTL, explaining together 80.2% of the total phenotypic variation. The SNPs associated with these QTL provide a promising resource for marker-assisted selection in existing breeding programs, facilitating the development of novel cultivars with increased resistance against two devastating fungal diseases of red clover.

A Novel AICDA Splice-Site Mutation in Two Siblings with HIGM2 Permits Somatic Hypermutation but Abrogates Mutational Targeting.

Hyper-IgM syndrome type 2 (HIGM2) is a B cell intrinsic primary immunodeficiency caused by mutations in AICDA encoding activation-induced cytidine deaminase (AID) which impair immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM). Whereas autosomal-recessive AID-deficiency (AR-AID) affects both CSR and SHM, the autosomal-dominant form (AD-AID) due to C-terminal heterozygous variants completely abolishes CSR but only partially affects SHM. AR-AID patients display enhanced germinal center (GC) reactions and autoimmune manifestations, which are not present in AD-AID, suggesting that SHM but not CSR regulates GC reactions and peripheral B cell tolerance. Herein, we describe two siblings with HIGM2 due to a novel homozygous AICDA mutation (c.428-1G > T) which disrupts the splice acceptor site of exon 4 and results in the sole expression of a truncated AID variant that lacks 10 highly conserved amino acids encoded by exon 4 (AID-ΔE4a). AID-ΔE4a patients suffered from defective CSR and enhanced GC reactions and were therefore indistinguishable from other AR-AID patients. However, the AID-ΔE4a variant only partially affected SHM as observed in AD-AID patients. In addition, AID-ΔE4a but not AD-AID patients revealed impaired targeting of mutational hotspot motives and distorted mutational patterns. Hence, qualitative defects in AID function and altered SHM rather than global decreased SHM activity may account for the disease phenotype in these patients.

A Non-destructive Method to Quantify Leaf Starch Content in Red Clover.

Grassland-based ruminant livestock production provides a sustainable alternative to intensive production systems relying on concentrated feeds. However, grassland-based roughage often lacks the energy content required to meet the productivity potential of modern livestock breeds. Forage legumes, such as red clover, with increased starch content could partly replace maize and cereal supplements. However, breeding for increased starch content requires efficient phenotyping methods. This study is unique in evaluating a non-destructive hyperspectral imaging approach to estimate leaf starch content in red clover for enabling efficient development of high starch red clover genotypes. We assessed prediction performance of partial least square regression models (PLSR) using cross-validation, and validated model performance with an independent test set under controlled conditions. Starch content of the training set ranged from 0.1 to 120.3 mg g-1 DW. The best cross-validated PLSR model explained 56% of the measured variation and yielded a root mean square error (RMSE) of 17 mg g-1 DW. Model performance decreased when applying the trained model on the independent test set (RMSE = 29 mg g-1 DW, R 2 = 0.36). Different variable selection methods did not increase model performance. Once validated in the field, the non-destructive spectral method presented here has the potential to detect large differences in leaf starch content of red clover genotypes. Breeding material could be sampled and selected according to their starch content without destroying the plant.

Levels of uPA and PAI-1 in breast cancer and its correlation to Ki67-index and results of a 21-multigene-array.

BACKGROUND: Conventional parameters including Ki67, hormone receptor and Her2/neu status are used for risk stratification for breast cancer. The serine protease urokinase plasminogen activator (uPA) and the plasminogen activator inhibitor type-1 (PAI-1) play an important role in tumour invasion and metastasis. Increased concentrations in tumour tissue are associated with more aggressive potential of the disease. Multigene tests provide detailed insights into tumour biology by simultaneously testing several prognostically relevant genes. With OncotypeDX®, a panel of 21 genes is tested by means of quantitative real-time polymerase chain reaction. The purpose of this pilot study was to analyse whether a combination of Ki67 and uPA/PAI-1 supplies indications of the result of the multigene test. METHODS: The results of Ki67, uPA/PAI-1 and OncotypeDX® were analysed in 25 breast carcinomas (luminal type, pT1/2, max pN1a, G2). A statistical and descriptive analysis was performed. RESULTS: With a proliferation index Ki67 of < 14%, the recurrence score (RS) from the multigene test was on average in the low risk range, with an intermediate RS usually resulting if Ki67 was > 14%. Not elevated values of uPA and PAI-1 showed a lower rate of proliferation (average 8.5%) than carcinomas with an increase of uPA and/or PAI-1 (average 13.9%); p = 0.054, Student's t-test. When Ki67 was > 14% and uPA and/or PAI-1 was raised, an intermediate RS resulted. These differences were significant when compared to cases with Ki67 < 14% with non-raised uPA/PAI-1 (p < 0.03, Student's t-test). Without taking into account the proliferative activity, an intermediate RS was also verifiable if both uPA and PAI-1 showed raised values. CONCLUSION: A combination of the values Ki67 and uPA/PAI-1 tended to depict the RS to be expected. From this it can be deduced that an appropriate analysis of this parameter combination may be undertaken before the multigene test in routine clinical practice. The increasing cost pressure makes it necessary to base the implementation of a multigene test on ancillary variables and to potentially leave it out if not required in the event of a certain constellation of results (Ki67 raised, uPA and PAI-1 raised).

Practical Consequences Resulting from the Analysis of a 21-Multigene Array in the Interdisciplinary Conference of a Breast Cancer Center.

During the multidisciplinary planning of postoperative therapy after breast cancer, borderline cases can arise with no clear rationale for or against adjuvant chemotherapy. In 50 hormone- receptor-positive, Her2neu-negative carcinomas of the breast with no or only minimal lymph node involvement (max. pT1a) we initiated an Oncotype DX® multigene assay in addition to the evaluation of usual parameters. In the oncology conference a vote for or against chemotherapy was taken on the basis of the conventional criteria for decision-making before the test results were available. The final recommendation was made after the multigene test. In 32 breast carcinomas (64%) a low recurrence score could be documented, while 26 (32%) showed an intermediate RS and 3 (6%) showed a high RS. In most cases the result of the test could validate the choice of therapy established using conventional criteria. In 5 cases the initial recommendation for adjuvant therapy was revised, and in 3 cases chemotherapy was secondarily recommended after evaluation of the test results. Conversely, in some cases a low or intermediate risk constellation did not argue against a recommendation for adjuvant chemotherapy. Altogether, the results of our study do not indicate that a multigene assay should be used as a routine diagnostic tool. Instead a thorough compilation and careful analysis of conventional parameters for therapeutic decision-making should take precedence, with special emphasis on histopathological and immunohistochemical results. In selected cases, however, a multigene assay can be a useful tool in the deliberation for or against a therapeutic pathway.

Repair of an Autologous Saphenous Vein Graft Aneurysm Ten Years after Renal Artery Reconstruction during Live Donor Renal Transplantation.

Saphenous vein graft (SVG) aneurysms (SVGA) after renal transplantation represents a rare vascular complication with subsequent challenging multidisciplinary treatment. We present a case of a 30-year-old female who received a live donor kidney transplantation for end-stage renal disease that was caused due to the hemolytic uremic syndrome. Postoperatively, an insufficient graft perfusion due to an arterial kinking was noted and repaired using an autologous SVG interposition. Ten years later, a 3-cm aneurysm of the SVG at the anastomotic site with the common iliac artery was discovered. Multidisciplinary surgical exploration with excision of the aneurysm-carrying vein graft and interposition of a new autologous SVG was successfully carried out with preservation of renal allograft's function. Treatment of SVGA after rental transplantation with a new autologous SVG is challenging but feasible, requiring a multidisciplinary approach in order to guarantee successful rates and to prevent allograft loss.