Understanding homologous recombination repair deficiency in biliary tract cancers: clinical implications and correlation with platinum sensitivity.

BACKGROUND: Biliary tract cancers (BTCs) exhibit high mortality rates and significant heterogeneity in both clinical and molecular characteristics. This study aims to molecularly characterize a cohort of patients with BTC, with a specific focus on genomic alterations within homologous recombination repair (HRR) genes in a real-world setting. PATIENTS AND METHODS: We carried out a retrospective analysis on 256 patients with BTC treated at five Austrian centers and one German comprehensive cancer center between 2016 and 2023 utilizing comprehensive genomic profiling platforms to assess HRR status and its correlation with clinical outcomes after platinum-based chemotherapy. RESULTS: A total of 67 patients (27.5%) exhibited HRR gene mutations (HRRm), with the most common pathogenic alterations in BAP1 (9%), ARID1A (7.8%), and ATM (6.1%). Time to failure of the first-line strategy (TFS) between patients with HRRm and non-HRRm treated with platinum agents was 7.9 and 6.7 months, respectively [hazard ratio (HR) 0.89; P = 0.49]. The overall survival (OS) estimates at 6, 18, and 24 months were 82%, 45%, and 39% in the HRRm group (median 16.01 months) and 81%, 42%, and 22% in the HRR group (median 15.68 months), respectively (Fleming-Harrington test P = 0.0004; log-rank P = 0.022). Significance did not persist in the multivariate analysis (HR 0.72; 95% confidence interval 0.489-1.059; P = 0.095). An interaction between HRRm status and molecular-informed therapeutic strategies in later lines was noted. In the second-line treatment, OS following an irinotecan-based regimen was comparable to re-exposure to platinum-based agents (12.36 versus 10.13 months; HR 0.92; P = 0.85). No better outcome was noted for patients with HRRm versus patients with non-HRRm with second-line platinum agents (HR 1.45; P = 0.35). CONCLUSIONS: Patients with HRRm with BTC showed a potential advantage in OS following platinum-based first-line chemotherapy, presumably attributed to enhanced opportunities for targetable coalterations. Further investigation is needed to outline HRR within the scope of BTCs and detail a clinically meaningful sensitivity to platinum agents or targeted approaches with poly (ADP-ribose) polymerase (PARP) inhibitors.

Bevacizumab mitigates codon-specific effects of trifluridine/tipiracil on efficacy outcome parameters in metastatic colorectal cancer.

BACKGROUND: Molecular informed therapy changed treatment patterns of metastatic colorectal cancer (mCRC). Recently KRAS G12, the most prevalent RAS mutation in mCRC, was investigated to be a negative predictive marker for the efficacy of trifluridine/tipiracil (FTD/TPI). Whether this proposed selectivity remains when FTD/TPI is combined with bevacizumab remains elusive. We aimed to describe the efficacy of FTD/TPI + bevacizumab depending on the RAS mutational status in a real-world population. PATIENTS AND METHODS: Patients from five different cancer centers in Austria who received FTD/TPI + bevacizumab in any treatment line having available information on their molecular profile were eligible. Data were retrospectively collected by chart review. Survival data were compared using log-rank test. Multivariate Cox regression models included several established covariates. RESULTS: One hundred and twenty-three patients with mCRC were included in this study. Median overall survival (OS) was highly similar in the RAS wild type (WT) [9.63 months (95% confidence interval [CI] 8.055-13.775 months)] and the RAS mutant cohorts [8.78 months (95% CI 8.055-11.014 months)], which was confirmed in a multivariable model adjusting for potential confounders; hazard ratio (HR): 1.05 (95% CI 0.618-1.785; P = 0.857). In addition, no effect of KRAS G12 status on patient outcome was observed. In detail, OS was 8.88 months (95% CI 7.332-12.921 months) in patients with KRAS G12 mutation, compared to 9.47 months (95% CI 8.088-11.375 months) in patients with RAS WT/no-KRAS G12 disease [HR: 0.822 (95% CI 0.527-1.282; P = 0.387)]. CONCLUSION: This real-world study indicates that the efficacy of FTD/TPI + bevacizumab is independent of RAS mutational status and that bevacizumab may therefore mitigate the potentially limited efficacy of FTD/TPI monotherapy in the KRAS G12-mutated population.

Sequence of the porcine full-length cDNA encoding ribosomal protein rpS12.

The full-length cDNA encoding the porcine ribosomal protein rpS12 was shown to be differentially expressed in endothelial and smooth muscle cells. A cDNA clone containing the entire rpS12 sequence was isolated from growth-stimulated capillary endothelial cells by use of subtractive hybridization and differential screening. The porcine rpS12 cDNA coding region exhibits 94% identity to the rat rpS12 and 92% to the human rpS12 cDNAs.

Expression of a chemotactic cytokine (MCP-1) in cerebral capillary endothelial cells in vitro.

A full-length cDNA encoding the porcine monocyte chemoattractant protein-1 (pMCPC-1) was isolated from growth-stimulated porcine cerebral capillary endothelial cells (cEC); the pMCP-1 cDNA showed 89% identity to human MCP-1 and was isolated by use of subtractive hybridization and differential screening of two phenotypically different sub-populations of cloned cEC. pMCP-1 was abundantly expressed in cEC grown in the presence of FCS, ECGF and heparin whereas lower expression was observed in cEC kept in FCS-supplemented medium only. As shown by Northern blot analysis, no pMCP-1 transcripts were present in total RNA derived from freshly isolated brain capillaries, large brain vessels or whole brain homogenate. MCP/JE expression was also demonstrated in ECGF/heparin-treated murine cEC. Astrocytes and smooth muscle cells grown in FCS-supplemented medium did not show MCP-1 expression. Treatment of porcine cEC with TNF-alpha increased pMCP-1 mRNA levels in a dose-dependent manner. These data further support the notion that cerebral capillary endothelial cells actively participate in processes of CNS host defense.

Incorporation of beta-galactosidase-expressing endothelial cells into the skeletal muscle microvascular bed of mice.

Cloned murine endothelial cells (cEC) were used as a carrier system for introducing a foreign gene into the microvascular bed of the hind limb of inbred mice. cEC were transfected with a beta-galactosidase-neo fusion construct, which enables both selection for DNA uptake in the presence of G 418 and the staining of cells for beta-galactosidase activity. Transfected cEC adhered and integrated readily into confluent monolayers of nontransfected cEC (up to 26% of total cell number). Seeding lacZ-transfected cEC on explanted arteries revealed rapid adhesion of the cells (within minutes) to the intact endothelium. After injection of 10(6) transfected EC via the femoral artery into the microvascular bed of the hind limb their presence was documented by beta-galactosidase staining after various time periods (1 h to 4 wk). Implanted cEC were detected in numerous elements of the microcirculation both in frozen sections and in squash preparations of the hind limb muscle and in the femoral bone up to 4 wk after the injection. The microvascular bed of skeletal muscle of the mouse as a recipient site for transduced syngeneic endothelial cells is, thus, a suitable experimental model to study various strategies for somatic gene therapy.

Comparison of gene expression of extracellular matrix molecules in brain microvascular endothelial cells and astrocytes.

By use of random-primed cDNA probes the expression of extracellular matrix molecules in cerebral microvascular endothelial cells (cEC) and in astrocytes from mouse brain was examined. Two phenotypically different batches of cloned cEC were used. Expression of major adhesive ECM molecules, constituting the endothelial basement membrane (i.e., fibronectin, laminin A, B and collagen IV) and of other attachment factors, such as SPARC (osteonectin), tenascin and thrombospondin 1, was examined. We have demonstrated that cEC of different morphology display variations in the expression of fibronectin (FN), thrombospondin 1 (TSP1) and collagen IV (C IV). Astrocytes were shown to contain FN, TSP1, TN and SPARC mRNA. Unexpectedly, SPARC mRNA could not be detected in any of the capillary endothelial cells examined. Therefore, we suggest that astrocytes are likely to be involved in endothelial differentiation and function in the central nervous system via ECM molecule secretion.

Use of genetically modified glial cells overexpressing laminin alpha1-chain peptides in neurite outgrowth studies.

1. C6 glioma cells were transfected with two constructs carrying C-terminal laminin alpha1-chain sequences of 117 and 114 bp length, respectively. These sequences are specifically known to code for peptides which have neurite-promoting activity. 2. The stable expression and secretion of the two peptides was detected by Northern and Western blot analysis. 3. Primary neuronal cultures derived from embryonic mouse forebrain were cocultured with these transfected cells and exhibited a substantial increase in neurite outgrowth and in survival time. Conditioned media from the transfected cells generated similar effects. 4. Organotypic cultures from embryonic mouse brain were used as a second system as being closer to the in vivo situation. Again, coculture of brain slices with transfected cells or treatment with laminin peptide-containing media increased neuronal outgrowth.

Transcription-independent activation of ornithine decarboxylase activity by heparin in cloned cerebral endothelial cells.

Heparin, a highly sulfated glycosaminoglycan, is known to be obligatory for long-term endothelial cell cultures; it potentiates the mitogenic activities of endothelial cell growth factors and prolongs the replicative life span of the cells. Here we have shown that besides its growth factor-supportive role, heparin exerts a specific action on cerebral capillary endothelial cells (cECs), unrelated to serum or growth factors, by increasing activity of ornithine decarboxylase (ODC; EC 4.1.1.17) in these cells. For our experiments we have used two different types of cloned cECs: type I cECs, grown in the presence of endothelial cell growth factor and heparin, and type II cECs, usually cultivated without growth factors. Heparin action on ODC activity was shown to be dose dependent within the range of 1-100 micrograms/ml. Increasing concentrations of or depletion of endothelial cell growth factor from type I cultures had no effect on ODC activity. The increase in enzyme activity was highest after 30 min to 1 h of heparin treatment. As evidenced by northern analysis, the heparin-mediated enhancement of ODC activity was not accompanied by changes of ODC mRNA levels. Studies of DNA replication revealed that in the absence of heparin-binding growth factors, heparin did not affect the proliferative activity of cloned cECs.