Fibroblast growth factor receptors in cancer: genetic alterations,diagnostics, therapeutic targets and mechanisms of resistance

Fibroblast growth factor receptors (FGFRs) are aberrantly activated through single-nucleotide variants, gene fusions and copy number amplifications in 5–10% of all human cancers, although this frequency increases to 10–30% in urothelial carcinoma and intrahepatic cholangiocarcinoma. We begin this review by highlighting the diversity of FGFR genomic alterations identified in human cancers and the current challenges associated with the development of clinical-grade molecular diagnostic tests to accurately detect these alterations in the tissue and blood of patients. The past decade has seen significant advancements in the development of FGFR-targeted therapies, which include selective, non-selective and covalent small-molecule inhibitors, as well as monoclonal antibodies against the receptors. We describe the expanding landscape of anti-FGFR therapies that are being assessed in early phase and randomised controlled clinical trials, such as erdafitinib and pemigatinib, which are approved by the Food and Drug Administration for the treatment of FGFR3-mutated urothelial carcinoma and FGFR2-fusion cholangiocarcinoma, respectively. However, despite initial sensitivity to FGFR inhibition, acquired drug resistance leading to cancer progression develops in most patients. This phenomenon underscores the need to clearly delineate tumour-intrinsic and tumour-extrinsic mechanisms of resistance to facilitate the development of second-generation FGFR inhibitors and novel treatment strategies beyond progression on targeted therapy.Subject terms: Cancer, Cancer     

Intake of arsenic from water, food composites and excretion through urine, hair from a studied population in West Bengal, India.

To evaluate the main intake source of arsenic by the villagers from arsenic-affected families in Jalangi and Domkol blocks in Mushidabad district, West Bengal-India, we determined the concentrations of arsenic in tube-well water and in food composites, mainly including vegetables and cereals collected from the surveyed families which were cultivated in that region. The daily dietary intakes of arsenic by the villagers were estimated and the excretions of arsenic through urine and hair were determined. The arsenic concentrations in hair and urine of the studied population living in mild (2.78 microg/L), moderate (30.7 microg/L) and high (118 microg/L) arsenic-affected families were 133, 1,391 and 4,713 microg/kg and 43.1, 244 and 336 microg/L, respectively. The linear regressions show good correlations between arsenic concentrations in water vs hair (r(2)=0.928, p<0.001) and water vs urine (r(2)=0.464, p<0.01). Approximately 29.4%, 58.1% and 62.1% of adult population from mild, moderate and high arsenic-affected families were suffering from arsenical skin manifestations. The mean arsenic concentrations of food composites (vegetables and cereals) in high arsenic-affected families are not significantly different from mild arsenic-affected families. The daily dietary intakes of arsenic from water and food composites of the studied population, living in high, moderate and mild arsenic-affected families were 568, 228 and 137 microg, respectively. The linear regressions show good correlations between arsenic concentrations in hair vs daily dietary intake (r(2)=0.452, p<0.001) and urine vs daily dietary intake (r(2)=0.134, p<0.001). The water for drinking contributed 6.07%, 26.7% and 58.1% of total arsenic in our study from mild, moderate and high arsenic-affected families. The result suggested that the contaminated water from high arsenic-affected families should be the main source for intake of arsenic. On contrary, the contribution of arsenic-contaminated food composites from mild and moderate arsenic-affected families might be the main source for intake of arsenic. The Food and Agriculture Organization/World Health Organization (FAO/WHO) provisional tolerable weekly intake (PTWI) values of arsenic in our study were 3.32, 5.75 and 12.9 microg/kg body weight/day from mild, moderate and high arsenic-affected families, respectively, which is higher than the recommended PTWI value of arsenic (2.1 microg/kg body weight/day).     

Presence of hepatitis B surface antigen mutant G145R DNA in the peripheral blood leukocytes of the family members of an asymptomatic carrier and evidence of its horizontal transmission

An asymptomatic carrier and all six of his family members were detected positive for HBV DNA in their peripheral blood leukocytes (PBL), by polymerase chain reaction. Direct sequencing of the amplified DNA revealed that the HBV DNA from the carrier and his wife was of subtype ayw. Interestingly, the amplified HBV DNA from the five other members of the family was found to be not only of subtype adw but also contained G to A mutation at nucleotide position 587. This indicates the presence of established vaccine escape mutant of the virus (G145R) and suggests two different sources of infection within the family. Southern blot hybridization of EcoR1 digested DNA from PBL indicated presence of HBV DNA, integrated into cellular DNA and also in the form of free viral DNA. The study not only establishes the persistence of surface mutant G145R HBV DNA, within the PBL of HBsAg negative individuals from the non-vaccinated random population, but also suggests possible horizontal transmission of the mutant among the family members although none of the family members has received immunoprophylaxis against HBV or had clinically apparent disease or any other known risk factors of HBV infection. As all of them were seronegative for HBsAg/antiHBc, the presence of G145R mutant in the PBL signaled possibility of spread of the vaccine escape mutant virus by blood transfusion, unsafe injection practices or through sexual root.     

Evaluation of Hybridization Capture Versus Amplicon‐Based Methods for Whole‐Exome Sequencing

Next‐generation sequencing has aided characterization of genomic variation. While whole‐genome sequencing may capture all possible mutations, whole‐exome sequencing remains cost‐effective and captures most phenotype‐altering mutations. Initial strategies for exome enrichment utilized a hybridization‐based capture approach. Recently, amplicon‐based methods were designed to simplify preparation and utilize smaller DNA inputs. We evaluated two hybridization capture‐based and two amplicon‐based whole‐exome sequencing approaches, utilizing both Illumina and Ion Torrent sequencers, comparing on‐target alignment, uniformity, and variant calling. While the amplicon methods had higher on‐target rates, the hybridization capture‐based approaches demonstrated better uniformity. All methods identified many of the same single‐nucleotide variants, but each amplicon‐based method missed variants detected by the other three methods and reported additional variants discordant with all three other technologies. Many of these potential false positives or negatives appear to result from limited coverage, low variant frequency, vicinity to read starts/ends, or the need for platform‐specific variant calling algorithms. All methods demonstrated effective copy‐number variant calling when evaluated against a single‐nucleotide polymorphism array. This study illustrates some differences between whole‐exome sequencing approaches, highlights the need for selecting appropriate variant calling based on capture method, and will aid laboratories in selecting their preferred approach.     

Myeloid Mixed Lineage Kinase 3 Contributes to Chronic Ethanol-Induced Inflammation and Hepatocyte Injury in Mice

Proinflammatory activity of hepatic macrophages plays a key role during progression of alcoholic liver disease (ALD). Since mixed lineage kinase 3 (MLK3)-dependent phosphorylation of JNK is involved in the activation of macrophages, we tested the hypothesis that myeloid MLK3 contributes to chronic ethanol-induced inflammatory responses in liver, leading to hepatocyte injury and cell death. Primary cultures of Kupffer cells, as well in vivo chronic ethanol feeding, were used to interrogate the role of MLK3 in the progression of liver injury. Phosphorylation of MLK3 was increased in primary cultures of Kupffer cells isolated from ethanol-fed rats compared to cells from pair-fed rats. Kupffer cells from ethanol-fed rats were more sensitive to LPS-stimulated cytokine production; this sensitization was normalized by pharmacological inhibition of MLK3. Chronic ethanol feeding to mice increased MLK3 phosphorylation robustly in F4/80⁺ Kupffer cells, as well as in isolated nonparenchymal cells. MLK3^−/− mice were protected from chronic ethanol-induced phosphorylation of MLK3 and JNK, as well as multiple indicators of liver injury, including increased ALT/AST, inflammatory cytokines, and induction of RIP3. However, ethanol-induced steatosis and hepatocyte apoptosis were not affected by MLK3. Finally, chimeric mice lacking MLK3 only in myeloid cells were also protected from chronic ethanol-induced phosphorylation of JNK, expression of inflammatory cytokines, and increased ALT/AST. MLK3 expression in myeloid cells contributes to phosphorylation of JNK, increased cytokine production, and hepatocyte injury in response to chronic ethanol. Our data suggest that myeloid MLK3 could be targeted for developing potential therapeutic strategies to suppress liver injury in ALD patients.Key words: Alcoholic liver disease (ALD), Kupffer cells, Necroptosis, Toll-like receptor 4 (TLR4), Cytokines     

Formation and uptake of arylhydroxylamine-haptenated proteins in human dendritic cells.

Bioactivation of sulfonamides and the subsequent formation of haptenated proteins is believed to be a critical step in the development of hypersensitivity reactions to these drugs. Numerous lines of evidence suggest that the presence of such adducts in dendritic cells (DCs) migrating to draining lymph nodes is essential for the development of cutaneous reactions to xenobiotics. Our objective was to determine the ability of human DCs to form drug-protein covalent adducts when exposed to sulfamethoxazole (SMX), dapsone (DDS), or their arylhydroxylamine metabolites [sulfamethoxazole hydroxylamine (S-NOH) and dapsone hydroxylamine (D-NOH)] and to take up preformed adduct. Naive and immature CD34+ KG-1 cells were incubated with SMX, DDS, or metabolites. Formation of haptenated proteins was probed using confocal microscopy and ELISA. Cells were also incubated with preformed adduct (drug-bovine serum albumin conjugate), and uptake was determined using confocal microscopy. Both naive and immature KG-1 cells were able to bioactivate DDS, forming drug-protein adducts, whereas cells showed very little protein haptenation when exposed to SMX. Exposure to S-NOH or D-NOH resulted in protein haptenation in both cell types. Both immature and naive KG-1 cells were able to take up preformed haptenated proteins. Thus, DCs may acquire haptenated proteins associated with drugs via intracellular bioactivation, uptake of reactive metabolites, or uptake of adduct formed and released by adjacent cells (e.g., keratinocytes).     

Detection of haptenated proteins in organotypic human skin explant cultures exposed to dapsone.

Bioactivation of parent drug to reactive metabolite(s) followed by protein haptenation has been suggested to be a critical step in the elicitation of cutaneous drug reactions. Although liver is believed to be the primary organ of drug bioactivation quantitatively, other organs including skin may also metabolize drugs. Cultured human epidermal keratinocytes and dermal fibroblasts have been shown to be capable of bioactivating sulfonamides and sulfones, giving rise to haptenated proteins. It is, however, unclear whether metabolic events in these isolated cells reflect bioactivation in vivo. Hence, split-thickness human skin explants were exposed to dapsone (DDS) or its arylhydroxylamine metabolite (dapsone hydroxylamine, D-NOH) and probed for protein haptenation. DDS and D-NOH were applied either epicutaneously or mixed in the medium (to mimic its entry into skin from the systemic circulation). DDS-protein adducts were readily detected in skin explants exposed to either DDS or D-NOH. Adducts were detected mainly in the upper epidermal region in response to epicutaneous application, whereas adducts were formed all over the explants when DDS/D-NOH were mixed in the culture medium. In addition, adducts were visible in HLA-DR+ cells, indicating their presence in the dendritic cell population in the skin. Our results demonstrate the ability of intact human skin to bioactivate DDS leading to protein haptenation.