ROS1 rearrangements in non-small cell lung cancer: screening by immunohistochemistry using proportion of cells staining without intensity and excluding cases with MAPK pathway drivers improves test performance.

Therapeutically actionable ROS1 rearrangements have been described in 1-3% of non-small cell lung cancer (NSCLC). Screening for ROS1 rearrangements is recommended to be by immunohistochemistry (IHC), followed by confirmation with fluorescence in situ hybridisation (FISH) or sequencing. However, in practise ROS1 IHC presents difficulties due to conflicting scoring systems, multiple clones and expression in tumours that are wild-type for ROS1. We assessed ROS1 IHC in 285 consecutive cases of NSCLC with non-squamous histology over a nearly 2-year period. IHC was scored with ROS1 clone D4D6 (n=270), clone SP384 (n=275) or both clones (n=260). Results were correlated with ROS1 break-apart FISH (n=67), ALK status (n=194), and sequence data of EGFR (n=178) and other drivers, where possible. ROS1 expression was detected in 161/285 cases (56.5%), including 13/14 ROS1 FISH-positive cases. There was no ROS1 expression in one ROS1 FISH-positive case in which sequencing detected an ALK-EML4 fusion, but not a ROS1 fusion. The other 13 ROS1 FISH-positive cases showed moderate to strong staining with both IHC clones. However, one case with a TPM3-ROS1 fusion would have been scored as negative with SP384 and D4D6 clones by some previous criteria. ROS1 expression was also detected in 58/285 cases (20.4%) that had driver mutations in genes other than ROS1. A sensitivity of 100% for detecting a ROS1 rearrangement by FISH was achieved by omitting intensity from the IHC scoring criteria and expression in >0% cells with D4D6 or in ≥50% cells with SP384. Excluding cases with driver events in any MAPK pathway gene (e.g., in ALK, EGFR, KRAS, BRAF, ERBB2 and MET) substantially reduced the number of cases proceeding to ROS1 FISH. Only 15.9% of MAPK-negative NSCLC would proceed to FISH for an IHC threshold of >0% cells with D4D6, with a specificity of 42.4%. For a threshold of ≥50% cells with SP384, only 18.5% of MAPK-negative cases would proceed to FISH, with a specificity of 31.4%. Based on our data we suggest an algorithm for screening for ROS1 rearrangements in NSCLC in which ROS1 FISH is only performed in cases that have been demonstrated to lack activating mutations in any MAPK pathway gene by comprehensive sequencing and ALK IHC, and show staining at any intensity in ≥50% of cells with clone SP384, or >0% cells with D4D6.

Diversity in exon 5 of HLA-C(∗)04:01:01G is significant in anthropological studies.

Polymorphisms in Human Leukocyte Antigen (HLA) class I genes are generally considered to be relevant only if they reside in exons 2 and 3 or if they affect the expression of the allele. HLA-C(∗)04:82 differs from the common HLA-C(∗)04:01:01 by having a 9 nucleotide, or 3 amino acid duplication, in exon 5. Having observed HLA-C(∗)04:82 in a New Zealand Maori stem cell patient, we have attempted to examine the prevalence of this allele in different ethnicities. Although our studies are in a limited number of patients and donors, they have revealed that, in the Pacific region, HLA-C(∗)04:82 appears to be the most common allele of the HLA-C(∗)04:01:01G group of alleles, notably in Filippinos and in Maori/Polynesians. In these populations this allele has characteristic HLA-ABCDRB1 haplotypes. Thus, our studies have shown that polymorphisms outside of the clinically important exons can be considered to be relevant in anthropological studies.

Ruthenium-catalyzed N-alkylation of amines and sulfonamides using borrowing hydrogen methodology.

The alkylation of amines by alcohols has been achieved using 0.5 mol % [Ru(p-cymene)Cl(2)](2) with the bidentate phosphines dppf or DPEphos as the catalyst. Primary amines have been converted into secondary amines, and secondary amines into tertiary amines, including the syntheses of Piribedil, Tripelennamine, and Chlorpheniramine. N-Heterocyclization reactions of primary amines are reported, as well as alkylation reactions of primary sulfonamides. Secondary alcohols require more forcing conditions than primary alcohols but are still effective alkylating agents in the presence of this catalyst.

A mechanistic investigation into the elimination of phosphonium salts from rhodium-TRIPHOS complexes under methanol carbonylation conditions.

Phosphine modified rhodium complexes are currently the topic of considerable research as methanol carbonylation catalysts, but often suffer from poor stability. This paper reports on an investigation into how coordination mode affects the elimination of phosphonium salts from rhodium complexes, namely [trans-RhCl(CO)(PPh3)2] 1, [RhCl(CO)(dppe)] 2, [RhCl(CO)(dppb)]2 3, [Rh(TRIPHOS)(CO)2]Cl 4. These complexes are all potential pre-catalysts for methanol carbonylation. The reaction of these complexes with methyl iodide at 140 degrees C under both N2 and CO atmospheres has been studied and has revealed clear differences in the stability of the corresponding Rh(III) complexes. In contrast to both monomeric 2 and dimeric 3 that react cleanly with CH3I to give stable Rh(III) acetyl complexes, 4 forms a novel bidentate complex after the elimination of the one arm of the ligand as a quaternised phosphonium salt. The structure of this complex has been determined spectroscopically and using X-ray crystallography. The mechanism of formation of this novel complex has been investigated using 13CH3I and strong evidence that supports a dissociative mechanism as the means of phosphine loss from the rhodium centre is provided.

Evaluation of C4 diphosphine ligands in rhodium catalysed methanol carbonylation under a syngas atmosphere: synthesis, structure, stability and reactivity of rhodium(I) carbonyl and rhodium(III) acetyl intermediates.

The carbonylation of methanol to acetic acid is a hugely important catalytic process, and there are considerable cost and environmental advantages if a process could be designed that was tolerant of hydrogen impurities in the CO feed gas, while eliminating by-products such as propionic acid and acetaldehyde altogether. This paper reports on an investigation into the application of rhodium complexes of several C(4) bridged diphosphines, namely BINAP, 1,4-bis(diphenylphosphino)butane (dppb), bis(diphenylphosphino)xylene (dppx) and 1,4-bis(dicyclohexylphosphino)butane (dcpb) as catalysts for hydrogen tolerant methanol carbonylation. An investigation into the structure, reactivity and stability of pre-catalysts and catalyst resting states of these complexes has also been carried out in order to understand the observations in catalysis. Rh(I) carbonyl halide complexes of each of the ligands have been prepared from both [Rh(2)(CO)(4)Cl(2)] and dimeric mu-Cl-[Rh(L)Cl](2) complexes. These Rh(I) carbonyl complexes are either dimeric with bridging phosphine ligands (dppb, dcpb, dppx) or monomeric chelate complexes. The reaction of the complexes with methyl iodide at 140 degrees C has been studied, which has revealed clear differences in the stability of the corresponding Rh(III) complexes. Surprisingly, the dimeric Rh(I) carbonyls react cleanly with MeI with rearrangement of the diphosphine to a chelate co-ordination mode to give stable Rh(III) acetyl complexes. The Rh acetyls for L=dppb and dppx have been fully characterised by X-ray crystallography. During the catalytic studies, the more rigid dppx and BINAP ligands were found to be nearly 5 times more hydrogen tolerant than [Rh(CO)(2)I(2)](-), as revealed by by-product analysis. The origin of this hydrogen tolerance is explained based on the differing reactivities of the Rh acetyls with hydrogen gas, and by considering the structure of the complexes.