Correlations of EGFR mutations and increases in EGFR and HER2 copy number to gefitinib response in a retrospective analysis of lung cancer patients

<p>Abstract</p> <p>Background</p> <p>Gefitinib, a small molecule tyrosine kinase inhibitor of the Epidermal Growth Factor Receptor (<it>EGFR</it>), has shown limited efficacy in the treatment of lung cancer. Recognized clinical predictors of response to this drug, specifically female, non-smoker, Asian descent, and adenocarcinoma, together suggest a genetic basis for drug response. Recent studies have addressed the relationship between response and either sequence mutations or increased copy number of specific receptor tyrosine kinases. We set out to examine the relationship between response and the molecular status of two such kinases, <it>EGFR </it>and <it>HER2</it>, in 39 patients treated with gefitinib at the BC Cancer Agency.</p> <p>Methods</p> <p>Archival patient material was reviewed by a pathologist and malignant cells were selectively isolated by laser microdissection or manual recovery of cells from microscope slides. Genomic DNA was extracted from 37 such patient samples and exons 18–24, coding for the tyrosine kinase domain of <it>EGFR</it>, were amplified by PCR and sequenced. <it>EGFR </it>and <it>HER2 </it>copy number status were also assessed using FISH in 26 samples. Correlations between molecular features and drug response were assessed using the two-sided Fisher's exact test.</p> <p>Results</p> <p>Mutations previously correlated with response were detected in five tumours, four with exon 19 deletions and one with an exon 21 missense L858R point mutation. Increased gene copy number was observed in thirteen tumours, seven with <it>EGFR </it>amplification, three with <it>HER2 </it>amplification, and three with amplification of both genes. In our study cohort, a correlation was not observed between response and <it>EGFR </it>mutations (exon 19 deletion p = 0.0889, we observed a single exon 21 mutation in a non-responder) or increases in <it>EGFR </it>or <it>HER2 </it>copy number (p = 0.552 and 0.437, respectively).</p> <p>Conclusion</p> <p>Neither mutation of <it>EGFR </it>nor increased copy number of <it>EGFR </it>or <it>HER2 </it>was diagnostic of response to gefitinib in this cohort. However, validation of these features in a larger sample set is appropriate. Identification of additional predictive biomarkers beyond <it>EGFR </it>status may be necessary to accurately predict treatment outcome.</p

Interaction of inflammation and hyperoxia in a rat model of neonatal white matter damage

Intrauterine infection and inflammation are major reasons for preterm birth. The switch from placenta-mediated to lung-mediated oxygen supply during birth is associated with a sudden rise of tissue oxygen tension that amounts to relative hyperoxia in preterm infants. Both infection/inflammation and hyperoxia have been shown to be involved in brain injury of preterm infants. Hypothesizing that they might be additive or synergistic, we investigated the influence of a systemic lipopolysaccharide (LPS) application on hyperoxia-induced white matter damage (WMD) in newborn rats. Three-day-old Wistar rat pups received 0.25 mg/kg LPS i.p. and were subjected to 80% oxygen on P6 for 24 h. The extent of WMD was assessed by immunohistochemistry, western blots, and diffusion tensor (DT) magnetic resonance imaging (MRI). In addition, the effects of LPS and hyperoxia were studied in an in vitro co-culture system of primary rat oligodendrocytes and microglia cells. Both noxious stimuli, hyperoxia, and LPS caused hypomyelination as revealed by western blot, immunohistochemistry, and altered WM microstructure on DT-MRI. Even so, cellular changes resulting in hypomyelination seem to be different. While hyperoxia induces cell death, LPS induces oligodendrocyte maturity arrest without cell death as revealed by TUNEL-staining and immunohistological maturation analysis. In the two-hit scenario cell death is reduced compared with hyperoxia treated animals, nevertheless white matter alterations persist. Concordantly with these in vivo findings we demonstrate that LPS pre-incubation reduced premyelinating-oligodendrocyte susceptibility towards hyperoxia in vitro. This protective effect might be caused by upregulation of interleukin-10 and superoxide dismutase expression after LPS stimulation. Reduced expression of transcription factors controlling oligodendrocyte development and maturation further indicates oligodendrocyte maturity arrest. The knowledge about mechanisms that triggered hypomyelination contributes to a better understanding of WMD in premature born infants

Inflammation and hyperoxia decreases fractional anisotropy in corpus callosum.

<p>Typical DT images are presented in (A) T₂W images (b0), fractional anisotropy (FA) and direction encoded color maps (DEC) of a VN (upper panel) and LH (lower panel) rat brains are shown. (B) Significant decrease of FA was observed in the corpus callosum of the injured groups (LN, VH, LH) compared with control group (VN) due to a significant increase of D⊥ (i.e. myelination defect). There were no significant differences between the injured groups providing evidence of similar damage in corpus callosum with or without hyperoxia after LPS challenge. Mean+SEM were presented with n = 6 animals per group. * p<0.05; VN: vehicle+normoxia, LN: LPS+normoxia, VH: vehicle+hyperoxia, LH: LPS+hyperoxia.</p

Primer and probe sequences.

<p>Primer and probe sequences.</p

Inflammation and hyperoxia decreases MBP expression.

<p>A significantly decrease of MBP protein expression as a sign of hypomyelination occurs in all experimental settings with similar severity. Results of densitometric western blot quantification of P3 LPS P6 hyperoxia experiment (A), results of densitometric western blot quantification of P6 LPS P6 hyperoxia experiment (B), a representative western blot series of P3 LPS P6 hyperoxia experiment (C), a representative western blot series of P3 LPS P6 hyperoxia experiment (D) and images of immunohistological stained sections against MBP (E) are shown. Immunohistological staining against MBP supports these findings and illustrates a profound reduction of MBP in external capsule with loss of processes and thinning up to fragmentation of the capsule in all treated groups. Scale bar = 100 µm. Brain hemisphere extracts were used for western blot analyses with n = 9–11 animals per group. Western blot values represent mean+SEM normalized ratios of the MBP bands to β-actin as an internal standard and VN group was set to 100%. * p<0.05, ** p<0.01, *** p<0.001 and ns p>0.05; VN: vehicle+normoxia, LN: LPS+normoxia, VH: vehicle+hyperoxia, LH: LPS+hyperoxia.</p

Immunohistological analysis of cell death and oligodendrocyte maturation.

<p>Hyperoxia but not LPS induce oligodendrocyte cell death revealed by TUNEL-staining. (A) LPS pre-treatment prior to hyperoxia reduce number of TUNEL-positive cells in CTX, WM and THL (LH, dashed bars). LPS application slightly increases number of TUNEL-positive cells in WM and THL but not in CTX (LN, grey bars), whereas hyperoxia affects all probed brain regions strongly (VH, black bars). TUNEL-positive cells were counted by two blinded observers in 5 sections per animal and 4 randomly assigned fields of each region per section. (B) A co-labeling with Olig2 and TUNEL shows that LPS do not induce oligodendrocyte death (LN, grey bar). Hyperoxia induces oligodendrocyte death (VH, black bar) and LPS pre-treatment reduces hyperoxia induced cell death (LH, dashed bar). (C) Hyperoxia (VH, black bar) but not LPS (LN, grey bar) causes a loss of O4-positive cells. Pups underlying the two-hit scenario exhibit a decreasing tendency of O4-positive cells (LH, dashed bar). (D) In all treated groups the number of APC-CC1-positive cells is decreased. (E) Representative images of O4- and APC-CC1 staining are shown. Scale bar = 100 µm. Mean+SEM were presented as TUNEL-positive cells/mm³ with n = 9–11 animals per group. * p<0.05, ** p<0.01and *** p<0.001; VN: vehicle+normoxia, LN: LPS+normoxia, VH: vehicle+hyperoxia, LH: LPS+hyperoxia.</p

Inflammation and hyperoxia increases cCasp3 expression.

<p>Systemic LPS application as well as exposure to 80% oxygen increases caspase-3 activation in the developing rat brain. Results of densitometric western blot quantification (A) and a representative western blot series are shown (B). Animals receiving the two-hit scenario (LH, dashed bar) showed neither a higher nor a faintly cCasp3 expression compared to LPS single-hit treated pups (LN, grey bar). Brain hemisphere extracts were used for western blot analyses with n = 9–11 animals per group. Western blot values represent mean+SEM normalized ratios of the cCasp3 bands to β-actin as an internal standard and VN group was set to 100%. * p<0.05, *** p<0.001 and ns p>0.05; VN: vehicle+normoxia, LN: LPS+normoxia, VH: vehicle+hyperoxia, LH: LPS+hyperoxia.</p