Impact of Reflex EGFR/ ALK Testing on Time to Treatment of Patients With Advanced Nonsquamous Non-Small-Cell Lung Cancer.

PURPOSE: Optimal first-line systemic therapy for patients with advanced nonsquamous (nonsq) non-small-cell lung cancer (NSCLC) requires confirmation of EGFR/ ALK status, which can delay treatment. We evaluated the impact of reflex testing, defined as pathologists initiating EGFR/ ALK testing at the time of diagnosis of nonsq NSCLC, on time to treatment (TTT). METHODS: We conducted a retrospective review of patients with nonsq NSCLC with medical oncology consultation at Sunnybrook Odette Cancer Centre between March 18, 2010 and April 30, 2014. Data were compared during routine and reflex testing. TTT was defined as the interval between the first medical oncology visit with advanced NSCLC and the initiation of systemic therapy. RESULTS: A total of 306 patients were included (n = 232 for routine testing, n = 74 for reflex testing). There was a trend to improvement in median TTT with reflex testing (36 days [interquartile range {IQR}, 16 to 71 days v 26 days [IQR, 8 to 41 days], P = .071). Omitting patients with intentional delays in systemic therapy for low-volume disease, poor performance status, comorbidity management, and/or radiation therapy, median TTT improved (34 days [IQR, 15 to 67 days] v 22 days [IQR, 8 to 42 days], P = .049). Time to optimal first-line systemic therapy according to published guidelines improved (median, 36 days [IQR, 16 to 91 days] v 24 days [IQR, 8 to 43 days], P = .036). There was no impact on receipt of any first-line systemic therapy (55% v 59%, P = .66). The quality of biomarker testing improved, with fewer unsuccessful tests ( EGFR, 14% v 4%, P = .039; and ALK, 17% v 3%, P = .037). CONCLUSION: Reflex testing of EGFR/ ALK improved the time to optimal systemic therapy and the quality of biomarker testing for patients with advanced nonsq NSCLC.

Abscopal Effects: Case Report and Emerging Opportunities.

The abscopal effect is a phenomenon observed in the treatment of metastatic cancer where localized irradiation of a particular tumor site causes a response in a site distant to the irradiated volume. The mechanisms of the abscopal effect are speculated to be of several origins, including distant effects on p53, elaboration of inflammatory agents including cytokines, and, most recently, secondary to immune mechanisms. In this case report, we present a rare report of a patient with hepatocellular carcinoma with lung metastases who, after receiving radiation treatment to the liver, had a treatment response in the liver and a complete response in the lung. Recent advances in the understanding of the primary role of immune-modulated cytotoxicity, especially with the success of immune checkpoint inhibitors, have the potential to turn the abscopal effect from a rare phenomenon into a tool to guide antineoplastic therapy and provide a new line of research.

Relationship between the number of positive fecal occult blood tests and the diagnostic yield of colonoscopy.

BACKGROUND: In 2007, Ontario launched a colon cancer screening program for average-risk individuals based on biennial fecal occult blood tests (FOBTs) on three fecal samples, followed by colonoscopy for individuals who tested positive. OBJECTIVE: To determine whether >1 positive screening FOBT was predictive of finding advanced neoplasia at colonoscopy. METHODS: A retrospective chart review of outpatient colonoscopic procedures performed at Hotel Dieu Hospital (Kingston, Ontario) in the first two years of the colon cancer screening program was conducted, focusing on endoscopic and pathological findings. RESULTS: Of 5556 individuals undergoing colonoscopy, 346 were referred for positive FOBT. Overall, 41 (11.8%) patients with a positive FOBT had colon cancer. In 16 (4.6%) cases, the number of positive FOBTs was not reported. For the 330 individuals in whom the number of positive tests was specified, 198, 71 and 61 cases had one, two and three positive results, respectively. Cancer was found at colonoscopy in 11 (5.6%), 11 (15.5%) and 18 (29.5%) of individuals with one, two and three positive FOBT results, respectively (OR 3.0 [95% CI 1.2 to 7.3] and 6.5 [95% CI 2.8 to 15.0] for two or three positive FOBTs compared with one; P=0.015 and P<0.001, respectively). High-risk adenomas (>1 cm in diameter, villous component and/or high-grade dysplasia) were found in 41 (20.8%), 29 (42.0%) and 25 (41.0%) individuals with one, two and three positive FOBTs, respectively (OR 2.8 [95% CI 1.5 to 5.0] and 2.4 [95% CI 1.3 to 4.5] for two or three positive FOBTs compared with one; P=0.001 and P=0.006, respectively). CONCLUSIONS: The diagnostic yield of colonoscopy varied directly with the number of positive FOBTs. This information may be useful in assigning scheduling priority for patients with positive FOBTs.

Nitric oxide activation of a potassium channel (BK(Ca)) in feline lower esophageal sphincter.

AIM: To assess the effect of nitric oxide (NO) on the large conductance potassium channel (BK(Ca)) in isolated circular (CM) and sling (SM) muscle cells and muscle strips from the cat lower esophageal sphincter (LES) to determine its regulation of resting tone and relaxation. METHODS: Freshly enzymatically-digested and isolated circular smooth muscle cells were prepared from each LES region. To study outward K+ currents, the perforated patch clamp technique was employed. To assess LES resting tone and relaxation, muscle strips were mounted in perfused organ baths. RESULTS: (1) Electrophysiological recordings from isolated cells: (a) CM was more depolarized than SM (-39.7 ± 0.8 mV vs -48.1 ± 1.6 mV, P < 0.001), and maximal outward current was similar (27.1 ± 1.5 pA/pF vs 25.7 ± 2.0 pA/pF, P > 0.05); (b) The NO donor sodium nitroprusside (SNP) increased outward currents only in CM (25.9 ± 1.9 to 46.7 ± 4.2 pA/pF, P < 0.001) but not SM (23.2 ± 3.1 to 27.0 ± 3.4 pA/pF, P > 0.05); (c) SNP added in the presence of the BK(Ca) antagonist iberiotoxin (IbTX) produced no increase in the outward current in CM (17.0 ± 2.8 vs 13.7 ± 2.2, P > 0.05); and (d) L-NNA caused a small insignificant inhibition of outward K+ currents in both muscles; and (2) Muscle strip studies: (a) Blockade of the nerves with tetrodotoxin (TTX), or BK(Ca) with IbTX had no significant effect on resting tone of either muscle; and (b) SNP reduced tone in both muscles, and was unaffected by the presence of TTX or IbTX. CONCLUSION: Exogenous NO activates BK(Ca) only in CM of the cat. However, as opposed to other species, exogenous NO-induced relaxation is predominantly by a non-BK(Ca) mechanism, and endogenous NO has minimal effect on resting tone.

Feline lower esophageal sphincter sling and circular muscles have different functional inhibitory neuronal responses.

The lower esophageal sphincter (LES) has a circular muscle component exhibiting spontaneous tone that is relaxed by nitric oxide (NO) and a low-tone sling muscle that contracts vigorously to cholinergic stimulation but with little or no evidence of NO responsiveness. This study dissected the responses of the sling muscle to nitrergic innervation in relationship to its cholinergic innervation and circular muscle responses. Motor responses were induced by electrical field stimulation (EFS; 1-30 Hz) of muscle strips from sling and circular regions of the feline LES in the presence of cholinergic receptor inhibition (atropine) or NO synthase inhibition [NG-nitro-L-arginine (L-NNA)+/-atropine]. This study showed the following. First, sling muscle developed less intrinsic resting tone compared with circular muscle. Second, with EFS, sling muscle contracted (most at 50% by 5 Hz. Third, on neural blockade with atropine or L-NNA+/-atropine, 1) sling muscle, although predominantly influenced by excitatory cholinergic stimulation, had a small neural NO-mediated inhibition, with no significant non-NO-mediated inhibition and 2) circular muscle, although little affected by cholinergic influence, underwent relaxation predominantly by neural release of NO and some non-NO inhibitory influence (at higher EFS frequency). Fourth, the sling, precontracted with bethanecol, could relax with NO and some non-NO inhibition. Finally, the tension range of both muscles is similar. In conclusion, sling muscle has limited NO-mediated inhibition to potentially augment or replace sling relaxation effected by switching off its cholinergic excitation. Differences within the LES sling and circular muscles could provide new directions for therapy of LES disorders.

Regional differences in cholinergic regulation of potassium current in feline esophageal circular smooth muscle.

Potassium channels are important contributors to membrane excitability in smooth muscles. There are regional differences in resting membrane potential and K(+)-channel density along the length of the feline circular smooth muscle esophagus. The aim of this study was to assess responses of K(+)-channel currents to cholinergic (ACh) stimulation along the length of the feline circular smooth muscle esophageal body. Perforated patch-clamp technique assessed K(+)-channel responses to ACh stimulation in isolated smooth muscle cells from the circular muscle layer of the esophageal body at 2 (distal)- and 4-cm (proximal) sites above the lower esophageal sphincter. Western immunoblots assessed ion channel and receptor expression. ACh stimulation produced a transient increase in outward current followed by inhibition of spontaneous transient outward currents. These ACh-induced currents were abolished by blockers of large-conductance Ca(2+)-dependent K(+) channels (BK(Ca)). Distal cells demonstrated a greater peak current density in outward current than cells from the proximal region and a longer-lasting outward current increase. These responses were abolished by atropine and the specific M(3) receptor antagonist 4-DAMP but not the M(1) receptor antagonist pirenzipine or the M(2) receptor antagonist methoctramine. BK(Ca) expression along the smooth muscle esophagus was similar, but M(3) receptor expression was greater in the distal region. Therefore, ACh can differentially activate a potassium channel (BK(Ca)) current along the smooth muscle esophagus. This activation probably occurs through release of intracellular calcium via an M(3) pathway and has the potential to modulate the timing and amplitude of peristaltic contraction along the esophagus.

Calcium source diversity in feline lower esophageal sphincter circular and sling muscle.

Within muscular equivalents of cat lower esophageal sphincter (LES), the circular muscle develops greater spontaneous tone, whereas the sling muscle is more responsive to cholinergic stimulation. Smooth muscle contraction involves a combination of calcium release from stores and of calcium entry via several pathways. We hypothesized that there are differences in the sources of Ca(2+) used for contraction in sling and circular muscles and that these differences could contribute to functional asymmetry observed within LES. Contraction of muscle strips from circular and sling regions of LES was assessed in the presence of TTX. In Ca(2+)-free Krebs, tone was inhibited to a greater degree in circular than sling muscle. L-type Ca(2+) channel blockade with nifedipine or verapamil inhibited tone in LES circular but not sling muscle. Sarcoplasmic reticulum (SR) Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA) caused greater increase in tone in sling than in circular muscle. The phospholipase C inhibitor U-73122 and the SR inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptor blocker 2-aminoethoxydiphenyl borate (2-APB) inhibited tone in circular and sling muscles, demonstrating that continuous release of Ca(2+) from Ins(1,4,5)P(3)-sensitive stores is important in tone generation in both muscles. In Ca(2+)-free Krebs, ACh-induced contractions (AChC) were inhibited to a greater degree in sling than circular muscles. However, nifedipine and verapamil greatly inhibited AChC in the circular but not sling muscle. Depletion of SR Ca(2+) stores with CPA or inhibition of Ins(1,4,5)P(3)-mediated store release with either U-73122 or 2-APB inhibited AChC in both muscles. We demonstrate that LES circular and sling muscles 1) use intracellular and extracellular Ca(2+) sources to different degrees in the generation of spontaneous tone and AChC and 2) use different Ca(2+) entry pathways. These differences hold the potential for selective modulation of LES tone in health and disease.

Potassium channel diversity within the muscular components of the feline lower esophageal sphincter.

We hypothesized that regional differences in electrophysiological properties exist within the musculature of the feline lower esophageal sphincter (LES) and that they may potentially contribute to functional asymmetry within the LES. Freshly isolated esophageal smooth muscle cells (SMCs) from the circular muscle and sling regions within the LES were studied under a patch clamp. The resting membrane potential (RMP) of the circular SMCs was significantly more depolarized than was the RMP of the sling SMCs, resulting from a higher Na+ and Cl- permeability in circular muscle than in sling muscle. Large conductance Ca2+-activated K+ (BKCa) set the RMP at both levels, since specific BKCa inhibitors caused depolarization; however, BKCa density was greatest in the circular region. A significant portion of the outward current was due to non-BKCa, especially in sling muscle, and likely delayed rectifier K+ channels (KDR). There was a large reduction in outward current with 4-aminopyridine (4-AP) in sling muscle, while BKCa blockers had a limited effect on the voltage-activated outward current in sling muscle. Differences in BKCa:KDR channel ratios were also manifest by a leftward shift in the voltage-dependent activation curve in circular cells compared to sling cells. The electrophysiological differences seen between the circular and sling muscles provide a basis for their different contributions to LES activities such as resting tone and neurotransmitter responsiveness, and in turn could impart asymmetric drug responses and provide specific therapeutic targets.

Regional differences in L-type Ca2+ channel expression in feline lower esophageal sphincter.

In humans and cats, muscle from the lower esophageal sphincter (LES) circular region exhibits greater spontaneous tone than LES sling muscle, whereas the sling muscle is much more responsive to cholinergic stimulation. Despite physiological and pharmacological evidence for the presence of L-type Ca2+ channel current (ICa,L) activity in LES circular muscle, the identity of this channel has not been demonstrated biochemically or electrophysiologically fingerprinted. Furthermore, there is no information on the channel's presence and role in the sling region of the LES. We hypothesized that regional differences in the expression of ICa,L between LES circular and sling muscles, if present, could contribute to the functional asymmetry observed within the LES. ICa,L expression was compared between circular and sling regions of the LES by Western blot analysis. The patch-clamp technique was used to study ICa,L. Muscle strip studies assessed ICa,L contribution to contractile activity. We found both protein expression of ICa,L and ICa,L density to be greater in LES circular muscle than sling muscle. ICa,L voltage- and time-dependent activation and inactivation curves were similar in cells from both regions. ICa,L blockade with nifedipine inhibited spontaneous tone and ACh-induced contractions only in circular muscle but was able to abolish depolarization (KCl)-induced contractions in both sling and circular muscles. In contrast, La3+ inhibited tone and ACh-induced contractions in muscles from both regions. Therefore, regional myogenic differences in ICa,L expression within the LES circular and sling muscle exist and provide one explanation for the differential contribution of sling and circular muscle to LES contractility.

SNAP-25 inhibits L-type Ca2+ channels in feline esophagus smooth muscle cells.

We recently reported that non-secretory gastrointestinal smooth muscle cells also possessed SNARE proteins, of which SNAP-25 regulated Ca(2+)-activated (K(Ca)) and delayed rectifier K(+) channels (K(V)). Voltage-gated, long lasting (L-type) calcium channels (L(Ca)) play an important role in excitation-contraction coupling of smooth muscle. Here, we show that SNAP-25 could also directly inhibit the L-type Ca(2+) channels in feline esophageal smooth muscle cells at the SNARE complex binding synprint site. SNARE proteins could therefore regulate additional cell actions other than membrane fusion and secretion, in particular, coordinated muscle membrane excitability and contraction, through their actions on membrane Ca(2+) and K(+) channels.