ALK Group Research Articles

Distinction of ALK fusion gene- and EGFR mutation-positive lung cancer with tumor markers.

It is difficult to predict gene mutations individually based on clinical background alone. Tumor markers may help to predict each gene mutation. Identifying tumor markers that can predict gene mutation will facilitate timely genetic testing and intervention. We selected 134 cases of advanced or recurrent ALK-positive and 172 cases of advanced or recurrent EGFR-positive lung cancer from our clinical database. The cutoff values for the tumor markers were defined as 5.0 ng/mL or higher for carcinoembryonic antigen (CEA) and 3.5 ng/mL or higher for soluble fragment of cytokeratin 19 (CYFRA21-1) in accordance with the institutional standards. A positive CYFRA21-1:CEA ratio was defined as 0.7 or higher. The CEA-positivity rate was 49% for ALK-positive lung cancers and 73% for EGFR-positive lung cancers, which was significantly different (p < 0.001). The CYFRA21-1 positivity rate was significantly higher in ALK-positive lung cancer (36%) compared with EGFR-positive lung cancer (23%) (p = 0.034). The median CYFRA21-1:CEA ratio was 0.395 for the ALK group, which was significantly higher compared with 0.098 for the EGFR group (p < 0.001). These trends were similar when excluding histology other than adenocarcinoma. The median time-to-treatment failure (TTF) for initial tyrosine kinase inhibitor (TKI) therapy was 308 days for the high CYFRA21-1:CEA ratio group and 617 days for the low CYFRA21-1:CEA ratio group for ALK-positive lung cancer (p = 0.100). A higher proportion of patients with ALK-positive lung cancer were CYFRA21-1 positive and had higher CYFRA21-1:CEA ratios compared with EGFR-positive lung cancer patients.

P11.02.B DO GENOMIC ALTERATIONS INFLUENCE THE DEVELOPMENT OF BRAIN METASTASES IN ADVANCED AND METASTATIC NON-SMALL CELL LUNG CANCER?

Abstract BACKGROUND Lung cancer is the most common cause of cancer deaths worldwide. Development of brain metastases (BM) in patients with advanced (Stage III) and metastatic (Stage IV) non-small cell lung cancer (NSCLC) is linked with poor prognosis. Identifying mutations associated with BM development could influence screening and determine targeted treatment paradigms. We aimed to establish BM prevalence and incidence in patients with advanced and metastatic NSCLC, stratified by genomic alterations. MATERIAL AND METHODS A systematic review and meta-analysis was conducted in accordance with PRISMA guidelines (PROSPERO ID CRD42022315915). Three databases were searched, and articles published between January 2000-May 2022 included (Search date 30th April 2022). Prevalence at diagnosis, and incidence of new BM per year were obtained, including patients with EGFR, ALK, KRAS, ROS1, RET, MET, and other genomic alterations. Pooled incidence rates were calculated using random effects models. RESULTS Of 960 articles identified, 65 were included in the study (24,784 patients). Pooled BM prevalence at diagnosis was 28.6% (45 studies, 95% Confidence Interval [CI] 26.1-31.0), and highest in patients with ALK (34.9%) and RET translocations (32.2%). With a median follow-up of 24 months, the per-year incidence of new BM was 12.5% in the wild-type group (14 studies, 95% CI 10.9-15.9). Incidence in the subgroups with different genomic alterations was 15.6% in the EGFR group (16 studies, 95% CI 11.4-21.2), 17.0% in the ALK group (6 studies, 95% CI 11.3-25.8), 9.8% in the KRAS group (4 studies, 95% CI 5.5-17.5), 12.5% in the ROS1 group (3 studies, 95% CI 5.7-27.6), and 11.8% in the RET group (2 studies, 95% CI 8.0-17.5). At the end of the follow-up period, a median of 55.0% of patients had BM (IQR 44.2-67.8). CONCLUSION This is the first study to show prevalence, and incidence of BM development in advanced and metastatic NSCLC, stratified by genomic alterations. Our data indicates a higher prevalence and incidence of BM in patients with targetable genomic alterations. This supports brain imaging at staging and follow-up, underlining the need for NSCLC therapies with brain penetrance and efficacy.

Differential Distribution of Brain Metastases from Non-Small Cell Lung Cancer Based on Mutation Status.

Non-small cell lung cancer (NSCLC) has a high rate of brain metastasis. The purpose of this study was to assess the differential distribution of brain metastases from primary NSCLC based on mutation status. Brain MRI scans of patients with brain metastases from primary NSCLC were retrospectively analyzed. Brain metastatic tumors were grouped according to mutation status of their primary NSCLC and the neuroimaging features of these brain metastases were analyzed. A total of 110 patients with 1386 brain metastases from primary NSCLC were included in this study. Gray matter density at the tumor center peaked at ~0.6 for all mutations. The median depths of tumors were 7.9 mm, 8.7 mm and 9.1 mm for EGFR, ALK and KRAS mutation groups, respectively (p = 0.044). Brain metastases for the EGFR mutation-positive group were more frequently located in the left cerebellum, left cuneus, left precuneus and right precentral gyrus. In the ALK mutation-positive group, brain metastases were more frequently located in the right middle occipital gyrus, right posterior cingulate, right precuneus, right precentral gyrus and right parietal lobe. In the KRAS mutation-positive patient group, brain metastases were more frequently located in the posterior left cerebellum. Our study showed differential spatial distribution of brain metastases in patients with NSCLC according to their mutation status. Information regarding distribution of brain metastases is clinically relevant as it could be helpful to guide treatment planning for targeted therapy, and for predicting prognosis.

New strategies for surface modification of poly (vinyl alcohol) toward click chemistry applications

AbstractIn this study, new synthesis strategies for poly (vinyl alcohol) (PVA) functionalized with azidation (Az) and alkynation (Alk) were carried out for surface functionality modification. These two functionalization routes utilize azide and alkyne groups, respectively, and offer simple way to enhance the PVA crosslinking capabilities toward producing new functional polymer‐based materials. The Az functionalized PVA was prepared in two steps and denoted by PVA‐Az, while Alk functionalized PVA was prepared in one step to produce PVA‐Alk. Surfaces modification of PVA by using Az and Alk functional groups was confirmed by spectral analysis (Fourier transform infrared and 13C‐NMR). Results showed that Alk prompted excellent chemical modifications on the surface of PVA while the thermal stability was enhanced by functionalizing the Az and Alk into PVA. Furthermore, X‐ray photoelectron spectroscopy investigations showed a successful functionalization and new functional groups addition to PVA surface. Scanning electron microscope images were used to analyze the surface morphology of PVA by incorporating the Az and Alk groups, which indicated rougher surfaces compared to that of PVA backbone. In terms of efficiency and simplicity, the techniques used in this study appear to be entirely satisfactory.

Predicting Survival Duration With MRI Radiomics of Brain Metastases From Non-small Cell Lung Cancer.

Background: Brain metastases are associated with poor survival. Molecular genetic testing informs on targeted therapy and survival. The purpose of this study was to perform a MR imaging-based radiomic analysis of brain metastases from non-small cell lung cancer (NSCLC) to identify radiomic features that were important for predicting survival duration.Methods: We retrospectively identified our study cohort via an institutional database search for patients with brain metastases from EGFR, ALK, and/or KRAS mutation-positive NSCLC. We segmented the brain metastatic tumors on the brain MR images, extracted radiomic features, constructed radiomic scores from significant radiomic features based on multivariate Cox regression analysis (p < 0.05), and built predictive models for survival duration.Result: Of the 110 patients in the cohort (mean age 57.51 ± 12.32 years; range: 22–85 years, M:F = 37:73), 75, 26, and 15 had NSCLC with EGFR, ALK, and KRAS mutations, respectively. Predictive modeling of survival duration using both clinical and radiomic features yielded areas under the receiver operative characteristic curve of 0.977, 0.905, and 0.947 for the EGFR, ALK, and KRAS mutation-positive groups, respectively. Radiomic scores enabled the separation of each mutation-positive group into two subgroups with significantly different survival durations, i.e., shorter vs. longer duration when comparing to the median survival duration of the group.Conclusion: Our data supports the use of radiomic scores, based on MR imaging of brain metastases from NSCLC, as non-invasive biomarkers for survival duration. Future research with a larger sample size and external cohorts is needed to validate our results.

Effect of Zanthoxylum alkylamides on glucose metabolism in streptozotocin-induced diabetic Sprague\u2013Dawley rats

This research aimed at investigating the hypoglycemic effect of Zanthoxylum alkylamides and whether TRPV1 receptor could participate in the glucose metabolism by using streptozotocin-induced diabetic rat model. The results showed that the blood glucose measured in the Zanthoxylum alkylamides treated group (ALK) showed significantly lower values than that in the model group (Model). Significant improvements in the oral glucose tolerance as well as plasma insulin and hepatic glycogen were also observed in the ALK group, when compared to the model group. However, the improving effects of Zanthoxylum alkylamides on glucose metabolism disorder in diabetic rats were markedly inhibited by capsazepine as the TRPV1 receptor antagonist. In addition, there were significant differences in the levels of mRNA and protein of phosphoenolpyruvate carboxylase (PEPCK), gucose-6-phosphatase (G6Pase), glucokinase (GK) and cannabinoid receptor l (CB1) in the livers of the ALK group compared to model group. Meanwhile, ALK group also exhibited a remarkable increase in the pancreatic-duodenal homeobox 1 (PDX-1), glucose transporter 2 (GLUT 2), GK levels and a significant decrease in the expression levels of CB1 in the pancreas, while the presence of capsazepine would affected the expression of these genes. These findings indicate that Zanthoxylum alkylamides could ameliorate the glucose metabolism disorder in diabetic rats. Furthermore, the TRPV1 receptor could participate in regulating the expressions of genes and proteins related to glucose metabolism and insulin secretion in the liver and pancreas, and takes a role in the hypoglycemic process of Zanthoxylum alkylamides.

Prediction of the molecular status in non-small cell lung cancer based on metastatic pattern: A free webtool powered by artificial intelligence.

9535 Background: Molecular characterization of metastatic lung adenocarcinomas is mandatory but might be hampered by the quantity of tissue, restricted access to molecular platforms or limited economical resources. Our aim was to develop a tool supported by the hypothesis that radiological patterns of pts could help predict the rate of positivity of the most common oncogenic drivers. Methods: We defined an algorithm based on a molecularly defined cohort of 656 pts with stage IV lung adenocarcinoma. Two radiologists centrally reviewed the baseline imaging. Clinical data were retrospectively collected. There were 135 EGFR mutations, 81 ALK fusions, 47 BRAF mutations, 141 KRAS mutations, and 146 pan-negative tumors for these 4 oncogenic drivers. Univariate correlation analyses were performed to define an algorithm predicting the molecular testing positivity based on the metastatic pattern. Subsequently, an online tool was developed. This study was approved by our institutional review board. Results: Metastatic patterns correlated with the genomic drivers when compared to the pan-negative group. In the EGFR group, pleural metastases were more frequent (32% vs. 20%; p = 0.021), whereas adrenal and node metastases less frequent (6% vs.23%; p &lt; 0.001 and 11% vs. 23% respectively; p = 0.011). In the ALK group, there were more brain and lung metastases (respectively 42% vs. 29%; p = 0.043 and 37% vs. 24% respectively; p = 0.037). In the BRAF group, pleural and pericardial metastases were more common (47% vs. 20%; p &lt; 0.001 and 11% vs. 3% respectively; p = 0.04) and bone metastases less common (21% vs. 42%; p = 0.011). Lymphangitis was more frequent in EGFR, ALK and BRAF groups (6%, 7% and 15% vs. 1%; p = 0,016, p = 0,009 and p &lt; 0,001 respectively). A free online access to the algorithm is now available after registration at http//tactic-ct.fr. Physicians enter age, sex, smoking status and the sites of metastases at diagnosis (present/absent/unknown). A mutation score is calculated, reflecting the % of chance to find an oncogenic driver. On the website, contributors can also enter new cases and an artificial intelligence will refine the algorithm and expand the number of oncogenic drivers. Conclusions: Our free access tool allows establishing a hierarchy in the molecular testing based on simple clinical and radiological information. Continual learning from new cases entered in the database will increase the sensitivity of the tool. This tool might save time, tumor tissue, economical resources and accelerate access to personalized treatment.

Association of metastatic pattern and molecular status in stage IV non-small cell lung cancer adenocarcinoma.

The aim of our study was to investigate the association between driver oncogene alterations and metastatic patterns on imaging assessment, in a large cohort of metastatic lung adenocarcinoma patients. From January 2010 to May 2017, 550 patients with stage IV lung adenocarcinoma with molecular analysis were studied retrospectively including 135 EGFR-mutated, 81 ALK-rearrangement, 47 BRAF-mutated, 141 KRAS-mutated, and 146 negative tumors for these 4 mutations (4N). After review of the complete imaging report by two radiologists (junior and senior) to identify metastatic sites, univariate correlation analyzes were performed. We found differences in metastatic tropism depending on the molecular alteration type when compared with the non-mutated 4N group: in the EGFR group, pleural metastases were more frequent (32% versus 20%; p = 0.021), and adrenal and node metastases less common (6% versus 23%; p < 0.001 and 11% versus 23%; p = 0.011). In the ALK group, there were more brain and lung metastases (respectively 42% versus 29%; p = 0.043 and 37% versus 24%; p = 0.037). In the BRAF group, pleural and pericardial metastases were more common (respectively 47% versus 20%; p < 0.001 and 11% versus 3%; p = 0.04) and bone metastases were rarer (21% versus 42%; p = 0.011). Lymphangitis was more frequent in EGFR, ALK, and BRAF groups (respectively 6%, 7%, and 15% versus 1%); p = 0.016; p = 0.009; and p < 0.001. The application of these correlations between molecular status and metastatic tropism in clinical practice may lead to earlier and more accurate identification of patients for targeted therapy. • Bone and brain metastasis are the most common organs involved in lung adenocarcinoma but the relative incidence of each metastatic site depends on the molecular alteration. • EGFR-mutated tumors preferentially spread to the pleura and less commonly to adrenals, ALK-rearrangement tumors usually spread to the brain and the lungs, whereas BRAF-mutated tumors are unlikely to spread to bones and have a serous (pericardial ad pleural) tropism. • These correlations could help in the clinical management of patients with metastatic lung adenocarcinoma.

Immune checkpoint inhibitors for patients with advanced lung cancer and oncogenic driver alterations: results from the IMMUNOTARGET registry.

BackgroundAnti-PD1/PD-L1 directed immune checkpoint inhibitors (ICI) are widely used to treat patients with advanced non-small-cell lung cancer (NSCLC). The activity of ICI across NSCLC harboring oncogenic alterations is poorly characterized. The aim of our study was to address the efficacy of ICI in the context of oncogenic addiction.Patients and methodsWe conducted a retrospective study for patients receiving ICI monotherapy for advanced NSCLC with at least one oncogenic driver alteration. Anonymized data were evaluated for clinicopathologic characteristics and outcomes for ICI therapy: best response (RECIST 1.1), progression-free survival (PFS), and overall survival (OS) from ICI initiation. The primary end point was PFS under ICI. Secondary end points were best response (RECIST 1.1) and OS from ICI initiation.ResultsWe studied 551 patients treated in 24 centers from 10 countries. The molecular alterations involved KRAS (n = 271), EGFR (n = 125), BRAF (n = 43), MET (n = 36), HER2 (n = 29), ALK (n = 23), RET (n = 16), ROS1 (n = 7), and multiple drivers (n = 1). Median age was 60 years, gender ratio was 1 : 1, never/former/current smokers were 28%/51%/21%, respectively, and the majority of tumors were adenocarcinoma. The objective response rate by driver alteration was: KRAS = 26%, BRAF = 24%, ROS1 = 17%, MET = 16%, EGFR = 12%, HER2 = 7%, RET = 6%, and ALK = 0%. In the entire cohort, median PFS was 2.8 months, OS 13.3 months, and the best response rate 19%. In a subgroup analysis, median PFS (in months) was 2.1 for EGFR, 3.2 for KRAS, 2.5 for ALK, 3.1 for BRAF, 2.5 for HER2, 2.1 for RET, and 3.4 for MET. In certain subgroups, PFS was positively associated with PD-L1 expression (KRAS, EGFR) and with smoking status (BRAF, HER2).Conclusions: ICI induced regression in some tumors with actionable driver alterations, but clinical activity was lower compared with the KRAS group and the lack of response in the ALK group was notable. Patients with actionable tumor alterations should receive targeted therapies and chemotherapy before considering immunotherapy as a single agent.

P2.01-84 Patterns of Central Nervous System Metastases in EGFR Mutated or ALK Rearranged Non Small Cell Lung Cancer Patients

Development of brain and leptomeningeal metastases has prognostic and therapeutic implications in oncogene driven non small cell lung cancer (NSCLC). A rising trend is being observed in the incidence of central nervous system (CNS) metastases in such populations, probably due to better imaging techniques as well as improved survival of patients. The presence of EGFR and ALK mutations could have a significant impact on the pattern of metastatic disease spread. Data from the Indian subcontinent on this subject is scarce. The information gained from this study may have clinical relevance in today’s era of first line use of second and third generation tyrosine kinase inhibitors (TKIs). 286 EGFR mutated (220) or ALK rearranged (66) NSCLC patients were selected for this retrospective analysis. All patients with clinically suspected CNS metastases underwent contrast enhanced MR imaging of brain. Two groups of patients were identified as per their driver mutation status. The CNS lesions were analysed with regards to number, size, site (parenchymal or meningeal) and nature (morphological heterogeneity and central necrosis) Incidence of brain metastases was found to be higher in the ALK group (24/66, 36.3 %) as compared to the EGFR group (68/220, 30.9%). Leptomeningeal spread in the ALK group was found to be less compared to the EGFR group (2/24, 8.3% as opposed to 18/68, 26.4%). Morphological heterogeneity and central necrosis in parenchymal lesions was more common in the ALK group (8/24, 33.3%) as opposed to EGFR group (2/68, 2.94%). The incidence, type and morphology of CNS lesions vary with the driver mutation status in NSCLC patients. This may have prognostic and therapeutic implications, especially in identifying patients who may benefit from upfront second or third generation TKI’s.

K+ deficiency caused defects in renal tubular cell proliferation, oxidative stress response, tissue repair and tight junction integrity, but enhanced energy production, proteasome function and cellular K+ uptake

ABSTRACTHypokalemia is a common electrolyte disorder in hospitalized patients and those with chronic diseases and is associated with renal tubular injury. Our recent expression proteomics study revealed changes in levels of several proteins in renal tubular cells during K+ deficiency. However, functional significance and mechanisms underlying such changes remained unclear. The present study, thus, aimed to investigate functional changes of renal tubular cells induced by K+ deficiency. MDCK cells were maintained in normal-K+ (ANK; [K+] = 5.0 mM), Low-K+ (ALK; [K+] = 2.5 mM), or K+-depleted (AKD; [K+] = 0 mM) medium. Cell count and cell death assay showed that ALK and AKD groups had marked decrease in cell proliferation without significant change in cell death. Other functional investigations revealed that AKD cells had significantly increased levels of carbonylated proteins (by OxyBlot assay), impaired tissue repair (by scratch assay), defective tight junction (by Western blotting, immunofluorescence staining and measuring transepithelial electrical resistance), increased intracellular ATP level (by ATP measurement), decreased levels of ubiquitinated proteins (by Western blotting), and increased level of Na+/K+-ATPase (by Western blotting), which was consistent with the increased cellular K+ uptake after K+ repletion. Our findings have shown that AKD caused defects in cell proliferation, oxidative stress response, tissue repair and tight junction integrity, but on the other hand, enhanced energy production, proteasome function and cellular K+ uptake. These findings may shed light onto cellular response to K+ deficiency and better understanding of both pathogenic and compensatory mechanisms in hypokalemic nephropathy.

Functional electrical stimulation improves quality of life by reducing intermittent claudication

ObjectiveTo determine if Functional Electrical Stimulation (FES) would improve ischemic pain, walking distance, and quality of life of patients with intermittent claudication. DesignSingle blind, randomized block, two factorial design. PatientsPatients diagnosed with Peripheral Artery Disease (PAD) and intermittent claudication (IC). Ankle Brachial Index ranged 0.4-0.9 on at least one leg. Patients were randomly assigned to experimental (FES+Walk, N=13) or control (WALK, N=14) groups. InterventionExperimental group patients received FES to the dorsiflexor and plantarflexor muscles while walking for 1h/day, six days/week for eight weeks. Control group patients received similar intervention without FES. A Follow-up period of both groups lasted eight weeks. Outcome measuresOutcome measures were taken at baseline (T0), after intervention (T1), and after follow-up (T2). Primary measures included Perceived Pain Intensity (PPI), Six minute walk (6MW), and Peripheral Arterial Disease Quality of Life (PADQOL). Secondary measures included Intermittent Claudication Questionnaire (ICQ) and Timed Up and Go (TUG). ResultsGroup by time interactions in PPI were significant (P<0.001) with differences of 27.9 points at T1 and 36.9 points at T2 favoring the FES+Walk group. Groups difference in Symptoms and Limitations in Physical Function of the PADQOL reached significance (T1=8.9, and T2=8.3 improvements; P=0.007). ICQ was significant (T1=9.3 and T2=13.1 improvements; P=0.003). Improvement in 6MW and TUG tests were similar between groups. Conclusions and relevanceWalking with FES markedly reduced ischemic pain and enhanced QOL compared to just walking. FES while walking may offer an effective treatment option for the elderly with PAD and Intermittent Claudication. Trial registrationNIH-NIA 1R21AG048001 https://projectreporter.nih.gov/project_info_description.cfm?aid=8748641&icde=30695377&ddparam=&ddvalue=&ddsub=&cr=1&csb=default&cs=ASC.https://clinicaltrials.gov/ct2/show/NCT02384980?term=David+Embrey&rank=1.

MicroRNA expression profiles and clinicopathological implications in lung adenocarcinoma according to EGFR, KRAS, and ALK status.

Lung adenocarcinoma has distinctive clinicopathological features that are related to specific genetic alterations, including EGFR and KRAS mutations and ALK rearrangement. MicroRNAs are small non-coding RNAs that post-transcriptionally regulate many important biological processes and influence cancer phenotypes. This study retrospectively investigated microRNA expression profiles, and their clinicopathological implications, in lung adenocarcinoma according to genetic status (EGFR, KRAS, ALK, and triple negative). A total of 72 surgically resected lung adenocarcinoma specimens (19 EGFR-mutated, 17 KRAS-mutated, 16 ALK-rearranged, and 20 triple negative cancers) were screened for 23 microRNAs using quantitative real-time reverse transcriptase polymerase chain reaction. We then evaluated the associations between the microRNA expressions and the cancers’ genetic and clinicopathological features. Eight microRNAs were associated with clinicopathological features, such as male sex and ever-smoker status (high miR-373-3p, miR-1343-3p, miR-138-1-3p, and miR-764; low miR-27b-3p) and vascular invasion (high miR-27b-3p; low miR-1343-3p and miR-764). Clustering and discriminant analyses revealed that the microRNA expression patterns in the ALK group were different from those in the EGFR and KRAS groups. Five microRNAs (high miR-1343-3p; low miR-671-3p, miR-103a-3p, let-7e, and miR-342-3p) were especially distinctive in the ALK group, compared to the EGFR and KRAS groups. Moreover, a significant association was observed between ALK-rearrangement, decreased miR-342-3p expression, and immunohistochemical loss of E-cadherin. Therefore, microRNA expression profiles appear to have distinctive clinicopathological implications in ALK-rearranged lung adenocarcinoma. Furthermore, the association of ALK rearrangement, decreased miR-342-3p expression, and E-cadherin loss might indicate that miR-342-3p is involved in the ALK-associated phenotypes and epithelial-mesenchymal transition.

High MET Receptor Expression But Not Gene Amplification in ALK 2p23 Rearrangement Positive Non–Small-Cell Lung Cancer

Overexpression of MET receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) and MET gene amplification have been well-documented in non-small-cell lung cancer (NSCLC). Activated MET signaling plays an important role in human cancer tumorigenesis, metastasis, and drug resistance. However, the deregulation of MET/HGF pathway in NSCLC harboring ALK gene rearrangement (ALK[+]), which is sensitive to dual ALK and MET inhibitor Crizotinib, has not been reported. We performed systematic analysis of MET/HGF expression by immunohistochemistry (IHC) and MET gene amplification by dual color, dual hapten bright field in situ hybridization in 19 ALK(+) and 73 ALK(-) NSCLC tumor tissues from those who had clinical ALK rearrangement test done at the Cleveland Clinic from August 2010 to January 2013. IHC scoring was interpreted on a standard four-tier system. The percentage of MET IHC score 0, 1+, 2+, and 3+ were 5.5%, 27.8%, 50.0%, and 16.7% in ALK(+) group, compared with 28.8%, 33.9%, 23.7%, and 13.6% in ALK(-) group, respectively. The MET high expression (IHC score 2 or 3) was significantly higher in ALK(+) group statistically (66.7% versus 37.3%, p = 0.03). HGF-high expression (IHC score 2 or 3) was 33.3% in ALK(+) and 15.8% in ALK(-) (p = 0.17). We identified eight cases in ALK(-) and one case in ALK(+) tumor who had MET gene amplification (18.4% versus 7.1%, p = 0.43) by dual color, dual hapten bright field in situ hybridization. No significant correlation between MET protein receptor expression and gene amplification was identified. Our study demonstrated for the first time that MET receptor expression, but not MET gene amplification, is significantly increased in ALK(+) NSCLC. MET gene amplification is a relatively rare event in this unique population compared with ALK(-) NSCLC.

Corneal radius of curvature after anterior lamellar versus penetrating keratoplasty

Keratoplasty may induce major spherical refractive error related to abnormal corneal radius of curvature (CRC). Two hundred and thirty-eight consecutive eyes of 238 patients with clear graft and at least one postoperative Orbscan examination performed after suture removal (average follow-up time, 86months) were retrospectively analyzed. Anterior lamellar keratoplasties (ALK group, n = 119) and penetrating keratoplasties (PK group, n = 119) were matched for preoperative diagnosis and lens status. The average postoperative, suture-out, Orbscan 3-mm CRC was 7.17mm with a wide 95% confidence interval [6.26mm; 8.37mm]. It was 7.05mm in the ALK group and 7.31mm in the PK group (p < 0.01). In the ALK group, this figure was 7.00mm for oversized grafts and 7.67mm for non-oversized grafts (p < 0.001). CRC values were significantly lower for eyes with keratoconus (7.00mm) or stromal scar after infectious keratitis (7.06mm) compared with stromal scar after trauma (7.74mm) or stromal dystrophies (8.17mm). Values were significantly lower for big-bubble ALKs (6.92mm) and manual dissection-ALKs (7.14mm) compared with PKs (7.31mm) and microkeratome-assisted ALKs (7.45mm). The average Orbscan 3-mm SimK cylinder, irregularity, and refractive power symmetry index were, respectively, 4.7 D/4.8 D/1.9 D for ALKs and 5.2 D/4.8 D/1.8 D for PKs (p = 0.99). The CRC is lower after ALK compared with PK, and features important variability. In eyes with ALK, non-oversized grafts result in postoperative CRC close to normal values and corneal diseases associated with stromal thinning and DALK result in lower postoperative CRC.

Abstract 2374: Tumoral MET expression and plasma HGF level in patients with ALK 2p23 fusion driven lung cancer.

Abstract Background. MET is the tyrosine kinase receptor for the ligand hepatocyte growth factor (HGF). MET kinase has been identified as a novel promising target in non-small cell lung lung cancer (NSCLC). Crizotinib, a recently FDA-approved ALK inhibitor for NSCLC harboring ALK 2p23 fusion (ALK+), was initially developed as a MET inhibitor. MET expression level is a potential biomarker for response to MET inhibitors. The role of MET/HGF pathway in ALK+ NSCLC is unknown. Methods. A cohort of 51 NSCLC patients tested for ALK fusion at Cleveland Clinic (2000 to 2012), including 15 ALK(+) and 36 ALK(-) subjects, were included in the study. Immunohistochemistry (IHC) stain against MET was performed using a monoclonal CONFIRM anti-Total c-MET antibody (SP44, Ventana). Staining was performed on a Ventana Benchmark XT automated immunostainer (Ventana Medical Systems). A four-tier (0, 1+, 2+, 3+) scoring system and H-score (the sum of each intensity score x %) were used. Thirteen ALK(+) patients treated on crizotinib therapy were prospectively followed and a total of 32 blood plasma samples were collected (1) prior to crizotinib initiation, (2) during responding period and (3) at disease progression, for HGF level measurement using HGF ELISA kit (R&amp;D). Statistical analysis was performed using Fisher exact test and Wilcoxon rank sum test in JMP. Results. Of the 51 tumor tested for MET IHC, 39 were adenocarcinoma (15 ALK+ and 24 ALK-), 6 squamous cell, 4 large cell carcinoma and 2 other NSCLC. None received any MET inhibitor when tissue was collected. The percentage of negative MET IHC (score 0 or 1+ in 50% tumor cells) was not statistically significant between ALK(+) and ALK(-) groups (40% vs 30%, p=0.5). The plasma HGF levels in ALK(+) patients varies from undetectable to 14000pg/ml. The median (25th-75th quantiles) level in patients prior to crizotinib therapy (n=5), during responding period (CR/PR/SD, n=12) and at disease progression (n=5) were 2137(767-12100)pg/ml, 1629(785-3430)pg/ml and 4064(1108-7308)pg/ml. The plasma HGF level showed a trend of decreasing during clinical response to crizotinib and elevation upon disease progression, albeit not reaching statistical significance in this small study cohort (p&amp;gt;0.05). Conclusions. MET expression is commonly seen in NSCLC, and no significant difference were observed between fusion ALK(+) and ALK(-) NSCLC. The baseline plasma HGF concentration varied significantly among ALK(+) NSCLC patients, but showed a trend correlating with therapeutic response and resistant progression, to crizotinib therapy. Prospective study with larger cohorts is warranted to further evaluate the role of MET/HGF as biomarker in ALK targeted therapy. Citation Format: Yan Feng, Eugen Minca, Wei Zhang, Lihong Yin, Nathan Pennell, Carol Farver, Raymond Tubbs, Angen Liu, Patrick Ma. Tumoral MET expression and plasma HGF level in patients with ALK 2p23 fusion driven lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2374. doi:10.1158/1538-7445.AM2013-2374

The Effects of Chronic Sodium Bicarbonate Ingestion and Interval Training in Highly Trained Rowers

Recent research has reported performance improvements after chronic NaHCO3 ingestion in conjunction with high-intensity interval training (HIT) in moderately trained athletes. The purpose of the current study was to determine the effects of altering plasma H+ concentration during HIT through NaHCO3 ingestion over 4 wk (2 HIT sessions/wk) in 12 Australian representative rowers (M ± SD; age 22 ± 3 yr, mass 76.4 ± 4.2 kg, VO(2peak) 65.50 ± 2.74 ml · kg(-1) · min(-1)). Baseline testing included a 2,000-m time trial and an incremental exercise test. After baseline testing, rowers were allocated to either a chronic NaHCO3 (ALK) or placebo (PLA) group. Starting 90 min before each HIT session, subjects ingested a 0.3-g/kg body mass dose of NaHCO3 or a placebo substance. Fingertip blood samples were taken throughout the study to analyze bicarbonate and pH levels. The ALK group did not produce any additional improvements in 2,000-m rowing performance time compared with PLA (p > .05). Magnitude-based inferential analysis indicated an unclear or trivial effect on 2,000-m power, 2,000-m time, peak power output, and power at 4 mmol/L lactate threshold in the ALK group compared with the PLA group. Although there was no difference between groups, during the study there was a significant mean (± SD) 2,000-m power improvement in both the ALK and PLA groups of 17.8 ± 14.5 and 15.2 ± 18.3 W, respectively. In conclusion, despite overall improvements in rowing performance after 4 wk of HIT, the addition of chronic NaHCO3 supplementation during the training period did not significantly enhance performance further.

Clinical characteristics of NSCLC harboring ALK 2p23 translocation at Cleveland Clinic.

e18085 Background: The ALK 2p23 translocation, ALK(+), is an important druggable target in 5-7% of non-small cell lung cancer (NSCLC). However, the break-part FISH assay is labor-intensive. Better understanding of the target population’s clinical features and alternative screening tests are desirable to enable cost-effective patient selection for molecular therapy. Methods: NSCLC patients (N=120) seen at the Cleveland Clinic (CC) who had clinical screening ALK 2p23 FISH (Abbott Molecular) were included in this retrospective analysis. Biopsy specimens were also tested using immunohistochemistry (IHC) for ALK overexpression via clone D5F3 (Cell Signaling Tech.) with OptiView ultrasensitive detection (Ventana Medical Sys.). Clinical data were extracted from electronic medical records. Comparison was performed using Fisher’s exact test, Wilcoxon rank sum test or log-rank test. Results: Of the 120 tumors tested, 34 (28.3%) were ALK(+) by FISH, predominantly adenocarcinomas (33/34). 97% of the samples were also tested by ALK-IHC, with a concordance rate of 99%. Comparing to ALK (-) group, ALK(+) patients were younger (median 53 vs 65, p&lt;0.01) and mostly never/light smokers (91% vs 43%, p&lt;0.01). ALK(+) tumors tended to have higher number of metastatic sites at diagnosis, especially for liver metastasis (26.5% vs 10.5%, p=0.04). Interestingly, venous thrombosis (DVT/PE) was also significantly more common in ALK(+) patients (35.3% vs 16.3%, p=0.03). 17 (50%) patients were treated with crizotinib in the ALK(+) group. Two cases with positive screening ALK-IHC helped to identify FISH(+) tumor areas. Of those tumors with EGFR status available (n=103), 6 were EGFR mutation-positive, all being ALK(-). No significant OS difference was seen in ALK(+)/EGFR(-) patients (n=28) compared to ALK(-)/EGFR(-) patients (n=69). Conclusions: ALK 2p23 (+) NSCLC at CC was more commonly seen in younger patients and never/light smokers. They tended to have greater number of metastases, especially in the liver, and significantly higher risk of venous thrombosis. ALK-ultrasensitive IHC using the D5F4 clone helped to identify FISH(+) tumor areas and may be considered a cost-effective screening test demonstrating high concordance with ALK FISH.

Identification of Genes Upregulated in ALK-Positive and EGFR/KRAS/ALK-Negative Lung Adenocarcinomas

Activation of the EGFR, KRAS, and ALK oncogenes defines 3 different pathways of molecular pathogenesis in lung adenocarcinoma. However, many tumors lack activation of any pathway (triple-negative lung adenocarcinomas) posing a challenge for prognosis and treatment. Here, we report an extensive genome-wide expression profiling of 226 primary human stage I-II lung adenocarcinomas that elucidates molecular characteristics of tumors that harbor ALK mutations or that lack EGFR, KRAS, and ALK mutations, that is, triple-negative adenocarcinomas. One hundred and seventy-four genes were selected as being upregulated specifically in 79 lung adenocarcinomas without EGFR and KRAS mutations. Unsupervised clustering using a 174-gene signature, including ALK itself, classified these 2 groups of tumors into ALK-positive cases and 2 distinct groups of triple-negative cases (groups A and B). Notably, group A triple-negative cases had a worse prognosis for relapse and death, compared with cases with EGFR, KRAS, or ALK mutations or group B triple-negative cases. In ALK-positive tumors, 30 genes, including ALK and GRIN2A, were commonly overexpressed, whereas in group A triple-negative cases, 9 genes were commonly overexpressed, including a candidate diagnostic/therapeutic target DEPDC1, that were determined to be critical for predicting a worse prognosis. Our findings are important because they provide a molecular basis of ALK-positive lung adenocarcinomas and triple-negative lung adenocarcinomas and further stratify more or less aggressive subgroups of triple-negative lung ADC, possibly helping identify patients who may gain the most benefit from adjuvant chemotherapy after surgical resection.

Analysis of corneal epithelial cells with confocal microscopy after lamellar keratoplasty

Abstract Purpose the purpose of this study was to analyze morphometry of corneal epithelial cells in normal eyes and after anterior lamellar keratoplasty using confocal microscopy. Methods The combination of the Heidelberg Retina Tomograph II and the Rostock Cornea Module (HRT II/RCM) was used to evaluate the corneal epithelium of 10 eyes of patients who had undergone anterior lamellar keratoplasty (ALK group) and 10 normal eyes (control group). Three epithelial layers per cornea were examined: superficial cell layer, wing cell layer, and basal cell layer. A morphometric analysis software was used to determine area, perimeter, circularity, and Feret diameter of the corneal epithelial cells. Results The mean basal cell area in the central zone was significantly higher (p&lt; 0.001) after ALK than in the control group. After ALK, the mean basal cell area decreased with post‐operative time (rS = ‐ 0.42; p = 0.039) but it did not return to normal 24 months after transplantation. Comparison of both groups showed significant differences in cell perimeter, circularity, and Feret diameter of basal cells (p&lt; 0.001). Similar differences were found in wing cell layer. Only few superficial cells could be analyzed on corneal graft (no statistical analysis could be done). Conclusion This study imaged the living human basal epithelium of lamellar grafts, analyzed its structure and highlighted changes in epithelial cells density and morphology. We found an increase in basal cell density between 12 and 30 months. This could be explained by reinnervation of the cornea. Indeed, corneal nerves provide important protective and trophic functions. The basal cell area seems to be a relevant morphometric parameter to assess the corneal epithelium.