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Purpose While HER2 testing is well established in directing appropriate treatment for breast cancer, a small percentage of cases show equivocal results by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Alternative probes may be used in equivocal cases. We present a single community-based institution’s experience in further evaluating these cases. Patients and methods Between 2014 and 2016, 4255 samples were submitted for HER2 amplification testing by alternative probes, TP53, RAI1, and RARA. Of the patients tested by FISH, 505/3908 (12.9%) also had IHC data. Results Most (73.9%) FISH equivocal cases remained equivocal after IHC testing. However, 50.5% of equivocal cases were classified as HER2 amplified by alternative probes. Most cases were positive by more than one probe: 78% of positive cases by RAI1 and 73.9% by TP53. There was a significant difference between IHC and FISH alternative testing (p

// Maher Albitar 1 , Sucha Sudarsanam 1 , Wanlong Ma 1 , Shiping Jiang 1 , Wayne Chen 1 , Vincent Funari 1 , Forrest Blocker 1 and Sally Agersborg 1 1 NeoGenomics Laboratories, Aliso Viejo, CA, USA Correspondence to: Maher Albitar, email: maher.albitar@neogenomics.com Keywords: NSCLC; PD-L1; EGFR; MET; TP53 Received: October 06, 2017 Accepted: January 09, 2018 Published: February 08, 2018 ABSTRACT Background: The role of MET amplification in lung cancer, particularly in relation to checkpoint inhibition and EGFR WT, has not been fully explored. In this study, we correlated PD-L1 expression with MET amplification and EGFR , KRAS , or TP53 mutation in primary lung cancer. Methods: In this retrospective study, tissue collected from 471 various tumors, including 397 lung cancers, was tested for MET amplification by FISH with a MET /centromere probe. PD-L1 expression was evaluated using clone SP142 and standard immunohistochemistry, and TP53 , KRAS , and EGFR mutations were tested using next generation sequencing. Results: Our results revealed that PD-L1 expression in non-small cell lung cancer is inversely correlated with EGFR mutation (P=0.0003), and positively correlated with TP53 mutation (P=0.0001) and MET amplification (P=0.004). Patients with TP53 mutations had significantly higher MET amplification (P=0.007), and were more likely (P=0.0002) to be EGFR wild type. There was no correlation between KRAS mutation and overall PD-L1 expression, but significant positive correlation between PD-L1 expression and KRAS with TP53 co-mutation (P=0.0002). A cut-off for the ratio of MET : centromere signal was determined as 1.5%, and 4% of lung cancer patients were identified as MET amplified. Conclusions: This data suggests that in lung cancer both MET and TP53 play direct roles in regulating PD-L1 opposing EGFR . Moreover, KRAS and TP53 co-mutation may cooperate to drive PD-L1 expression in lung cancer. Adding MET or TP53 inhibitors to checkpoint inhibitors may be an attractive combination therapy in patients with lung cancer and MET amplification.

The safety of lithium-ion batteries (LIBs) remains the focus of engineering and scientific attention as its bending failure cannot be monitored by conventional means such as voltage and temperature due to its special failure mechanism. Therefore, it is of great significance to study the mechanical properties and failure mechanisms of LIBs during bending failure. In this paper, 18,650 LIBs are taken as the research object, and an in-situ experimental platform based on acoustic emission (AE) and three-dimensional digital image correlation (3D-DIC) methods is built to study the mechanical properties and failure mechanisms of LIBs in different states of charge (SOC) under bending loads. The results show that the bending modulus and stiffness of the battery increase with the increase of SOC. The main types of damage and the expansion process of the battery during the bending failure process are also obtained. The research results provide a theoretical basis for research on the bending failure of LIBs. Meanwhile, the experimental platform built in this article solves the failure of previous monitoring methods, including deformation and temperature during the bending failure of LIBs. In addition, this paper provides a new method for in-situ monitoring of LIBs.

Echinococcosis is a serious parasitic disease caused byEchinococcustapeworms. Protoscoleces are sometimes released during surgical treatment for hydatid cysts, causing the recurrence of echinococcosis. Protoscoleces may be susceptible to radiation therapy. In this studyEchinococcusprotoscoleces were culturedin vitroand then divided into four differentγ-ray irradiation dose groups (10 Gy, 20 Gy, 40 Gy, and 80 Gy) and a blank group. The protoscoleces were then implanted into the abdominal cavity of mice. Four months later, we observed that the incidence and weight of cysts declined with the increase of irradiation dose.γ-ray irradiation can suppress the generation ofEchinococcusoriginated from protoscolex, the reason of which is due to the damaging to the structure ofEchinococcus. Irradiation may prevent echinococcosis recurrence after surgical removal of hydatid cysts.

PURPOSE While HER2 testing is well established in directing appropriate treatment for breast cancer, a small percentage of cases show equivocal results by IHC and FISH. Alternative probes may be used in equivocal cases. We present a single community-based institution's experience in further evaluating these cases. PATIENTS AND METHODS Between 2014-2016, 4255 samples were submitted for HER2 amplification testing by alternative probes, TP53, RAI1, and RARA. Of patients tested by FISH, 505/3908 (12.9%) also had IHC data. RESULTS Most (73.9%) FISH equivocal cases remained equivocal after IHC testing. Duplicate testing by IHC using a different paraffin block resulted in poor reproducibility as 44% of IHC negative became equivocal and 40% of IHC equivocal became negative. Of the equivocal cases by IHC, 52.3% became positive by alternative FISH. However, 50.5% of cases equivocal by conventional FISH were classified as HER2 amplified by alternative probes. Most cases were positive by more than one probe; 78% of positive cases were positive by RAI1 and 73.9% by TP53. There was a significant difference between IHC and FISH alternative testing (P CONCLUSION The prevalence of double HER2 equivocal cases and the discrepancy between IHC and alternative FISH testing suggest that FISH alternative testing using both RAI1 and TP53 probes is necessary for conclusive classification. Due to the significant difference between IHC repeat testing, reflexing on IHC alone in this group of cases may not be reproducible. Because almost half of FISH equivocal cases converted to HER2 amplified upon alternative testing, clinical studies to determine benefit of anti-HER2 therapy in these patients is urgently needed. Alternative FISHIHC Score 0 NegativeIHC Score 1 NegativeIHC Score 2 EquivocalIHC Score 3 PositiveNo%No%No%No%Negative1463.65454.517847.7436.4Positive836.44545.519552.3763.6Total22100.099100.0373100.011100.0 Citation Format: Sally Agersborg, Christopher Mixon, Thanh Nguyen, Sramila Aithal, Forrest Blocker, Lawrence Weiss, Robert Gasparini, Shiping Jiang, Wayne Chen, Gregory Hess, Maher Albitar. Immunohistochemistry and alternative FISH testing in breast cancer with HER2 equivocal amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4524.

e15604Background: Microsatellite instability (MSI) and DNA mismatch repair defect (dMMR) are dependable predictive biomarkers of colorectal cancer (CRC) response to checkpoint inhibitors. We explor...

3022Background: DNA mismatch repair deficiency (dMMR) can be tested by immunohistochemistry (IHC) or microsatellite instability (MSI). While either IHC and MSI is adequate for establishing Lynch sy...

To study the early change of bone matrix and soft tissue around articulation in adjuvant-induced arthritic (AIA) rats non-invasively by X-ray phase-contrast imaging (XPCI), a new imaging method.Adjuvant-induced arthritis was established in male Sprague-Dawley (SD) rats (n=6, age 40 days) by subcutaneous injection of Freund's complete adjuvant (FCA) into the left hindpaw. In vivo XPCI evaluation of the early soft tissue and bone changes in AIA rats was consecutively performed and correlated with changes in volumes of right hindpaws and body weights. In comparison, the changes in the AIA rats were also evaluated with absorption-contrast imaging using the same X-ray source as XPCI and conventional radiography at the same time. After the imaging evaluation, AIA rats were subjected to histological examination.There was significant difference between the score of XPCI and the other two methods in demonstrating soft tissue (P0.01), bone details (P0.01) and lesions (P0.001). By day 10 after subcutaneous injection of FCA, bone changes in the right hindpaw were not obvious, but swelling of soft tissue appeared. By day 12, bone erosion in the articular facet and the area around the articular facet, was detected, along with osteoporosis, and swelling of soft tissue was aggravated. By day 14 bone erosions became fused and expanded, especially in the margin area around the articular facet. At day 16 bone erosion still existed. Joint interspaces seemed wider than normal, and swelling of soft tissue was significant. By day 18 periosteal new bone formation was seen definitely, destruction of bone decreased, bone density around the articular was enhanced, and swelling of soft tissue was relieved. XPCI could clearly distinguish all these alterations, which could not be demonstrated by absorption-contrast imaging and conventional radiography. During the test period, the volume of the right hindpaw and the body weight of the AIA rats also changed significantly compared with the normal rat. Histological examination confirmed that adjuvant-induced arthritis had occurred in all rats of the adjuvant group.Osteoporosis, bone erosion and periosteal new bone formation take place at the early stage of adjuvant-induced arthritis. XPCI can evaluate non-invasively these subtle bone changes that are "blind areas" for conventional radiography.

Introduction: MET gene activation has been reported to be associated with resistance to EGFR inhibitors in lung cancer. Resistance to EGFR inhibitors is also reported to be associated with upregulation of PD-L1. We studied PD-L1 expression levels with MET gene amplification and mutation in EGFR, KRAS, or TP53 in a large number of primary untreated lung cancers. Methods: Tissue samples collected from 397 core biopsies or resections from lung cancers were studied for MET gene amplification by fluorescent in-situ hybridization (FISH) using MET (7q31) probe and centromere 7 as a control. Signals were quantified. PD-L1 expression on the same samples was evaluated using clone SP142 and standard immunohistochemistry (IHC) procedure. Samples were also sequenced using next generation sequencing (NGS) for mutations in TP53, KRAS, and EGFR. Results: PD-L1 expression was detected in 166 patients (42%). Twenty seven (7%) had expression between 1% and 5%, 61 (15%) between 1% and 20%, 92 (23%) between 1% and 50%, and 105 (26%) had PD-L1 > 50% in tumor cells. Ratio of MET: centromere signals was > 1.5 in 16 (4%) of patients. Patients with MET ratio > 1.5% had significantly higher (P=0.004) percentage of PD-L1 as a continuous variable as well as when cut-off points of 5% (P=0.01), 20% (P=0.0006), and 50% (P=0.01) were used. Patients with EGFR mutation had significantly lower levels of PD-L1 expression (P=0.003). When a cut-off point of 50% is used for PD-L1 expression, the EGFR-mutant cases had significantly less number of positive cases (P=0.0003). There was no correlation between the presence of High MET: Ratio and EGFR mutation. There was no correlation between KRAS mutation and overall PD-L1 expression (P=0.4). There was no correlation between MET ratio and KRAS mutation. Patients with TP53 mutation had significantly higher MET ratio when a cut-off at 1.5 is used (P=0.01). Also there was significant correlation between TP53 mutation and overall PD-L1 expression (P=0.0001). This remained significant when cut-off point of 50% or 5% are used (P=0.0004 and P=0.007, respectively). TP53 mutation was significantly more common in EGFR wild-type cases (P=0.0002). Conclusions: Extra copies of MET gene as detected by FISH testing in therapy-naïve lung cancer patients is associated with higher expression of PD-L1, while EGFR-mutant lung cancers had significantly lower expression of PD-L1 when clone SP142 is used and NGS is used for detecting EGFR mutations. Patients with TP53 mutation had strikingly high expression of PD-L1 using SP142 clone and higher copy number of MET gene. This data suggests that in lung cancer, both MET and TP53 genes play a direct role in regulating PD-L1 expression opposing the EGFR gene, which appears to suppress PD-L1 expression. KRAS gene may not be involved in PD-L1 expression in lung cancer. Citation Format: Maher Albitar, Sucha Sudarsanam, Wanlong Ma, Shiping Jiang, Wayne Chen, Vincent Funari, Steven Brodie, Sally Agersborg. Correlation between MET gene amplification and TP53 mutation in upregulating PD-L1 expression in EGFR wild-type lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5618. doi:10.1158/1538-7445.AM2017-5618

e14500 Background: PD-L1 expression as detected by immunohistochemistry (IHC) is significantly lower in colorectal cancers (CRC) when compared with lung cancer or other types of cancer. We explored if mutations in the RAS/RAF gene family, TP53 or PIK3CA can define a subgroup of CRC that express PD-L1. Methods: Tissue samples collected from 107 patients with CRC were studied for the expression of PD-L1 using clone SP142. The same samples were also tested for mutations in NRAS, KRAS, HRAS, BRAF, TP53, and PIK3CA using Next Generation Sequencing (NGS). Results: Of the 107 CRC samples only 15 (14%) showed PD-L1 positive tumor cells (≥1%) and 8 of the 15 (7.5% of total) had PD-L1 in ≤5% of tumor cells. Detected mutations in these samples were as follows: TP53 65%, KRAS 49.5%, PI3KCA 22.5%, NRAS 5%, HRAS 1%, and BRAF 17%. There was no correlation between PD-L1 expression and mutation status in any of the RAS/RAF genes. There was also no correlation between TP53 mutation and PD-L1 expression. This was true irrespective if PD-L1 expression is considered as a continuous variable or when cut-off points of 5%, 20%, or 50% were used. However, patients without any mutation in RAS or TP53 had significantly (P = 0.005) more expression of PD-L1 when cut-off point of 5% is used. This remained true if PD-L1 expression is considered as a continuous variable (P = 0.04). There was no correlation between PIK3CA and PD-L1 expression. Conclusions: PD-L1 expression is significantly more common in CRC that lack mutations in RAS or TP53. PD-L1 expression is detected in 31% of patients with wild-type RAS/TP53 as compared with 12% in patients with RAS/TP53 mutations (P = 0.04). If a cut-off point of 5% is used, 31% of RAS/TP53-wild-type CRC were positive for PD-L1, while only 6% of RAS/TP53- mutant CRC were positive for PD-L1 (P = 0.005). This suggests that in CRC without RAS/TP53 mutation, the PD-L1 may play a more important role in oncogenesis. Exploring immunotherapy in this group of CRC patients might be justified.

e21050 Background: BRAF mutation and PD-L1 expression appear independent in melanoma. We have established that PD-L1 clones 22C3 and 28.8 show almost identical results. In this study we correlated between BRAF mutation and PD-L1 expression as detected by 22C3/28.8 and SP142 in melanoma clinical samples. Methods: Melanoma samples were tested for PD-L1 expression and BRAF mutation. IHC testing for PD-L1 22C3 or 28.8 were tested using FDA-approved kits as recommended. Testing with SP142 (Spring Biosciences; LDT) was performed using standard techniques. BRAF testing in combination with SP142 PD-L1 testing was performed using next generation sequencing covering exons 11 and 15. Samples tested with 22C3 and 28.8 clones were tested using FDA-cleared BRAF kits (Cobas, Therascreen) testing V600 only. Results: 68 samples were tested for PD-L1 expression with clone 22C3, 67 with 28.8, and 56 with SP142. There was no overall statistical difference between the three clones in PD-L1 expression (P = 0.41). For combined 22C3 and 28.8, 61 of 135 cases (45%) had PD-L1 expression and 31 (23%) had BRAF mutation detected using FDA kits (V600). There was no statistical correlation between PD-L1 expression and BRAF mutation in this group (P = 0.9) at any cut-off. In cases tested with SP142 clone, BRAF mutation was detected in 31 cases (55%) by NGS, higher than using the FDA kit (P < 0.0001). In V600 mutations by NGS, 14 cases (25%) were positive, similar FDA kits. In SP142 cases, PD-L1 was positive in 45% of cases. There was no correlation between BRAF mutation and PD-L1 using SP142 expression as a continuous variable (P = 0.59) or cut-off points of 5% or 50%. When cut-off of 20% was used, significant inverse correlation with BRAF mutation (P = 0.004) was identified and was maintained if V600 codon only was evaluated. Conclusions: There is no correlation between BRAF mutation and PD-L1 expression as detected using clones 22C3 and 28.8. With SP142, a negative correlation between PD-L1 expression and BRAF mutation is identified with a 20% cut-off. The rate of BRAF mutations practically doubles when NGS is used and exons 11 and 15 are included in the testing, in contrast to FDA approved testing.

e15064Background: Current recommendations for colorectal cancer testing include KRAS and NRAS for anti-EGFR therapy, and BRAF mutational analysis with microsatellite instability (MSI) testing for p...

e23146Background: Companion diagnostic tests are currently recommended for the prescription of multiple therapeutic agents in oncology. Most of these companion tests are FDA-cleared kits. We compar...

11557Background: Expression of PD-L1 is, in general, associated with response to immunotherapy. However, it is believed that additional intrinsic factors play a role in determining the potential of...

A high-resolution three-dimensional (3D) imaging technology has been developed, which is a combination of x-ray holography and computerized tomography (CT) technology called holographic computerized tomography (HCT). The theory and experimental techniques on biological specimens with the use of synchrotron radiation are discussed. Projections at different angles are reconstructed with the numerical method of in-line holography, and then the reconstructed data with a higher lateral resolution are used to restore the 3D image by the CT technique. With this method, the degradation caused by the diffraction of x rays is canceled, and 3D images with high resolution of micrometer magnitude in both the lateral and the longitudinal directions are obtained.