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Supplementary Table S1. ALK inhibitors in clinical development. Supplementary Table S2. Summary of clinical characteristics of ALK-positive patients undergoing biopsies at the time of ALK inhibitor resistance. Supplementary Table S3. Paired, post-crizotinib and post second-generation ALK inhibitor biopsies. Supplementary Table S4. Treatment course of ALK-positive patients undergoing post-ceritinib biopsies. Supplementary Table S5. Treatment course of ALK-positive patients undergoing post-alectinib biopsies. Supplementary Table S6. Treatment course of ALK-positive patients undergoing post-brigatinib biopsies. Supplementary Table S7. Resistant biopsies with {greater than or equal to}2 resistance mutations. Supplementary Table S8. Mutations in ceritinib-resistant biopsies. Supplementary Table S9. Mutations in alectinib-resistant biopsies. Supplementary Table S10. Mutations in brigatinib-resistant biopsies. Supplementary Table S11. Single-nucleotide variants identified in patient-derived cell lines. Supplementary Table S12. EMT in ceritinib-resistant biopsies. Supplementary Table S13. Cells seeded for survival assays. Supplementary Table S14. Cells seeded for growth assays.

Supplementary Figure S1. ALK G1202R versus ALK G1202del. Supplementary Figure S2. ALK G1202del in ALK-rearranged lung cancer. Supplementary Figure S3. Swimmer's plot - alectinib. Supplementary Figure S4. Evaluation of MGH075-2E in drug screen. Supplementary Figure S5. MGH067-1 harbors an ALK rearrangement. Supplementary Figure S6. MGH021-5A. Supplementary Figure S7. MGH051-2C and MGH084-1D. Supplementary Figure S8. MGH049-1A and MGH075-2E. Supplementary Figure S9. MGH034-2A.

Purpose:Acquired resistance to next-generation ALK tyrosine kinase inhibitors (TKIs) is often driven by secondary ALK mutations. Here, we investigated utility of plasma genotyping for identifying ALK resistance mutations at relapse on next-generation ALK TKIs.Experimental Design:We analyzed 106 plasma specimens from 84 patients with advanced ALK-positive lung cancer treated with second- and third-generation ALK TKIs using a commercially available next-generation sequencing (NGS) platform (Guardant360). Tumor biopsies from TKI-resistant lesions underwent targeted NGS to identify ALK mutations.Results:By genotyping plasma, we detected an ALK mutation in 46 (66%) of 70 patients relapsing on a second-generation ALK TKI. When post-alectinib plasma and tumor specimens were compared, there was no difference in frequency of ALK mutations (67% vs. 63%), but plasma specimens were more likely to harbor ≥2 ALK mutations (24% vs. 2%, P = 0.004). Among 29 patients relapsing on lorlatinib, plasma genotyping detected an ALK mutation in 22 (76%), including 14 (48%) with ≥2 ALK mutations. The most frequent combinations of ALK mutations were G1202R/L1196M and D1203N/1171N. Detection of ≥2 ALK mutations was significantly more common in patients relapsing on lorlatinib compared with second-generation ALK TKIs (48% vs. 23%, P = 0.017). Among 15 patients who received lorlatinib after a second-generation TKI, serial plasma analysis demonstrated that eight (53%) acquired ≥1 new ALK mutations on lorlatinib.Conclusions:ALK resistance mutations increase with each successive generation of ALK TKI and may be underestimated by tumor genotyping. Sequential treatment with increasingly potent ALK TKIs may promote acquisition of ALK resistance mutations leading to treatment-refractory compound ALK mutations.

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Purpose:Most ALK-positive lung cancers will develop ALK-independent resistance after treatment with next-generation ALK inhibitors. MET amplification has been described in patients progressing on ALK inhibitors, but frequency of this event has not been comprehensively assessed.Experimental Design:We performed FISH and/or next-generation sequencing on 207 posttreatment tissue (n = 101) or plasma (n = 106) specimens from patients with ALK-positive lung cancer to detect MET genetic alterations. We evaluated ALK inhibitor sensitivity in cell lines with MET alterations and assessed antitumor activity of ALK/MET blockade in ALK-positive cell lines and 2 patients with MET-driven resistance.Results:MET amplification was detected in 15% of tumor biopsies from patients relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients treated with a second-generation ALK inhibitor in the first-line setting were more likely to develop MET amplification than those who had received next-generation ALK inhibitors after crizotinib (P = 0.019). Two tumor specimens harbored an identical ST7-MET rearrangement, one of which had concurrent MET amplification. Expressing ST7-MET in the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was reversed with dual ALK/MET inhibition. MET inhibition resensitized a patient-derived cell line harboring both ST7-MET and MET amplification to ALK inhibitors. Two patients with ALK-positive lung cancer and acquired MET alterations achieved rapid responses to ALK/MET combination therapy.Conclusions:Treatment with next-generation ALK inhibitors, particularly in the first-line setting, may lead to MET-driven resistance. Patients with acquired MET alterations may derive clinical benefit from therapies that target both ALK and MET.

Objective: To evaluate the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in colostrum from women who tested positive for the virus. Methods: Between March and September 2020 we obtained bilateral colostrum samples collected on spot cards within 48 hours of delivery from 15 new mothers who had previously tested positive for SARS-CoV-2. Four of 15 women provided liquid colostrum, which was used for validating results obtained from spot cards. Archived bilateral colostrum samples collected from 8 women during 2011–2013 were used as pre-coronavirus disease 2019 (COVID-19) controls. All samples were tested for reactivity to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein using an enzyme-linked immunosorbent assay that measures SARS-CoV-2 RBD-specific IgA, IgG, and IgM and for levels of 10 inflammatory cytokines (interferon-gamma [IFN-γ], tumor necrosis factor-alpha, interleukin [IL]-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13) using a multiplex electrochemiluminescent sandwich assay. Results: Our validation studies indicate that the levels of SARS-CoV-2-specific antibodies and the associated cytokines measured in liquid colostrum are comparable to levels eluted from spot cards. Bilateral colostrum samples from 73%, 73%, and 33% of the 15 COVID-19 mothers exhibited IgA, IgG, and IgM reactivity to RBD, respectively. In addition, symptomatic COVID-19 mothers had statistically significant elevated levels of 4 of the 10 inflammatory markers (IFN-γ, IL-4, IL-6, and IL-12) compared to asymptomatic COVID-19 mothers. Conclusions: A strong humoral immune response is present in the colostrum of women who were infected with SARS-CoV-2 before delivering. The evolution and duration of the antibody response, as well as dynamics of the cytokine response, remain to be determined. Our results also indicate that future large-scale studies can be conducted with milk easily collected on paper spot cards.

Histologic transformation from non-small cell to small cell lung cancer has been reported as a resistance mechanism to targeted therapy in EGFR-mutant and ALK fusion-positive lung cancers. Whether small cell transformation occurs in other oncogene-driven lung cancers remains unknown. Here we analyzed the genomic landscape of two pre-mortem and 11 post-mortem metastatic tumors collected from an advanced, ROS1 fusion-positive lung cancer patient, who had received sequential ROS1 inhibitors. Evidence of small cell transformation was observed in all metastatic sites at autopsy, with inactivation of RB1 and TP53, and loss of ROS1 fusion expression. Whole-exome sequencing revealed minimal mutational and copy number heterogeneity, suggestive of “hard” clonal sweep. Patient-derived models generated from autopsy retained features consistent with small cell lung cancer and demonstrated resistance to ROS1 inhibitors. This case supports small cell transformation as a recurring resistance mechanism, and underscores the importance of elucidating its biology to expand therapeutic opportunities.

Purpose: Most ALK-positive lung cancers will develop ALK-independent resistance after treatment with next-generation ALK inhibitors. MET amplification has been described in patients progressing on ALK inhibitors, but frequency of this event has not been comprehensively assessed. Experimental Design: We performed FISH and/or next-generation sequencing on 207 posttreatment tissue (n = 101) or plasma (n = 106) specimens from patients with ALK-positive lung cancer to detect MET genetic alterations. We evaluated ALK inhibitor sensitivity in cell lines with MET alterations and assessed antitumor activity of ALK/MET blockade in ALK-positive cell lines and 2 patients with MET-driven resistance. Results: MET amplification was detected in 15% of tumor biopsies from patients relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients treated with a second-generation ALK inhibitor in the first-line setting were more likely to develop MET amplification than those who had received next-generation ALK inhibitors after crizotinib (P = 0.019). Two tumor specimens harbored an identical ST7-MET rearrangement, one of which had concurrent MET amplification. Expressing ST7-MET in the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was reversed with dual ALK/MET inhibition. MET inhibition resensitized a patient-derived cell line harboring both ST7-MET and MET amplification to ALK inhibitors. Two patients with ALK-positive lung cancer and acquired MET alterations achieved rapid responses to ALK/MET combination therapy. Conclusions: Treatment with next-generation ALK inhibitors, particularly in the first-line setting, may lead to MET-driven resistance. Patients with acquired MET alterations may derive clinical benefit from therapies that target both ALK and MET.

Purpose: Acquired resistance to next-generation ALK tyrosine kinase inhibitors (TKIs) is often driven by secondary ALK mutations. Here, we investigated utility of plasma genotyping for identifying ALK resistance mutations at relapse on next-generation ALK TKIs. Experimental Design: We analyzed 106 plasma specimens from 84 patients with advanced ALK-positive lung cancer treated with second- and third-generation ALK TKIs using a commercially available next-generation sequencing (NGS) platform (Guardant360). Tumor biopsies from TKI-resistant lesions underwent targeted NGS to identify ALK mutations. Results: By genotyping plasma, we detected an ALK mutation in 46 (66%) of 70 patients relapsing on a second-generation ALK TKI. When post-alectinib plasma and tumor specimens were compared, there was no difference in frequency of ALK mutations (67% vs. 63%), but plasma specimens were more likely to harbor ≥2 ALK mutations (24% vs. 2%, P = 0.004). Among 29 patients relapsing on lorlatinib, plasma genotyping detected an ALK mutation in 22 (76%), including 14 (48%) with ≥2 ALK mutations. The most frequent combinations of ALK mutations were G1202R/L1196M and D1203N/1171N. Detection of ≥2 ALK mutations was significantly more common in patients relapsing on lorlatinib compared with second-generation ALK TKIs (48% vs. 23%, P = 0.017). Among 15 patients who received lorlatinib after a second-generation TKI, serial plasma analysis demonstrated that eight (53%) acquired ≥1 new ALK mutations on lorlatinib. Conclusions: ALK resistance mutations increase with each successive generation of ALK TKI and may be underestimated by tumor genotyping. Sequential treatment with increasingly potent ALK TKIs may promote acquisition of ALK resistance mutations leading to treatment-refractory compound ALK mutations.

Introduction Circulating tumor DNA analysis is an emerging genotyping strategy that can identify tumor-specific genetic alterations in plasma including mutations and rearrangements. Detection of ROS1 fusions in plasma requires genotyping approaches that cover multiple breakpoints and target a variety of fusion partners. Compared to other molecular subsets of NSCLC, experience with detecting ROS1 genetic alterations in plasma is limited. Methods To describe the spectrum of ROS1 fusions in NSCLC and determine sensitivity for detecting ROS1 fusions in plasma, we queried the Guardant Health plasma dataset and an institutional tissue database and compared plasma findings to tissue results. In addition, we used the Guardant360 NGS assay to detect potential genetic mediators of resistance in plasma from patients with ROS1-positive NSCLC who were relapsing on crizotinib. Results We detected seven distinct fusion partners in plasma, most of which (n = 6 of 7) were also represented in the tissue dataset. Fusions pairing CD74 with ROS1 predominated in both cohorts (plasma: n = 35 of 56, 63%; tissue: n = 26 of 52, 50%). There was 100% concordance between the specific tissue- and plasma-detected ROS1 fusion for seven patients genotyped with both methods. Sensitivity for detecting ROS1 fusions in plasma at relapse on ROS1-directed therapy was 50%. Six (33%) of 18 post-crizotinib plasma specimens harbored ROS1 kinase domain mutations, five of which were ROS1 G2032R. Two (11%) post-crizotinib plasma specimens had genetic alterations (n = 1 each BRAF V600E and PIK3CA E545K) potentially associated with ROS1-independent signaling. Conclusions Plasma genotyping captures the spectrum of ROS1 fusions observed in tissue. Plasma genotyping is a promising approach to detecting mutations that drive resistance to ROS1-directed therapies.

BackgroundColostrum provides an immune sharing between a mother and her infant. The transfer in colostrum of antibodies against SARS-CoV-2 and the elicited cytokines may provide crucial protection to the infant. There is limited literature on the immune response to SARS-CoV-2 present in colostrum.ObjectiveTo evaluate the presence of antibodies specific to SARS-CoV-2 and the associated cytokines in colostrum from women who tested positive for the virus.Study DesignBetween March and September 2020 we obtained bilateral colostrum samples collected on spot cards within 48 hours of delivery from 15 new mothers who had previously tested positive for SARS-CoV-2. Five of these 15 COVID-19 positive women also provided bilateral liquid colostrum within 1-2 days of providing the spot card samples. Archived bilateral colostrum samples collected from 8 women during 2011-2013 were used as pre-COVID-19 controls. All samples were tested for reactivity to the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein using an ELISA that measures SARS-CoV-2 RBD-specific IgA, IgG, and IgM, and for concentrations of 10 inflammatory cytokines (IFNγ, TNFα, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13) using a multiplex electrochemiluminescent sandwich assay.ResultsBilateral colostrum samples from 73%, 73% and 33% of the 15 COVID-19 mothers exhibited IgA, IgG, and IgM reactivity to RBD respectively. Colostrum samples from two of the 8 pre-pandemic controls showed IgA and IgG reactivity to RBD. Additionally, COVID-19 mothers had significantly higher levels of 9 of the 10 inflammatory markers (all except IFNγ) as compared to the pre-COVID-19 controls. Comparable results were obtained with both the spot card-eluates and liquid samples.ConclusionsA strong humoral immune response is present in the colostrum of women who were infected with SARS-CoV-2 before delivering. High levels of 9 inflammatory markers were also present in the colostrum. The evolution and duration of the antibody response, as well as dynamics of the cytokine response, remain to be determined. Our results also indicate that future large-scale studies can be conducted with milk easily collected on paper spot cards.

Geophysical, geochemical, and geological investigations have attributed the stable behaviour of Earth's continents to the presence of strong and viscous cratons underlying the continental crust. The cratons are underlain by thick and cold mantle keels, which are composed of melt-depleted and low density peridotite residues [1]. Progressive melt extraction increases the magnesium number Mg# in the residual peridotite, thereby making the roots of cratons chemically buoyant [2, 3] and counteracting their negative thermal buoyancy. Recent global models have shown the self-consistent production of Archean continental crust by two-step mantle differentiation [4]. These models exhibit intense recycling of crust with delamination and eclogitic dripping in the first 500 million years and this behaviour is similar to the "plutonic-squishy lid'' that has been suggested for the early Earth. However, no stable continents form and no major regime transition from "vertical tectonics'' towards "horizontal tectonics'' is observed. This points to the missing ingredient of cratonic lithosphere in these models, which could act as a stable basement for the crustal material to accumulate on and may help initiate plate tectonics. Based on the bulk FeO and MgO content of the residual peridotites, it has been proposed that cratonic mantle formed by hot shallow melting with mantle potential temperature, which was higher by 200-300 °C than present-day [5]. We will introduce Fe-Mg partitioning between mantle peridotite and melt to track the Mg# variation through melting, and parametrise craton formation using the corresponding P-T formation conditions. Grain-size evolution, which has been shown to influence mantle rheology [6] is another mechanism that may contribute towards cratonic strength and will be explored using self-consistent global geodynamic models. [1] Boyd, F. R. High-and low-temperature garnet peridotite xenoliths and their possible relation to the lithosphere- asthenosphere boundary beneath Africa. In Nixon, P. H. (ed.) Mantle Xenolith, 403–412 (John Wiley & Sons Ltd., 1987).[2] Jordan, T. H. Mineralogies, densities and seismic velocities of garnet lherzolites and their geophysical implications. In The Mantle Sample: Inclusion in Kimberlites and Other Volcanics, 1–14 (American Geophysical Union, Washington, D. C., 1979).[3] Schutt, D. L. & Lesher, C. E. Effects of melt depletion on the density and seismic velocity of garnet and spinel lherzolite. Journal of Geophysical Research 111 (2006).[4] Jain, C., Rozel, A. B., Tackley, P. J., Sanan, P. & Gerya, T. V. Growing primordial continental crust self-consistently in global mantle convection models. Gondwana Research 73, 96–122 (2019).[5] Lee, C.-T. A. & Chin, E. J. Calculating melting temperatures and pressures of peridotite protoliths: Implications for the origin of cratonic mantle. Earth and Planetary Science Letters 403, 273–286 (2014).[6] Hall, C. E. & Parmentier, E. M. Influence of grain size evolution on convective instability. Geochemistry, Geophysics, Geosystems 4, 469 (2003).

Rearranged during transfection proto-oncogene (RET) fusions represent a potentially targetable oncogenic driver in non-small cell lung cancer (NSCLC). Imaging features and metastatic patterns of advanced RET fusion-positive (RET+) NSCLC are not well established. Our goal was to compare the imaging features and patterns of metastases in RET+, ALK+ and ROS1+ NSCLC. Patients with RET+, ALK+, or ROS1+ NSCLC seen at our institution between January 2014 and December 2018 with available pre-treatment imaging were identified. The clinicopathologic features, imaging characteristics, and the distribution of metastases were reviewed and compared. We identified 215 patients with NSCLC harboring RET, ALK, or ROS1 gene fusion (RET = 32, ALK = 116, ROS1 = 67). Patients with RET+ NSCLC were older at presentation compared to ALK+ and ROS1+ patients (median age: RET = 64 years, ALK = 51 years, p &lt, 0.001, ROS = 54 years, p = 0.042) and had a higher frequency of neuroendocrine histology (RET = 12%, ALK = 2%, p = 0.025, ROS1 = 0%, p = 0.010). Primary tumors in RET+ patients were more likely to be peripheral (RET = 69%, ALK = 47%, p = 0.029, ROS1 = 36%, p = 0.003), whereas lobar location, size, and density were comparable across the three groups. RET+ NSCLC was associated with a higher frequency of brain metastases at diagnosis compared to ROS1+ NSCLC (RET = 32%, ROS1 = 10%, p = 0.039. Metastatic patterns were otherwise similar across the three molecular subgroups, with high incidences of lymphangitic carcinomatosis, pleural metastases, and sclerotic bone metastases. RET+ NSCLC shares several distinct radiologic features and metastatic spread with ALK+ and ROS1+ NSCLC. These features may suggest the presence of RET fusions and help identify patients who may benefit from further molecular genotyping.

Introduction The current standard initial therapy for advanced ALK receptor tyrosine kinase (ALK)–positive NSCLC is a second-generation ALK tyrosine kinase inhibitor (TKI) such as alectinib. The optimal next-line therapy after failure of a second-generation ALK TKI remains to be established; however, standard options include the third-generation ALK TKI lorlatinib or platinum/pemetrexed-based chemotherapy. The efficacy of platinum/pemetrexed-based chemotherapy has not been evaluated in cases that are refractory to second-generation TKIs. Methods This was a retrospective study performed at three institutions. Patients were eligible if they had advanced ALK-positive NSCLC refractory to one or more second-generation ALK TKI(s) and had received platinum/pemetrexed-based chemotherapy. Results Among 58 patients eligible for this study, 37 had scans evaluable for response with measurable disease at baseline. The confirmed objective response rate to platinum/pemetrexed-based chemotherapy was 29.7% (11 of 37 patients; 95% confidence interval [CI]: 15.9% – 47.0%), with median duration of response of 6.4 months (95% CI: 1.6 months – not reached). The median progression-free survival for the entire cohort was 4.3 months (95% CI: 2.9 – 5.8 months). Progression-free survival was longer in patients who received platinum/pemetrexed in combination with an ALK TKI compared to those who received platinum/pemetrexed alone (6.8 months vs. 3.2 months, respectively; hazard ratio = 0.33; p = 0.025). Conclusions Platinum/pemetrexed-based chemotherapy shows modest efficacy in ALK-positive NSCLC after failure of second-generation ALK TKIs. The activity may be higher if administered with an ALK TKI, suggesting a potential role for continued ALK inhibition.

BACKGROUND: Acquired resistance to next-generation ALK tyrosine kinase inhibitors (TKIs) is often driven by secondary ALK mutations. Here we investigated utility of plasma genotyping for identifying ALK resistance mutations at relapse on next-generation ALK TKIs. METHODS: We analyzed 106 plasma specimens from 84 patients with advanced ALK-positive lung cancer treated with second- and third-generation ALK TKIs using a commercially available next-generation sequencing (NGS) platform (Guardant360). Tumor biopsies from TKI-resistant lesions underwent targeted NGS to identify ALK mutations. RESULTS: By genotyping plasma, we detected an ALK mutation in 46 (66%) of 70 patients relapsing on a second-generation ALK TKI. When post-alectinib plasma and tumor specimens were compared, there was no difference in frequency of ALK mutations (67% vs 63%), but plasma specimens were more likely to harbor ≥2 ALK mutations (24% vs 2%, p=0.004). Among 29 patients relapsing on lorlatinib, plasma genotyping detected an ALK mutation in 22 (76%), including 14 (48%) with ≥2 ALK mutations. The most frequent combinations of ALK mutations were G1202R/L1196M and D1203N/1171N. Detection of ≥2 ALK mutations was significantly more common in patients relapsing on lorlatinib compared to second-generation ALK TKIs (48% vs 23%, p=0.017). Among 15 patients who received lorlatinib after a second-generation TKI, serial plasma analysis demonstrated that 8 (53%) acquired ≥1 new ALK mutations on lorlatinib. CONCLUSIONS: ALK resistance mutations increase with each successive generation of ALK TKI and may be underestimated by tumor genotyping. Sequential treatment with increasingly potent ALK TKIs may promote acquisition of ALK resistance mutations leading to treatment-refractory compound ALK mutations.

Advanced, anaplastic lymphoma kinase (ALK)–positive lung cancer is currently treated with the first-generation ALK inhibitor crizotinib followed by more potent, second-generation ALK inhibitors (e.g., ceritinib and alectinib) upon progression. Second-generation inhibitors are generally effective even in the absence of crizotinib-resistant ALK mutations, likely reflecting incomplete inhibition of ALK by crizotinib in many cases. Herein, we analyzed 103 repeat biopsies from ALK-positive patients progressing on various ALK inhibitors. We find that each ALK inhibitor is associated with a distinct spectrum of ALK resistance mutations and that the frequency of one mutation, ALKG1202R, increases significantly after treatment with second-generation agents. To investigate strategies to overcome resistance to second-generation ALK inhibitors, we examine the activity of the third-generation ALK inhibitor lorlatinib in a series of ceritinib-resistant, patient-derived cell lines, and observe that the presence of ALK resistance mutations is highly predictive for sensitivity to lorlatinib, whereas those cell lines without ALK mutations are resistant.Significance: Secondary ALK mutations are a common resistance mechanism to second-generation ALK inhibitors and predict for sensitivity to the third-generation ALK inhibitor lorlatinib. These findings highlight the importance of repeat biopsies and genotyping following disease progression on targeted therapies, particularly second-generation ALK inhibitors. Cancer Discov; 6(10); 1118–33. ©2016 AACR.See related commentary by Qiao and Lovly, p. 1084.This article is highlighted in the In This Issue feature, p. 1069

ROS proto-oncogene 1 (ROS1) rearrangements are a known molecular target in non-small-cell lung cancer (NSCLC). Our goal was to determine whether ROS1-rearranged NSCLC has imaging features and patterns of metastasis, which differ from those of anaplastic lymphoma kinase (ALK)-rearranged or epidermal growth factor receptor (EGFR)-mutant NSCLC.We retrospectively identified patients with metastatic ROS1-rearranged, ALK-rearranged, or EGFR-mutant NSCLC from January 2014 to June 2018 and included those with pretreatment imaging studies available. We assessed the imaging features of the primary tumor and the distribution of metastases in these patients. The Wilcoxon rank-sum test and Fisher exact test were used to compare the imaging features.We identified 257 patients (167 women and 90 men; median age, 56 years; range, 19-90 years) with metastatic NSCLC (ROS1, 53; ALK, 87; EGFR, 117). Compared with ALK-rearranged or EGFR-mutant NSCLC, ROS1-rearranged NSCLC was less likely to present with extrathoracic metastases (ROS1, 49%; ALK, 75%; EGFR, 72%; P .01), including brain metastases (ROS1, 9%; ALK, 25%; EGFR, 40%; P .04). Compared with EGFR-mutant NSCLC, ROS1-rearranged tumors were more likely to exhibit imaging features of lymphangitic carcinomatosis (ROS1, 42%; EGFR, 12%; P .01) and less likely to have air bronchograms in the primary tumor (ROS1, 2%; EGFR, 28%; P .01). ROS1-rearranged tumors were also more likely to present with distant nodal metastases (ROS1, 15%; EGFR, 2%; P .01) and sclerotic-type bone metastases (ROS1, 17%; EGFR, 6%; P .01).Although considerable overlap exists in the imaging features of ROS1-rearranged, ALK-rearranged, and EGFR-mutant NSCLC, we found that ROS1-rearranged NSCLC has certain distinct imaging features and patterns of spread.

The teleost major histocompatibility (MH) class II receptor presents peptides from exogenous sources to CD4+ T cells, leading to the initiation of the adaptive immune response. The genes encoding MH class II have been identified in a number of teleost species, but not in walleye, an important recreational fish and commercial fishery in North America. In this study, we cloned and characterized the sequences encoding walleye MH class II α and β chains. These sequences contained all of the domains typical for functional MH class II α and β chain proteins, and aligned with other teleost sequences of MH class II. The walleye MH class II α amino acid sequence, along with other members of the Supraorder Percomorpharia, contains a high concentration of methionine residues in the beginning of the leader peptide. Southern blotting indicated that there is more than one gene copy for both MH class II α and β, while northern blotting analysis of both genes showed that expression of these genes is greatest in lymphoid tissues and at potential entry points for pathogens. These results help to further the understanding of MH class II receptors in teleosts, and could prove useful in the study of disease issues in walleye such as dermal sarcoma virus.

Immune checkpoint inhibitors (ICIs) are standard therapies in advanced NSCLC. Although genotype-directed tyrosine kinase inhibitors represent the standard of care for subsets of oncogene-driven NSCLC, patients may receive ICIs during their disease course. The impact of sequential ICI and tyrosine kinase inhibitor therapy on the risk of hepatotoxicity has not been described.Patients with advanced ALK receptor tyrosine kinase (ALK)-driven, ROS1-driven, or MET proto-oncogene, receptor tyrosine kinase (MET)-driven NSCLC treated with crizotinib, with or without preceding ICI therapy, were identified. The cumulative incidences of crizotinib-associated grade 3 or higher increases in transaminase level (per the Common Terminology Criteria for Adverse Events, version 4.0) were compared.We identified 453 patients who had NSCLC with an oncogenic alteration in ALK receptor tyrosine kinase gene (ALK), ROS1, or MET proto-oncogene, receptor tyrosine kinase gene (MET) and were treated with crizotinib (11 with and 442 without prior ICI therapy). Among the 11 patients treated with an ICI followed by crizotinib, five (cumulative incidence 45.5% [95% confidence interval (CI): 14.9-72.2]) experienced development of a grade 3 or 4 increase in alanine transaminase level and four (cumulative incidence 36.4% [95% CI: 10.0-64.2]) experienced development of a grade 3 or 4 increase in aspartate transaminase level. In comparison, among the 442 patients who received crizotinib only, a grade 3 or 4 increase in alanine transaminase level occurred in 34 patients (cumulative incidence 8.1% [95% CI: 5.7-11.0, p0.0001]) and a grade 3 or 4 increase in aspartate transaminase level occurred in 14 (cumulative incidence 3.4% [95% CI: 1.9-5.5, p0.0001]). There were no grade 5 transaminitis events. All cases of hepatotoxicity after sequential ICI and crizotinib use were reversible and nonfatal, and no case met the Hy's law criteria.Sequential ICI and crizotinib treatment is associated with a significantly increased risk of hepatotoxicity. Careful consideration and monitoring for hepatotoxicity may be warranted in patients treated with crizotinib after ICI therapy.

Objectives Reports of the prognostic significance of ALK-rearrangement in resected non-small cell lung cancer (NSCLC) have been contradictory. We aimed to determine the prognosis of early-stage ALK-positive lung cancers relative to KRAS- and EGFR-mutant lung cancers. Material and methods We reviewed medical records of patients with resected NSCLC harboring an ALK rearrangement (n = 29) or a driver mutation in EGFR (n = 255) or KRAS (n = 480). Recurrence-free survival (RFS) was estimated for each genotype with the differences reported as a hazard ratio (HR). Results Among the 764 patients, 555 (73%), 101 (13%), and 108 (14%) had stage I, II, and III NSCLC, respectively. ALK-positive patients were distributed across all stages: 10 (34%) stage I, 6 (21%) stage II, and 13 (45%) stage III. Median RFS was not reached for EGFR-mutant patients, 24.3 months (95%CI 11.4–65.3) for ALK-positive patients, and 72.9 months (95%CI 59.7 to undefined) for KRAS-mutant patients. When adjusted for stage, ALK-positive NSCLC remained associated with worse RFS compared to EGFR-mutant (HR 1.8, 95%CI: 1.1-3.1), but not when compared to KRAS-mutant (HR 1.3, 95%CI: 0.8-2.1) NSCLC. Conclusions In this large series of resected NSCLC, ALK rearrangements were associated with a trend toward inferior disease outcomes compared to other clinically relevant genomic subsets. These data support the need for clinical trials evaluating use of ALK inhibitors among ALK-positive patients with localized or locally-advanced disease.

Plants and fungi provide a vast range of natural products including pharmaceuticals with diverse chemical structures and a broad array of biological activities. In the past few decades, there has been a worldwide resurgence of interest in the study and use of medicinal plants and fungi in the health-care system. Several medicinal plants and fungi are involved in the industrial processing of profitable products used in human medicines. However, due to severe constraints on the availability of desired plant and fungus materials, different strategies, including tissue culture, have been extensively studied. The present chapter focuses on the application of various in vitro culture systems in the production of bioactive secondary metabolites in medicinal herbs and fungi in Taiwan. All the species described in this article are sources of traditional Chinese medicines. The review article includes the research work carried out in our laboratory on the production of plant secondary metabolites in callus cultures of Saussurea involucrata, Solanum melongena, and Salvia miltiorrhiza and in cell suspension cultures of Taxus mairei, Gnetiana davidii var. formosana, and Angelica dahurica var. formosana. Also, plant metabolites were obtained from in vitro shoots of Glossogyne tenuifolia, Saussurea involucrata, and Polygonum multiflorum, aerial parts of Scrophularia yoshimurae, tubers of Corydalis yanhusuo, tissue culture plants, and hairy roots of Gentiana scabra. Also, we could achieve the production of terpenoids in Antrodia cinnamomea, a medicinal fungus, and tanshinones in Salvia miltiorrhiza using T-DNA activation-tagging technique (Agrobacterium tumefaciens-mediated transformation).

9067 Background: Second-generation (gen) ALK tyrosine kinase inhibitors (TKIs) are standard first- and second-line therapies in patients (pts) with advanced ALK+ non-small cell lung cancer (NSCLC). After progression on second-gen TKI(s), standard options include platinum (PT)-based chemotherapy (chemo) or the third-gen ALK TKI lorlatinib. The efficacy of PT-based chemo is established in treatment-naive pts but is undefined in pts who have failed prior ALK TKIs. Here we evaluate the efficacy of PT/pemetrexed (pem)-based chemo in pts with ALK+ NSCLC refractory to second-gen TKIs. Methods: A retrospective study was performed at three institutions. Pts were eligible if they had advanced ALK+ NSCLC refractory to ≥1 second-gen TKI, and received PT-pem-based chemo. Medical records and imaging were reviewed to determine outcomes. Results: Among 55 eligible pts, chemo regimens included: PT/pem (31/55, 56%), PT/pem/bevacizumab (bev) (6/55, 11%), PT/pem/PD-1 inhibitor (3/55, 5%), PT/pem with ALK TKI (8/55, 15%), PT/pem/bev with TKI (6/55, 11%), and PT/pem/PD-1 inhibitor with TKI (1/55, 2%). Pts had received one (6/55, 11%), two (38/55, 69%), or more (11/55, 20%) prior TKIs. Six pts (11%) previously received adjuvant or neoadjuvant chemo. Radiographic data for response evaluation was available for 39 pts. Among 36 pts with measurable baseline disease, confirmed ORR was 31% (11/36; 95% CI, 16-48%); 13 (36%) had stable disease. The median duration of response was 5.4 months (95% CI, 1.5-7.1 months). The median progression-free survival (PFS) for the entire cohort was 4.0 months (95% CI, 2.8-4.6 months). Chemo (PT/pem +/- bev or PD-1 inhibitor) plus ALK TKI (n = 15) was associated with a significant increase in PFS compared to chemo without TKI (n = 40) (median PFS 6.8 vs 3.2 months; HR 0.306; p = 0.002). Similarly, PT/pem plus ALK TKI (n = 8) was associated with increased PFS compared to PT/pem without TKI (n = 31) (median PFS 6.8 vs 2.9 months; HR 0.358; p = 0.036). Conclusions: The efficacy of PT-pem-based chemo is limited after failure of second-gen ALK TKIs but may be higher in pts who receive chemo plus ALK TKI, suggesting a potential role for ongoing ALK inhibition. The modest benefit of PT-pem-based chemo highlights the need for other therapeutic strategies for pts refractory to second-gen TKIs.

3079 Background: ALK tyrosine kinase inhibitors (TKIs) are effective in treating advanced anaplastic lymphoma kinase (ALK) fusion-positive non-small-cell lung cancers (NSCLC), and specific ALK variants are associated with the development of resistance to specific TKIs. Humans struggle to harness the full potential of the highly complex next-generation sequencing bioinformatics pipeline output. As a consequence, the decision to report a variant remains difficult, and we considered the discrete nature of the data and the binary decision (report vs. not-report) as an ideal setting to apply an artificial intelligence (AI) approach for variant reporting. Methods: We assessed diagnostic performance of an AI model in calling ALK-resistance mutations in n = 50 consecutive ALK fusion positive patients who relapsed on TKI-therapy and underwent repeat biopsy at MGH. The random forest model was derived from independent datasets (training and validation) capturing the reporting decision on > 36,000 variants with ~500 features per variant resulting in a matrix of > 18 million data points. The model output is a contiguous prediction score from 0 (not report) to 1 (report) and a visual drill-down functionality allows exploration of the underlying features that contributed to the decision. Results: Examination of n = 76 tests from n = 50 patients with a total of n = 130 reported variants (and = 115 not reported variants) included a total of n = 31 ALK point mutations: p.1156(n = 2), p.1171(n = 8), p.1174(n = 2), p.1180(n = 2), p.1196(n = 1), p.1198(n = 1), p.1202(n = 8), p.1203(n = 1), p.1204(n = 1), p.1206(n = 1), p.1269(n = 4). Setting a screening threshold of the model at > 10% for reporting showed only one false-negative (p.Ile1171Asn) variant and 96.7% sensitivity. The average model score for ALK variants was 0.664 (range: 0.08–0.98; median 0.8) and did not show significant differences from other reported variants (0.636; 0–1; 0.7; t-test 0.66). The model would have called n = 18 of the non-reported control variants (average 0.07; range < 0.001–0.64; P < 0.0001) and was 84% specific. Review of the drill-down function identified prior call frequency, allelic ratio, and predicted transcript consequences as common model features. Importantly, the model is currently agnostic to the medical literature and does not take clinical parameters (e.g. TKI type) into account, which may further improve performance. Conclusions: Applying artificial intelligence to large discrete datasets is one approach to help identify clinically relevant variants in the setting of ALK resistance in ALK-fusion positive NSCLC.

Plants are exploited as a potential source for the large-scale production of noble gold nanoparticles in the recent years owing to their various potential applications in nanobiotechnology and nanomedicine. The present work describes green biosynthetic procedures for the production of gold nanoparticles for the first time by using an aqueous extract of the Dysosma pleiantha rhizome. The biosynthesized gold nanoparticles were confirmed and characterized by ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, and scanning electron microscopy equipped with energy dispersive spectroscopy. The results revealed that aqueous extract of D. pleiantha rhizome has potential to reduce chloroauric ions into gold nanoparticles and the synthesized gold nanoparticles were showed spherical in shape with an average of 127nm. Further, we investigated the anti-metastatic activity of biosynthesized gold nanoparticles against human fibrosarcoma cancer cell line HT-1080. The results showed that the biosynthesized gold nanoparticles were non-toxic to cell proliferation and, also it can inhibit the chemo-attractant cell migration of human fibrosarcoma cancer cell line HT-1080 by interfering the actin polymerization pathway. Thus, the usage of gold nanoparticles biosynthesized from D. pleiantha rhizome can be used as a potential candidate in the drug and gene delivery to metastatic cancer.

Medicinal plants have played a significant role in maintaining human health and improving the quality of life for thousands of years. In the past few decades, there has been an exponential rise in demand for herbal medicines, which in turn has resulted in an overexploitation and dwindling supply of medicinal plants in the wild. Therefore, it is imperative to explore all possible modes of plant propagation and large-scale cultivation. Taiwan is the home of many highly valued medicinal herbs used in the traditional Chinese medicines. In our laboratories, for more than two decades, tissue culture techniques have been used successfully for the propagation of several medicinally important plant species. The present article reviews the studies carried out on in vitro propagation of selected medicinal plants in Taiwan (Glossogyne tenuifolia, Saussurea involucrata, Gentiana scabra, and Drynaria fortunei). The article also includes the studies on induction of somatic embryogenesis in medicinal herb Peucedanum japonicum and influence of different ventilation closures on hyperhydricity (vitrification) in cultures of Scrophularia yoshimurae, Bupleurum kaoi, Gentiana scabra, and Glossogyne tenuifolia.

This study describes the development of an efficient and reliable activation tagging system for the medicinal fungus Antrodia cinnamomea. For successful Agrobacterium tumefaciens-mediated transformation, different parameters were considered. The Agrobacterium concentration of 5 × 108 cfu ml−1, 1 m m acetosyringone, 25-d-old mycelia at 0.2 g ml−1, and co-culture period of 6 d were found to be the most optimal conditions for enhancing the transformation efficiency. The mitotic stability of transferred DNA (T-DNA) was demonstrated by growing eight randomly selected putative transformants in malt extract agar medium for five subcultures. Insertion of T-DNA into the genome of transformants was confirmed by PCR and Southern hybridization. Results showed that 88 % of the mutants contained a single T-DNA insertion. Two of the mutants were observed with different triterpenoid profiles compared with the untransformed cultures. Our results suggest a new functional genomics approach to tag the triterpenoid biosynthesis genes in A. cinnamomea.

We developed and characterized 8 polymorphic microsatellite loci or simple sequence repeats from the expressed sequence tags database of the medicinal fungus, Antrodia cinnamomea. Variability was assessed in a panel of 23 strains collected from various regions of Taiwan. The number of alleles per polymorphic locus ranged from two to seven loci with an average of 4.375 alleles per locus. The observed and expected heterozygosity values ranged from 0.130 to 0.783 and from 0.122 to 0.809, respectively. No significant Hardy–Weinberg equilibrium was detected (P < 0.01) at all loci. The EST-SSR markers developed in the present study can be used for strain identification and population genetic studies in A. cinnamomea.

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 × Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.

8536 Background: ALK rearrangements are important oncogenic drivers in NSCLC. However, the prognostic implications of these rearrangements are unclear due to 1) conflicting results from small series of patients (pts) with early-stage ALK+ NSCLC, and 2) use of highly effective ALK tyrosine kinase inhibitors (TKI) in the metastatic setting. To assess the prognostic significance of ALK rearrangements in resected NSCLC, we performed a retrospective analysis of survival outcomes among pts with resected ALK+, EGFR+, or KRAS+ NSCLC treated at two institutions. Methods: We reviewed charts of pts that underwent resection for stage 1-3 NSCLC at Massachusetts General Hospital or Memorial Sloan Kettering Cancer Center between 1/2009 and 12/2012. Recurrence-free survival (RFS) was estimated for each genotype. Results: Among 764 pts (480 KRAS+, 255 EGFR+, 29 ALK+), we identified 555 (73%), 101 (13%), and 108 (14%) pts with stage I, 2, and 3 NSCLC, respectively. ALK+ pts were distributed across all stages: 10 (34%) stage I, 6 (21%) stage 2, and 13 (45%) stage 3 NSCLCs. Chemotherapy was administered to 14 ALK+ ( 0% stage 1, 67% stage 2, 77% stage 3), 45 EGFR+ (3% stage 1, 44% stage 2, 81% stage 3), and 96 KRAS+ pts (4% stage 1, 56% stage 2, 71% stage 3), respectively, for early-stage NSCLC. Thirteen (7%) stage 1 EGFR+ patients received adjuvant EGFR TKI. Although median RFS was not reached for EGFR+ pts, it was 24.3 months (95%CI 11.4 to 65.3) for ALK+ pts and 72.9 months (95%CI 59.7 to undefined) for KRAS+ pts. RFS for ALK+ NSCLC was significantly shorter than the other groups (HR 2.9, 95%CI: 1.75-4.89 vs. EGFR and HR 1.8, 95%CI: 1.12-2.93 vs. KRAS). When adjusted for stage, ALK+ NSCLC remained associated with worse RFS compared to EGFR+ NSCLC (HR 1.8, 95%CI: 1.09-3.12), but not when compared to KRAS+ NSCLC (HR 1.30, 95%CI: 0.79-2.12). Conclusions: Early stage ALK+ NSCLC is associated with shorter RFS than EGFR+ NSCLC. The propensity for relapse and the significant anti-tumor activity of ALK TKIs in pts with metastatic NSCLC suggest that enrollment of patients on trials of adjuvant ALK TKIs should be prioritized.

Major histocompatibility (MH) gene polymorphism has been used to type populations of humans, mice, and fish. Walleye ( Stizostedion vitreum) comprise an economically important fishery in Lake Erie, but whether those in the western basin form a single population or separate shoal- and river-breeding populations is not known. To develop MH gene markers for use in defining their population structure, we constructed a head kidney cDNA library from which five full-length class I heavy-chain clones were isolated and sequenced. Although they came in roughly three sizes, 1300, 1400, and over 2000 bp, the clones all exhibited a high degree of sequence similarity to each other and to known teleost MH class I cDNAs in the area encoding the extracellular domains, but showed dramatic differences in their transmembrane and cytoplasmic domains. One clone had an AG repeat that eliminated the hydrophobicity of the transmembrane domain, indicating that it may encode a secreted class I receptor. The other four clones encode three distinctly different cytoplasmic domains. The two clones that encode the same cytoplasmic domain resemble those of the known teleost MH class I sequences the most. Southern blotting indicated that there were four copies of the gene present in the walleye genome. Northern blotting showed that class I MH genes are expressed in most tissues and mRNAs of all three size classes can be detected. A preliminary survey of the polymorphism of these genes indicates that they will provide useful markers for differentiating fish stocks.

In the present study, a novel plant transformation system for Doritaenopsis and Phalaenopsis has been developed. The pollen-mediated activation tagging system was established by artificial pollination. The pollens, co-cultured with Agrobacterium tumefaciens strain EHA105 harbouring an activation tagging vector (pTAG-8), were used for pollination. In order to optimize the transformation efficiency, several factors (concentration of A. tumefaciens, concentration of acetosyringone during co-cultivation and the duration of co-cultivation) known to influence Agrobacterium-mediated DNA transfer were examined. A concentration of 0.5-1 x 10(8) CFU/ml for A. tumefaciens, 0.1 mM acetosyringone, and 6 hrs of co-culture period were found to be the optimal condition for high transformation efficiency. Integration of T-DNA into the genome of putative transgenic plants was confirmed by PCR and DNA blot analyses. Single copy of the transgene was observed in all transgenic plants analyzed. Most of the transgenic plants had a morphologically normal phenotype and the overall capsule formation efficiency was similar to control plant. Our results showed a new approach of genetic transformation in orchids and this method can be employed for genetic improvement of the orchids.

In the present study, a novel plant transformation system for Doritaenopsis and Phalaenopsis has been developed. The pollen-mediated activation tagging system was established by artificial pollination. The pollens, co-cultured with Agrobacterium tumefaciens strain EHA105 harbouring an activation tagging vector (pTAG-8), were used for pollination. In order to optimize the transformation efficiency, several factors (concentration of A. tumefaciens , concentration of acetosyringone during co-cultivation and the duration of co-cultivation) known to influence Agrobacterium -mediated DNA transfer were examined. A concentration of 0.5-1 x 10 8 CFU/ml for A. tumefaciens, 0.1 mM acetosyringone, and 6 hrs of co-culture period were found to be the optimal condition for high transformation efficiency. Integration of T-DNA into the genome of putative transgenic plants was confirmed by PCR and DNA blot analyses. Single copy of the transgene was observed in all transgenic plants analyzed. Most of the transgenic plants had a morphologically normal phenotype and the overall capsule formation efficiency was similar to control plant. Our results showed a new approach of genetic transformation in orchids and this method can be employed for genetic improvement of the orchids.

Background Bitter gourd (Momordica charantia L.) is a common tropical vegetable that has been used in traditional or folk medicine to treat diabetes. Wild bitter gourd (WBG) ameliorated metabolic syndrome (MetS) in animal models. We aimed to preliminarily evaluate the effect of WBG supplementation on MetS in Taiwanese adults. Methods A preliminary open-label uncontrolled supplementation trial was conducted in eligible fulfilled the diagnosis of MetS from May 2008 to April 2009. A total of 42 eligible (21 men and 21 women) with a mean age of 45.7 ± 11.4 years (23 to 63 years) were supplemented with 4.8 gram lyophilized WBG powder in capsules daily for three months and were checked for MetS at enrollment and follow-up monthly. After supplementation was ceased, the participants were continually checked for MetS monthly over an additional three-month period. MetS incidence rate were analyzed using repeated-measures generalized linear mixed models according to the intention-to-treat principle. Results After adjusting for sex and age, the MetS incidence rate (standard error, p value) decreased by 7.1% (3.7%, 0.920), 9.5% (4.3%, 0.451), 19.0% (5.7%, 0.021), 16.7% (5.4%, 0.047), 11.9% (4.7%, 0.229) and 11.9% (4.7%, 0.229) at visit 2, 3, 4, 5, 6, and 7 compared to that at baseline (visit 1), respectively. The decrease in incidence rate was highest at the end of the three-month supplementation period and it was significantly different from that at baseline (p = 0.021). The difference remained significant at end of the 4th month (one month after the cessation of supplementation) (p = 0.047) but the effect diminished at the 5th and 6th months after baseline. The waist circumference also significantly decreased after the supplementation (p < 0.05). The WBG supplementation was generally well-tolerated. Conclusion This is the first report to show that WBG improved MetS in human which provides a firm base for further randomized controlled trials to evaluate the efficacy of WBG supplementation.

There has been a renewed interest in the use of herbal medicines throughout the world due to toxicities and health hazards associated with synthetic drugs and antibiotics. The World Health Organization estimates that up to 80% of people still rely mainly on traditional remedies such as herbs for their medicine, resulting in the increasing demand for medicinal plants. About 85% of traditional medicines involve the use of plant extracts. However, a large number of medicinal plants are needed to be investigated for their possible pharmaceutical value. Many pharmaceutical industries harness wildly growing plant populations for the supply of raw materials for extraction of medicinally important compounds. Many of the medicinal plants are severely threatened owing to illicit and indiscriminate collection and destruction of natural habitat. Advanced biotechnological methods of culturing plant cells and tissue provide an alternative means for rapid propagation and conservation of rare and endangered and/or commercially important medicinal plants. This chapter reviews the work carried out by our group in Taiwan on in vitro propagation of Pinellia ternata , Momordica charantia , Gentiana scabra var Bunge and Taraxacum formosanum and production of secondary metabolites from the callus of Stephania tetrandra , hairy root of Salvia miltiorrhiza and cell suspension culture of Gentiana davidii and Dioscorea doryophora .

We have developed analytical methods to measure the biological functions of pVGI.1(VEGF2), a naked plasmid DNA product containing vascular endothelial growth factor 2 used in clinical trials for coronary artery diseases (CAD) and peripheral artery diseases (PAD). After being injected into muscles, vascular endothelial growth factor 2 (VEGF-2), presumably expressed in muscle tissues, binds to the endothelial cell receptors VEGFR2 or VEGFR3, triggering the downstream responses including cell proliferation and vascularization. As it is important to make sure clinical material is biological active, we developed a quantitative assay first to measure the receptor binding activity of the pVGI.1(VEGF2) gene product expressed by the transfected host cells, and then a qualitative assay to confirm the cell proliferation promoting activity of the expressed protein. In both assays the signals were plotted directly against input DNA concentrations used to transfect the host cells. We confirmed specificity for both assays. In addition, we demonstrated acceptable levels of spike recovery (86.7-116%), precision (largest relative standard deviation (RSD)=19.3%), linearity and range (60-140% relative potency, 15 - 35 µg/mL) for the quantitative assay. We intend to use the potency assays for routine lot release and stability studies.

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 x Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.