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Different subtypes of non-small cell lung cancer (NSCLC) are associated with different patterns of metastatic spread. Anatomic location of lesions in the chest may influence patterns of cancer growth and the shrinkage to therapy. Consequently, lesion location could affect apparent response rates per RECIST. We sought to explore this and develop, as needed, treatment response assessments less affected by the location.Cases of advanced oncogene-addicted NSCLC (EGFR, ALK, and ROS1) with pre- and on-therapy imaging during initial targeted therapy were identified. Lesions located in the lung parenchyma, pleural space or intra-thoracic lymph nodes were identified and analyzed separately from each other by RECIST 1.1 (unidimensional measurements) and by a novel MAX methodology (bidimensional measurements) which takes the axis with the greatest absolute percentage change on therapy in each location as the representative measurement.Three hundred three patients with 446 unidimensional measured lesions were included for RECIST analysis. Two hundred forty nine patients with 386 bidimensional measured lesions were included for MAX analysis, as well as the analysis comparing RECIST and MAX. Intrathoracic location significantly impacted percentage shrinkage and the response rate per RECIST. The response rates for pleural, intra-parenchymal and nodal lesions were 34.1%, 49.6%, and 68.3%, respectively (P = .0002). The MAX methodology both increased the apparent treatment effect and made it consistent between intrathoracic locations. For pleural, parenchymal and nodal lesions, the MAX calculated response rate were 83.7%, 72.2%, and 75.4%, respectively (P-value = .24).Intrathoracic lesion location affects RECIST-based treatment effectiveness estimations. The MAX methodology neutralizes location effect when examining impact of treatment and should be explored further.

Purpose: A phase Ib/II clinical trial was conducted to evaluate the safety and efficacy of the combination of all-trans retinoic acid (ATRA) with pembrolizumab in patients with stage IV melanoma. Patients and Methods: Anti–PD-1 naïve patients with stage IV melanoma were treated with pembrolizumab plus supplemental ATRA for three days surrounding each of the first four pembrolizumab infusions. The primary objective was to establish the MTD and recommended phase II dose (RP2D) of the combination. The secondary objectives were to describe the safety and toxicity of the combined treatment and to assess antitumor activity in terms of (i) the reduction in circulating myeloid-derived suppressor cell (MDSC) frequency and (ii) progression-free survival (PFS). Results: Twenty-four patients were enrolled, 46% diagnosed with M1a and 29% with M1c stage disease at enrollment. All patients had an ECOG status ≤1, and 75% had received no prior therapies. The combination was well tolerated, with the most common ATRA-related adverse events being headache, fatigue, and nausea. The RP2D was established at 150 mg/m2 ATRA + 200 mg Q3W pembrolizumab. Median PFS was 20.3 months, and the overall response rate was 71%, with 50% of patients experiencing a complete response, and the 1-year overall survival was 80%. The combination effectively lowered the frequency of circulating MDSCs. Conclusions: With a favorable tolerability and high response rate, this combination is a promising frontline treatment strategy for advanced melanoma. Targeting MDSCs remains an attractive mechanism to enhance the efficacy of immunotherapies, and this combination merits further investigation. See related commentary by Olson and Luke, p. 1167

Defining clinically relevant MET amplification levels in non-small cell lung cancer (NSCLC) remains challenging. We hypothesize that oncogene overlap and MET amplicon size decline with increase in MET plasma copy number (pCN), thus enriching for MET-dependent states.We interrogated cell-free DNA NGS results of 16,782 patients with newly diagnosed advanced NSCLC to identify those with MET amplification as reported using Guardant360. Co-occurring genomic mutations and copy number alterations within each sample were evaluated. An exploratory method of adjusting for tumor fraction was also performed and amplicon size for MET was analyzed when available.MET amplification was detected in 207 (1.2%) of samples. pCN ranged from 2.1 to 52.9. Of these, 43 (20.8%) had an overlapping oncogenic driver, including 23 (11.1%) METex14 skipping or other MET mutations. The degree of (non-MET) oncogene overlap decreased with increases in pCN. Patients with MET pCN ≥ 2.7 had lower rates of overlapping drivers compared to those with MET pCN2.7 (6.1% vs. 16.3%, P = .033). None of the 7 patients with pCN6.7 had an overlapping driver. After adjusting for tumor fraction, adjusted pCN (ApCN) was also lower for those with overlapping drivers than those without (median ApCN 4.9 vs. 7.3, P =.024). There was an inverse relationship between amplicon size and pCN.We propose that a high MET pCN and/or ApCN, together with the absence of overlapping oncogenic drivers and small MET amplicon size, will enrich for patients most likely to derive benefit from MET targeted therapy.

Background Ependymoma (EPN) posterior fossa group A (PFA) has the highest rate of recurrence and the worst prognosis of all EPN molecular groups. At relapse, it is typically incurable even with re-resection and re-irradiation. The biology of recurrent PFA remains largely unknown, however, the increasing use of surgery at first recurrence has now provided access to clinical samples to facilitate a better understanding of this. Methods In this large longitudinal international multicenter study, we examined matched samples of primary and recurrent disease from PFA patients to investigate the biology of recurrence. Results DNA methylome derived copy number variants (CNVs) revealed large scale chromosome gains and losses at recurrence. CNV changes were dominated by chromosome 1q gain and/or 6q loss, both previously identified as high-risk factors in PFA, which were present in 23% at presentation but increased to 61% at 1 st recurrence. Multivariate survival analyses of this cohort showed that cases with 1q gain or 6q loss at 1 st recurrence were significantly more likely to recur again. Predisposition to 1q+/6q- CNV changes at recurrence correlated with hypomethylation of heterochromatin-associated DNA at presentation. Cellular and molecular analyses revealed that 1q+/6q- PFA had significantly higher proportions of proliferative neuroepithelial undifferentiated progenitors and decreased differentiated neoplastic subpopulations. Conclusions This study provides clinically and preclinically-actionable insights into the biology of PFA recurrence. The hypomethylation predisposition signature in PFA is a potential risk-classifier for trial stratification. We show that the cellular heterogeneity of PFAs evolves largely because of genetic evolution of neoplastic cells.

This clinical trial combined fulvestrant with the anti-androgen enzalutamide in women with metastatic ER+/HER2− breast cancer (BC). Eligible patients were women with ECOG 0–2, ER+/HER2− measurable or evaluable metastatic BC. Prior fulvestrant was allowed. Fulvestrant was administered at 500 mg IM on days 1, 15, 29, and every 4 weeks thereafter. Enzalutamide was given at 160 mg po daily. Fresh tumor biopsies were required at study entry and after 4 weeks of treatment. The primary efficacy endpoint of the trial was the clinical benefit rate at 24 weeks (CBR24). The median age was 61 years (46–87); PS 1 (0–1); median of 4 prior non-hormonal and 3 prior hormonal therapies for metastatic disease. Twelve had prior fulvestrant, and 91% had visceral disease. CBR24 was 25% (7/28 evaluable). Median progression-free survival (PFS) was 8 weeks (95% CI: 2–52). Adverse events were as expected for hormonal therapy. Significant (p PIK3CA and/or PTEN mutations. Baseline levels of phospho-proteins in the mTOR pathway were more highly expressed in biopsies of patients with shorter PFS. Fulvestrant plus enzalutamide had manageable side effects. The primary endpoint of CBR24 was 25% in heavily pretreated metastatic ER+/HER2− BC. Short PFS was associated with activation of the mTOR pathway, and PIK3CA and/or PTEN mutations were associated with an increased hazard of progression. Thus, a combination of fulvestrant or other SERD plus AKT/PI3K/mTOR inhibitor with or without AR inhibition warrants investigation in second-line endocrine therapy of metastatic ER+ BC.

Introduction Historical reservations regarding radiosurgery (SRS) for small-cell-lung-cancer (SCLC) brain metastases (BrM) include concerns for short-interval/diffuse CNS-progression, poor prognoses, and increased neurological mortality specific to SCLC histology. We compared SRS outcomes for SCLC and non-small-cell-lung-cancer (NSCLC) where SRS is well established. Methods Multicenter first-line SRS outcomes for SCLC and NSCLC from 2000-2022 were retrospectively collected (N=892-SCLC/N=4,785-NSCLC). Data from the prospective JLGK0901 SRS trial were analyzed as a comparison cohort (N=98-SCLC/N=794-NSCLC). OS and CNS-progression were analyzed using Cox-Proportional-Hazard and Fine-Gray models, respectively, with multivariable (MV) adjustment (including age/sex/performance-status/year/extracranial disease/BrM-number/BrM-volume). Mutation-stratified analyses were performed in propensity score-matched (PSM) retrospective cohorts of EGFR/ALK-positive-NSCLC, mutation-negative-NSCLC, and SCLC. Results OS was superior with NSCLC over SCLC in the retrospective dataset (median-OS, 10.5 vs 8.6 months, MV-p Conclusion After SRS, SCLC was associated with shorter OS compared to NSCLC. CNS progression occurred earlier in SCLC overall but was similar in patients matched on baseline characteristics. Neurological mortality, lesions at CNS-progression, and leptomeningeal-progression were comparable. These findings may better inform clinical decision-making for SCLC patients.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is an immune checkpoint expressed in regulatory T (Treg) cells and activated T lymphocytes. Despite its potential as a treatment strategy for melanoma, CTLA-4 inhibition has limited efficacy. Using data from The Cancer Genome Atlas (TCGA) melanoma database and another dataset, we found that decreased CTLA4 mRNA was associated with a poorer prognosis in metastatic melanoma. To investigate further, we measured blood CTLA4 mRNA in 273 whole-blood samples from an Australian cohort and found that it was lower in metastatic melanoma than in healthy controls and associated with worse patient survival. We confirmed these findings using Cox proportional hazards model analysis and another cohort from the US. Fractionated blood analysis revealed that Treg cells were responsible for the downregulated CTLA4 in metastatic melanoma patients, which was confirmed by further analysis of published data showing downregulated CTLA-4 surface protein expression in Treg cells of metastatic melanoma compared to healthy donors. Mechanistically, we found that secretomes from human metastatic melanoma cells downregulate CTLA4 mRNA at the post-transcriptional level through miR-155 while upregulating FOXP3 expression in human Treg cells. Functionally, we demonstrated that CTLA4 expression inhibits the proliferation and suppressive function of human Treg cells. Finally, miR-155 was found to be upregulated in Treg cells from metastatic melanoma patients compared to healthy donors. Our study provides new insights into the underlying mechanisms of reduced CTLA4 expression observed in melanoma patients, demonstrating that post-transcriptional silencing of CTLA4 by miRNA-155 in Treg cells may play a critical role. Since CTLA-4 expression is downregulated in non-responder melanoma patients to anti-PD-1 immunotherapy, targeting miRNA-155 or other factors involved in regulating CTLA4 expression in Treg cells without affecting T cells could be a potential strategy to improve the efficacy of immunotherapy in melanoma. Further research is needed to understand the molecular mechanisms regulating CTLA4 expression in Treg cells and identify potential therapeutic targets for enhancing immune-based therapies.

Representativeness of study participants

Figure legend Supplemental Figure 1

Bronchial dysplasia (BD), a presumed precursor of pulmonary squamous cell carcinoma (SCC), rarely progresses to invasive cancer. A high-risk cohort at the University of Colorado provided an opportunity to directly sample airway epithelium at mapped sites on successive bronchoscopies. We have hypothesized that persistent dysplastic lesions showing a similar or higher level of dysplasia on follow-up biopsy, are associated with increased risk for the development of SCC. Endoscopic biopsies from 188 high-risk subjects were histologically classified according to the current WHO classification for BD using a numeric histology score ranging from 1 to 8 representing normal bronchial mucosa through invasive lung cancer. Differences in follow-up histology scores were compared between sites classified by clinical, histologic, and immunohistochemical variables. Subjects with a higher frequency of sites that persist or progress to high-grade dysplasia (≥37.5% persist/progress, N = 35 versus N = 114) show a significant association with development of incident invasive SCC (adjusted HR, 7.84; 95% confidence interval, 1.56–39.39), and those with incident lung SCC have adjusted mean follow-up histology scores 1.55 U higher than in subjects without lung cancer. Current smoking, elevated Ki67 growth fraction, histologic features of angiogenic squamous dysplasia (ASD) and higher histology score in baseline biopsies are significantly associated with increased follow-up histology scores. These results show that persistent BD is associated with the development of invasive SCC. Furthermore, increased expression of Ki67, the presence of angiogenic change and degree of baseline atypia are associated with persistence of BD. Cancer Prev Res; 9(1); 96–104. ©2015 AACR.

Supplementary figure 1. (A) Changes in other circulating cytokines. Colored box denotes the time when the patients were being treated with ATRA. (B) example gating strategy for CD8+ T cell activation.

Resistance to immunotherapy is a significant challenge, and the scarcity of human models hinders the identification of the underlying mechanisms. To address this limitation, we constructed an autologous humanized mouse (aHM) model with hematopoietic stem and progenitor cells (HSPC) and tumors from 2 melanoma patients progressing to immunotherapy. Unlike mismatched humanized mouse (mHM) models, generated from cord blood–derived HSPCs and tumors from different donors, the aHM recapitulates a patient-specific tumor microenvironment (TME). When patient tumors were implanted on aHM, mHM, and NOD/SCID/IL2rg−/− (NSG) cohorts, tumors appeared earlier and grew faster on NSG and mHM cohorts. We observed that immune cells differentiating in the aHM were relatively more capable of circulating peripherally, invading into tumors and interacting with the TME. A heterologous, human leukocyte antigen (HLA-A) matched cohort also yielded slower growing tumors than non–HLA-matched mHM, indicating that a less permissive immune environment inhibits tumor progression. When the aHM, mHM, and NSG cohorts were treated with immunotherapies mirroring what the originating patients received, tumor growth in the aHM accelerated, similar to the progression observed in the patients. This rapid growth was associated with decreased immune cell infiltration, reduced interferon gamma (IFNγ)–related gene expression, and a reduction in STAT3 phosphorylation, events that were replicated in vitro using tumor-derived cell lines.Implications:Engrafted adult HSPCs give rise to more tumor infiltrative immune cells, increased HLA matching leads to slower tumor initiation and growth, and continuing immunotherapy past progression can paradoxically lead to increased growth.

Purpose:A phase Ib/II clinical trial was conducted to evaluate the safety and efficacy of the combination of all-trans retinoic acid (ATRA) with pembrolizumab in patients with stage IV melanoma.Patients and Methods:Anti–PD-1 naïve patients with stage IV melanoma were treated with pembrolizumab plus supplemental ATRA for three days surrounding each of the first four pembrolizumab infusions. The primary objective was to establish the MTD and recommended phase II dose (RP2D) of the combination. The secondary objectives were to describe the safety and toxicity of the combined treatment and to assess antitumor activity in terms of (i) the reduction in circulating myeloid-derived suppressor cell (MDSC) frequency and (ii) progression-free survival (PFS).Results:Twenty-four patients were enrolled, 46% diagnosed with M1a and 29% with M1c stage disease at enrollment. All patients had an ECOG status ≤1, and 75% had received no prior therapies. The combination was well tolerated, with the most common ATRA-related adverse events being headache, fatigue, and nausea. The RP2D was established at 150 mg/m2 ATRA + 200 mg Q3W pembrolizumab. Median PFS was 20.3 months, and the overall response rate was 71%, with 50% of patients experiencing a complete response, and the 1-year overall survival was 80%. The combination effectively lowered the frequency of circulating MDSCs.Conclusions:With a favorable tolerability and high response rate, this combination is a promising frontline treatment strategy for advanced melanoma. Targeting MDSCs remains an attractive mechanism to enhance the efficacy of immunotherapies, and this combination merits further investigation.See related commentary by Olson and Luke, p. 1167

Characteristics of patients experiencing clinical benefit

Table S1. Expanded HSPC engraftment into mouse cohorts Table S2. Human immune cell presence in HM tissues Table S3a. CUHM003 mutations present at diagnosis b. Additional CUHM003 mutations present at progression c. CUHM005 mutations present at diagnosis d. Additional CUHM005 mutations present at progression Table S4. Enrichment of immunity-related genes in patient and HM tumors Table S5a. Genes expressed only in the CUHM003 patient and mHM model b. Genes expressed only in the CUHM003 patient and aHM model c. Genes expressed only in the CUHM005 patient and mHM model d. Genes expressed only in the CUHM005 patient and aHM model Table S6. Changes in cancer pathway GSEA expression in patients, their NSG, mHM, and aHM models Table S7. Changes in GSEA hallmark expression between mouse models Table 8a. Genes differentially expressed in CUHM003 after PD1 treatment b. Genes differentially expressed in CUHM005 after PD1 treatment

Supplemental figure S2. Univariable analysis comparing frequency of SCC in cases grouped by highest baseline (BL) histology score (HS) or mean baseline histology score using a cutoff of HS > 5 (moderate dysplasia).

Supplementary figure 2. Changes in other clinical hematologic measures. Colored box denotes the time when the patients were being treated with ATRA. * Denotes p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

SUPPLEMENTAL TABLE S1. BIOPSY AND CLINCAL CHARACTERISTICS. SUPPLEMENTAL TABLE S2. BASELINE HISTOLOGY AND OUTCOME.

Supplemental figure S1. Univariable analysis comparing frequency of SCC using four different definitions of persistent bronchial dysplasia (PBD; RBD = regressive BD) for sites with any baseline diagnosis (HS 1-7).

Aurora A (A) and Aurora B (B) kinase gene expression in SCLC cell lines.

Supplemental figure S3. Sites that show HGD in follow-up biopsies are more frequent in subjects that have prevalent (Prev) or develop incident (Inc) squamous cell carcinoma (SCC) as compared to those without cancer (No CA). *p < 0.05.

All adverse events experienced by patients, including those unrelated to treatment

Trial inclusion/exclusion criteria

Establishing recommended phase II dose (RP2D) and dose limiting toxicities (DLTs)

BACKGROUND: Women diagnosed with breast cancer prior to age 45 years (

Hydrogen transfer (H-transfer) is an important elementary reaction in chemistry and bioscience. It is often facilitated by the hydrogen bonds between the H-donor and acceptor. Here, at room temperature and high pressure, we found that solid 2-butyne experienced a concerted two-in-two-out intermolecular CH···π H-transfer, which initiated the subsequent polymerization. Such double H-transfer goes through an aromatic Hückel six-membered ring intermediate state via intermolecular CH···π interactions enhanced by external pressure. Our work shows that H-transfer can occur via the CH···π route in appropriate conformations under high pressure, which gives important insights into the H-transfer in solid-state hydrocarbons.

PURPOSE: Radiation pneumonitis remains a major limitation in the radiation therapy treatment of patients with lung cancer. Functional avoidance radiation therapy uses functional imaging to reduce pulmonary toxic effects by designing radiation therapy plans that reduce doses to functional regions of the lung. Lung functional imaging has been developed that uses 4-dimensional computed tomography (4DCT) imaging to calculate 4DCT-based lung ventilation (4DCT-ventilation). A phase 2 multicenter study was initiated to evaluate 4DCT-ventilation functional avoidance radiation therapy. The study hypothesis was that functional avoidance radiation therapy could reduce the rate of grade ≥2 radiation pneumonitis to 12% compared with a 25% historical rate, with the trial being positive if ≤16.4% of patients experienced grade ≥2 pneumonitis. METHODS AND MATERIALS: Lung cancer patients receiving curative-intent radiation therapy (prescription doses of 45–75 Gy) and chemotherapy were accrued. Patient 4DCT scans were used to generate 4DCT-ventilation images. The 4DCT-ventilation images were used to generate functional avoidance plans that reduced doses to functional portions of the lung while delivering the prescribed tumor dose. Pneumonitis was evaluated by a clinician at 3, 6, and 12 months after radiation therapy. RESULTS: Sixty-seven evaluable patients were accrued between April 2015 and December 2019. The median prescription dose was 60 Gy (range, 45–66 Gy) delivered in 30 fractions (range, 15–33 fractions). The average reduction in the functional volume of lung receiving ≥20 Gy with functional avoidance was 3.5% (range, 0%–12.8%). The median follow-up was 312 days. The rate of grade ≥2 radiation pneumonitis was 10 of 67 patients (14.9%; 95% upper CI, 24.0%), meeting the phase 2 criteria. CONCLUSIONS: 4DCT-ventilation offers an imaging modality that is convenient and provides functional imaging without an extra procedure necessary. This first report of a multicenter study of 4DCT-ventilation functional avoidance radiation therapy provided data showing that the trial met phase 2 criteria and that evaluation in a phase 3 study is warranted.

Background: The clinical implications of the androgen receptor (AR), particularly in the context of aromatase inhibitor (AI) refractory metastatic breast cancer (MBC), are unclear. While AR is associated with more indolent primary tumors, in the absence of low estradiol/blocked ER, AR can exert a pro-survival signal. Thus, in a phase II trial of Fulvestrant (Fulv) plus Enzalutamide (Enza) in ER+/Her2- MBC (NCT02953860) we analyzed serial biopsies pre- and post-treatment. Methods: Eligible patients were women with ECOG 0-2, ER+/Her2- MBC. Fulv at 500 mg IM days 1, 15, 29 and every 4 weeks thereafter and Enzaat 160 mg po daily on a continual basis were administered. Biopsies were required at study entry and at ~4 weeks on therapy. The clinical benefit rate at 24 weeks (CBR24) was the primary endpoint for efficacy. We performed mutational analysis using a modified Archer VariantPlex Solid Tumor Assay to detect mutations in ESR1 exon 8 and 67 other gene hotspots. We examined estrogen, progesterone, androgen and glucocorticoid receptor protein by IHC and multiplex for immune cells and PD-L1. Frozen cores were utilized to perform reverse phase protein array (RPPA) based protein pathway activation analysis of over 150 proteins from LCM-enriched tumor in baseline and post-treatment metastatic biopsies. Comparisons of Responders (progression free survival (PFS) equal to or longer than 24 weeks) and Non-Responders were performed using moderated t-tests on log2 transformed data. Results: 32 patients were eligible, median age was 61 years (46-87), and 90.6% had visceral disease with an average of 4 prior non-hormonal therapies and 3 prior hormonal agents (including 37.5% with prior Fulv). PFS >24 weeks was observed in ~22% of patients treated with Fulv plus Enza, including 42% of those who had prior Fulv. When stratified by both AR and ER protein levels, median time to progression was 59 days (95% CI: 55 to Inf) when both targets were high (greater than or equal to 10%), but only 14 days (95% CI: 13 to Inf) when both were less than 10%. Metastases with ESR1 mutations in the ligand binding domain had significantly higher levels of ER and PR protein than those with wild type ESR1 (p24 weeks was observed in 22% of patients treated with Fulv plus Enza, including 42% who had prior Fulv treatment, suggesting contribution of the anti-androgen. Response was significantly better when metastases were >10% for ER and AR. Poor response to Fulv plus Enza was significantly associated with mTOR pathway activation and patients with PIK3CA and or PTEN mutated metastases had a significantly shorter PFS. Mutation status also affected hormone receptor expression and immune infiltrates. The increase in PD-L1 protein following treatment with Fulv plus Enza warrants further pre-clinical investigation into whether the addition of anti-androgen can enhance efficacy of checkpoint inhibitor therapy in ER+ metastatic disease resistant to standard endocrine therapy approaches. Citation Format: Jennifer Richer, Nicole Spoelstra, Alyse Winchester, Julia Wulfkuhlue, Rosa Gallagher, Sharon Sams, Gregory Vidal, Peter Kabos, Jennifer Diamond, Elena Shagisultanova, Anosheh Afghahi, Jose Mayordomo, Tessa McSpadded, Gloria Crawford, Virginia Borges, Dexiang Gao, Emanuel Petricoin, Anthony Elias. Response of persistent metastatic ER+/Her2- breast cancer treated with fulvestrant plus enzalutamide [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-17-01.

XLSX file - 66KB, Table S1. Compiled and tabulated ponatinib IC50 values, FGF and FGFR expression, FGFR1 gene copy number and characteristics of the 58 lung cancer cell lines.

PDF file - 786KB, Figure S1. FGFR1 immunoblots of extracts from lung cancer cell lines representative of the 58 cell lines employed in the studies. Figure S2. Images and quantification of clonogenic growth assays in selected lung cancer cell lines following shRNA-mediated FGFR1 silencing. Figure S3. Dose response curve for inhibition of Colo699 cell growth by the FGF ligand trap, FP-1039. Figure S4. Venn diagram of the overlap of FGFR1, FGF2 and FGF9 expression in ponatinib-sensitive lung cancer cell lines and splicing status of FGFR2 in H1581 and H1048 cells. Figure S5. Graph of ROC analysis of FGFR1 GCN and mRNA expression in a TMA comprised of 21 lung cancer cell lines. Figure S6. Kaplan-Meier analysis of overall survival of lung cancer patients with increased FGFR1 GCN or high mRNA. Figure S7. Venn diagram showing overlap of FGF2 and FGF9 expression with FGFR1 mRNA positivity in primary lung squamous cell carcinomas and adenocarcinomas analyzed by the Cancer Genome Atlas.

XLSX file - 106KB, Table S5. Tabulated FGFR1, FGF2 and FGF9 mRNA expression values, FGFR1 CNV values and mutation status of TCGA lung adenocarcinomas and squamous cell carcinomas.

PDF file - 90KB, Table S2. Tabulated expression values for selected FGFs in lung cancer cell lines from the Cancer Cell Line Encyclopedia. Table S3. Tabulated FGFR1 gene copy number values determined by SISH and associated patient data for the surgical lung cancer cohort. Table S4. Tabulated FGFR1 mRNA expression values determined by ISH and associated patient data for the surgical lung cancer cohort.

PDF file - 191K, Western blotting for phosphorylated (Y1068) EGFR, total EGFR, pERK and total ERK in NSCLC cells treated with DMSO, gefitinib and/or Wnt pathway inhibitors, qPCR analyses of Axin-2 mRNA levels in cells in cells treated with canonical Wnt pathway inhibitors, western blotting for activated beta-catenin in stable cell lines, and determination of the ability of activated beta-catenin expression to mitigate the impact of gefitinib and/or XAV939 treatment in HCC4006 NSCLC cells

PDF file - 222K, Demonstration that beta-catenin or TCF-4 knockdown results in enhanced sensitivity of NSCLC cells to gefitinib treatment, that beta-catenin or TCF-4 knockdowns reduce the expression of the corresponding protein, that there is minimal correlation between the expression of canonical Wnt target genes and gefitinib sensitivity for a panel of NSCLC cell lines, that there is no significant correlation of SLuGs with gefitinib induced genes, and that SLuGs are not enriched in lung cancer specific genes

PDF file - 105K, Graphical representation of shRNA counts obtained by deep sequencing in the screen for synthetic lethality with gefitinib in H322C and HCC4006 NSCLC cells

XLS file - 683K, The table contains all screening results from the shRNA screen, including results for each of the two cell lines (H322C and HCC4006) and overlapping hits (SLuG2s). The shRNA sequences, the frequency that these sequences were detected in the different replicates, and the associated significance are provided

PDF file - 235K, Validation of shRNA-mediated knockdown of tankyrase-1 expression, demonstration of enhanced NSCLC cell killing by combined inhibition of tankyrase and EGFR in multiple NSCLC cell lines, and specificity of this synergistic cell killing for NSCLC cells with EGFR activation

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XLS file - 34K, Lists of primers and probes for real-time RT-PCR, shRNA sequences and antibodies are provided

PDF file - 240K, Demonstration that CK1alpha or CK2alpha knockdown results in enhanced sensitivity of NSCLC cells to gefitinib treatment, that these knockdowns reduce the expression of the corresponding proteins, that treatment with the CK1� activator pyrvinium pamoate acts synergistically with gefitinib to eliminate NSCLC cells, and specificity of this synergistic cell killing for NSCLC cells following EGFR inhibition

Lung cancer is the leading cause of death worldwide. Adenocarcinomas, the most common histologic subtype of non-small cell lung cancer (NSCLC), are frequently associated with activating mutations in the epidermal growth factor receptor (EGFR) gene. Although these patients often respond clinically to the EGFR tyrosine kinase inhibitors erlotinib and gefitinib, relapse inevitably occurs, suggesting the development of escape mechanisms that promote cell survival. Using a loss-of-function, whole genome short hairpin RNA (shRNA) screen, we identified that the canonical Wnt pathway contributes to the maintenance of NSCLC cells during EGFR inhibition, particularly the poly-ADP-ribosylating enzymes tankyrase 1 and 2 that positively regulate canonical Wnt signaling. Inhibition of tankyrase and various other components of the Wnt pathway with shRNAs or small molecules significantly increased the efficacy of EGFR inhibitors both in vitro and in vivo. Our findings therefore reveal a critical role for tankyrase and the canonical Wnt pathway in maintaining lung cancer cells during EGFR inhibition. Targeting the Wnt-tankyrase-β-catenin pathway together with EGFR inhibition may improve clinical outcome in patients with NSCLC. Cancer Res; 72(16); 4154–64. ©2012 AACR.

PDF file - 76K, Second xenograft experiment utilizing a different shRNA for TNKS1

Continuous long-term eddy covariance (EC) measurements of CO2 fluxes (NEE) in a variety of terrestrial ecosystems are critical for investigating the impacts of climate change on ecosystem carbon cycling. However, due to a number of issues, approximately 30–60% of annual flux data obtained at EC flux sites around the world are reported as gaps. Given that the annual total NEE is mostly determined by variations in the NEE data with time scales longer than one day, we propose a novel framework to perform gap filling in NEE data based on machine learning (ML) and time series decomposition (TSD). The novel framework combines the advantages of ML models in predicting NEE with meteorological and environmental inputs and TSD methods in extracting the dominant varying trends in NEE time series. Using the NEE data from 25 AmeriFlux sites, the performance of the proposed framework is evaluated under four different artificial scenarios with gap lengths ranging in length from one hour to two months. The combined approach incorporating random forest and moving average (MA-RF) is observed to exhibit better performance than other approaches at filling NEE gaps in scenarios with different gap lengths. For the scenario with a gap length of seven days, the MA-RF improves the R2 by 34% and reduces the root mean square error (RMSE) by 55%, respectively, compared to a traditional RF-based model. The improved performance of MA-RF is most likely due to the reduction in data variability and complexity of the variations in the extracted low-frequency NEE data. Our results indicate that the proposed MA-RF framework can provide improved gap filling for NEE time series. Such improved continuous NEE data can enhance the accuracy of estimations regarding the ecosystem carbon budget.