bluuebunny/crossref_metadata_2025 huggingface dataset:
Estrogen receptor alpha (ESR1) gene amplification status and clinical outcome in tamoxifen-treated postmenopausal patients with endocrine-responsive early breast cancer: An analysis of the prospective ABCSG-6 trial.
Authors: Singer Christian F. , Holst Frederik , Steurer Stefan , Burandt E C , Samonigg Hellmut , Jakesz Raimund , Stoeger Herbert , Greil Richard , Sauter Guido , Filipits Martin , Simon Ronald , Gnant Michael ,   ABCSG
Published: March 2020

10501

Background: Estrogen receptor alpha (ERα) expression is a prognostic parameter in breast cancer and predicts response to endocrine therapy. One of the factors important for protein expression is amplification of its encoding gene ESR1. We have investigated the value of ESR1 amplification in predicting the long-term clinical outcome in tamoxifen-treated postmenopausal women with endocrine-responsive breast cancer. Methods: 394 patients who had been randomized into the tamoxifen-only arm of the prospectively designed endocrine ABCSG-06 trial and in whom FFPE tumor tissue was available were included in this analysis. Immunohistochemical ERα expression was evaluated both locally and centrally using the Allred score, while ESR1 gene amplification status was evaluated by FISH analysis using the ESR1/CEN6 ratio. Results: ESR1 copy number gains were detected in 187 of 394 (47%) tumor specimen and was associated with favorable clinical outcome. At a median follow-up of 10 years, women with intratumoral ESR1 copy number gains had a significantly longer distant recurrence-free survival (adjusted HR for relapse 0.48; 95% CI 0.28-0.83; p=0.009) and breast cancer-specific survival (adjusted HR for death 0.46; CI 0.46-0.71; p=0.006) when compared to women with normal ESR1 copy numbers. Immunohistochemical ERα protein expression, evaluated by Allred score, was significantly correlated with ESR1 copy number alterations (p<0.0001; Chi-Square test), but did itself not allow to discriminate between patients with poor and good prognosis. Conclusions: ESR1 amplification status is an independent and powerful predictor for long-term distant recurrence-free and breast cancer-specific survival in postmenopausal women with endocrine-responsive early-stage breast cancer who received 5 years of tamoxifen.
Next-generation RNA sequencing reveals transcriptomic changes after brief exposure to preoperative nab-paclitaxel, bevacizumab, and trastuzumab.
Authors: Varadan Vinay , Kamalakaran Sitharthan , Janevski Angel , Banerjee Nila , Lezon-Geyda Kimberly , Bossuyt Veerle , Flowers-Poole Dava , Sinclair Natalie Faye , Abu-Khalaf Maysa M. , Sikov William M. , Dimitrova Nevenka , Harris Lyndsay
Published: March 2020

10508

Background: Identification of differentially expressed transcripts after brief exposure to preoperative therapy can help determine likely response markers. We quantify and compare differential gene and isoform expression using RNA-seq on patient samples with 10 day exposure to one dose of trastuzumab, bevacizumab or nab-paclitaxel. Methods: We sequenced transcriptomes of 23 pairs of core biopsy RNA from breast cancers pre/post 10 day exposure to therapy. Paired-end sequencing was done on the Illumina GAII platform using amplified total RNA with 74bp read length, yielding data on transcript abundance for a total of 22,160 genes and 34,449 transcripts. Differential expression of transcripts between pre/post samples was estimated assuming Poisson-distributed read-counts, followed by multiple testing correction and enrichment analysis of 185 KEGG pathways. Results: PAM50-based clustering showed individual samples cluster together, demonstrating that tumor subtypes do not change over the 10-day treatment (SABCS 2011). We identified genes that were significantly differentially expressed (p<0.05; FDR<0.1) in at least 60% of samples within each therapy arm: 780 genes in trastuzumab, 302 in bevacizumab, and 176 in nab-paclitaxel. Surprisingly, only THAP11 and TINF2 were common amongst them. THAP11 is involved in stem cell maintenance and TINF2 is important for regulation of telomere length. Immune system and metabolism-related pathways were commonly affected (p<0.05) across all arms. The bevacizumab arm showed significant down-regulation of angiogenesis-associated genes: ESM1 and VEGFR2 in > 80% of samples. The nab-paclitaxel arm exhibited changes in TGF-beta signaling, Nod-like receptor and Wnt signaling. The trastuzumab arm exhibited consistent alteration of ErbB2 and mTOR pathways, with SOX11 and TOP2B downregulated in every sample. Conclusions: This is the first study to compare gene expression with brief exposure across therapies using RNA-seq technology. The unique aspects of transcriptional response to each treatment underscore the need for specific markers of therapeutic response to nab-paclitaxel, bevacizumab and trastuzumab.
Analysis of the intratumoral heterogeneity of PIK3CA mutant alleles in breast cancer (BC): Implications for the luminal (LUM) phenotype.
Authors: De Mattos-Arruda Leticia , Cortes Javier , Aura Claudia , Oliveira Mafalda , Navarro Alejandro , Caratu Ginevra , Sanchez-Pla Alex , Gregori Josep , Saura Cristina , Tabernero Josep , Seoane Joan , Vivancos Ana
Published: March 2020

10511

Background: The hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway may confer endocrine therapy resistance and is an attractive target for LUM patients (pts). However, PI3KCA mutations could be heterogeneously distributed within the tumor as different genetic clones and this could have therapeutic implications. Our aim was to assess the frequency of PIK3CA mutant alleles in different BC phenotypes. Methods: DNA was obtained from 75 consecutive BC FFPE samples and was profiled with the OncoCarta Panel v1.0 (Sequenom). Frequencies of mutant alleles (% mutant allele, mA) of PIK3CA mutations were extracted from the MassARRAY spectrum data. The viable tumor area (TA) was scored by H&E. Pts were stratified by BC phenotypes: ER+/HER2- (LUM); HER2+ (HER2); triple negative (TN). Results: 25.3% of pts had PIK3CA mutations. The mean PIK3CA mA (p=0.04) and mean TA (p=0.07) differed among phenotypes. LUM tumors demonstrated greater frequencies of PIK3CA mutation (38% vs.15%, p=0.05) and mean PIK3CA mA (31% vs.17%, p=0.01) than non-LUM.There was a significant linear correlation between mA and TAfor LUM tumors (r=0.91); when HER2 and TN tumors were also considered this relation was less pronounced (r=0.66). Overall, LUM pts, median age 55 (42-84), had significantly better clinical outcomes (p=0.00024), whilst analyses of prognosis did not differ among LUM pts (PIK3CA mutant vs. wild-type, p=0.68) nor mutant pts (LUM vs. non-LUM, p=0.36). Outcomes will be presented. To assess the intratumoral heterogeneity of PIK3CA mutations, we determined the mA/TA ratio. The mA/TA ratio should be 0.5 for a homogeneous distribution of the heterozygous PIK3CA mutation. The ratio of LUM patients was close to 0.5, while for non-LUM it was lower, suggesting a non-homogeneous distribution of PIK3CA mutant alleles in non-LUM tumors. Conclusions: Our analysis indicates that LUM tumors tend to be homogeneous regarding PIK3CA mutation as compared to HER2 and TN. This suggests that PIK3CA oncogenic activation could be an early hit for tumor initiation in LUM tumors, which could be more specifically targetable; thus, pts would derive greater benefit from PI3K-inhibitors plus hormonal therapy.
Clinical characteristics and chemotherapy options of triple-negative breast cancer: Role of classical CMF regimen.
Authors: Hur Min Hee , Kang Tae Ho , Im Ra Joo , Kim Se Won , Yoon Chan Seok , Ko Seung Sang , Lee Haekyung , Lee Ji Hyun , Kang Sung-Soo
Published: March 2020

1053

Background: Triple-negative breast cancer is a high risk breast cancer that lacks the benefit of specific targeted therapy. We investigated the clinicopathologic characteristics between triple-negative breast cancers (TNBC) and non-TNBC. And we also analyzed the effect of chemotherapy options such as classical CMF regimen, anthracycline-based, or taxane-based chemotherapy. Methods: A total of 826 invasive breast cancer patients were evaluated from March 2003 to December 2008. We investigated them retrospectively, who had the median follow-up for 88 months. We examined the differences between TNBC compared with non-TNBC in relation to the clinicopathologic parameters, chemotherapy regimen, overall survival (OS). Results: 156 (18.9%) cases among 826 patients were triple negative breast cancers. There were significantly positive associations with younger age (below 35 years), large tumor (>2cm), high stage, poorly differentiated nuclear grade, poorly histologic grade in TNBC. Positive lymph node, lymphovascular invasion were not significantly different between TNBC and non- TNBC. A total of 677 patients were treated with chemotherapy. In TNBC patients, 142 (93.4%) patients were treated with chemotherapy more than in 535 (61.4%) of non- TNBC patients. The chemotherapy in TNBC patients was composed of classical CMF (47.9%), anthracycline-based regimen (25.4%), taxane-based regimen (26.8%). 19 cases (12%) of TNBC experienced locoregional or systemic metastases. 48 (7.2%) patients of non- TNBC did local or systemic metastases. Patients with TNBC had worse 5-year OS than with non-TNT (95.7% vs 98.6%, p=0.01). Interestingly, patients treated with CMF regimen were better 5-year OS than with anthracycline-based, or taxane-based regimen in TNBC (100% vs 96.9% vs 89.2%, p=0.001). There was no survival difference among chemotherapy regimens in non-TNBC patients. Conclusions: Patients with TNBC have poor prognosis compared with non-TNBC. Classical CMF regimen for TNBC patients may be more effective than anthracycline-based or taxane-based regimens.
Profiling of circulating tumor cells isolated from 105 metastatic gastric cancer patients revealed HER2 overexpression/activation for potential use in clinical setting.
Authors: Hazra Saswati , Lee Jeeyun , Kim Phillip Sangwook , Kim Kyoung-Mee , Liu Limin , Webster Edvelyn , Fithian Andrew , Singh Sharat , Kang WonKi
Published: March 2020

10535

Background: Gastric cancer (GCA) is the second leading cause of cancer mortality in the world. Survival of patients with advanced GCA treated with chemotherapy remains low. New targeted therapies are urgently needed. There is mounting evidence of the role of HER2 overexpression in patients with GCA, and it has been highly correlated to poor outcomes with more aggressive disease. The ability to accurately determine HER2 status by testing circulating tumor cells (CTCs) may improve patient treatment by allowing ongoing assessment of HER2 status during treatment and/or identifying additional patients who could potentially benefit from HER2- targeted therapy. Methods: The Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) was utilized to determine the expression and activation (phosphorylation) levels of HER2 in CTCs isolated from blood specimens obtained from 105 metastatic GCA patients. Results: Utilizing the CEER platform, the levels of HER2 expression and phosphorylation were determined for CTCs isolated from metastatic GCA patients. Evaluable CTCs were found in 33% (35/105) of enrolled patients. Out of 35 patients, 7 patients (20%) have high HER2 over expression, 6 patients (17%) have moderate HER2 expression and 11 patients (31%) have HER2 activation (phospho positive) with no HER2 over-expression. Conclusions: When CTCs were present, the CEER assay identified varying levels of HER2 involvements in 68% of metastatic GCA patients. HER2 positive CTCs could serve as a prognostic and/or predictive marker in patients with advanced GCA and CTC-HER2 profile shifts can be utilized to monitor the treatment efficacy.
Biomarkers of response to Akt inhibitor MK-2206 in breast cancer.
Authors: Sangai Takafumi , Akcakanat Argun , Chen Huiqin , Tarco Emily , Wu Yun , Do Kim-Anh , Miller Todd W , Arteaga Carlos L. , Mills Gordon B. , Gonzalez-Angulo Ana M. , Meric-Bernstam Funda
Published: March 2020

1054

Background: Akt significantly contributes to cancer pathogenesis. PTEN, a negative regulator of PI3K/Akt signaling, is mutated or decreased, and PIK3CA is frequently mutated in multiple cancer lineages. We hypothesized that MK-2206, an allosteric Akt inhibitor, would inhibit Akt signaling thus blocking cancer cell growth, and PTEN and/or PIK3CA mutations may confer MK-2206 sensitivity in breast cancer. Methods: After determining sensitivity to MK-2206 in16 cell lines, the effect on Akt signaling was tested by reverse-phase protein array analysis and western blotting. The effect to cell cycle progression and cell death were analyzed by flow cytometry. Using RNA knockdown technique, the effect of PTEN, PIK3CA and Akt on MK2206 sensitivity was tested. Anti-tumor effect in vivo with or without paclitaxel was tested using PTEN-mutant ZR75-1 breast cancer xenografts. Results: MK-2206 inhibited Akt signaling and cell cycle progression, and increased apoptosis in a dose-dependent manner in breast cancer cell lines. Cell lines with PTEN or PIK3CA mutations were more sensitive to MK-2206 (P=0.0337), however, a number of lines with PTEN/PIK3CA mutations were MK-2206-resistant. Small interfering RNA (siRNA) knockdown of PTEN in breast cancer cells increased Akt phosphorylation concordant with increased MK-2206 sensitivity. Stable transfection of PIK3CA E545K or H1047R mutant plasmids into normal-like MCF10A breast cells enhanced MK-2206 sensitivity. Cell lines that were less sensitive to MK-2206 had lower ratios of Akt1/Akt2 and had less growth inhibition with Akt siRNA knockdown. In ZR75-1 xenografts, MK-2206 treatment inhibited Akt signaling, cell proliferation, and tumor growth. In vitro, MK-2206 showed a synergistic interaction with paclitaxel in MK-2206-sensitive cell lines, and this combination had significantly greater antitumor efficacy than either agent alone in vivo. Conclusions: MK-2206 has antitumor activity alone and in combination with chemotherapy. This activity may be greater in tumors with PTEN loss or PIK3CA mutation, providing a strategy for patient enrichment in clinical trials. However, not all tumors with PIK3CA/PTEN aberrations are MK-2206-sensitive, emphasizing the need for additional markers of response.
Use of next-generation sequencing (NGS) to detect high frequency of targetable alterations in primary and metastatic breast cancer (MBC).
Authors: Pusztai Lajos , Yelensky Roman , Wang Bailiang , Avritscher Rony , Symmans William Fraser , Lipson Doron , Palmer Gary A. , Moulder Stacy L. , Stephens Philip , Wu Yun , Cronin Maureen T.
Published: March 2020

10559

Background: The aim of this study was to assess the frequency of genomic alterations in breast cancer potentially treatable with approved targeted agents or investigational drugs in clinical trials. NGS was performed in a CLIA setting (Foundation Medicine). Methods: DNA was extracted from needle biopsies of 33 pre-therapy primary and 17 MBCs (mean age 52 yrs; 58% ER+, 20% HER2+, 30% triple negative) obtained prospectively for biomarker discovery and preserved in RNAlater. Patients with MBC received an average of 7 drugs (range 5-17) including adjuvant therapy before biopsy for this research; 13 biopsies were from soft tissues, 3 from liver and 1 from bone. Sequencing was targeted to 3230 exons in 182 cancer-related genes and 37 introns in 14 genes often rearranged in cancer. Average median depth was >1200x. Results: All biopsies yielded sufficient DNA. NGS revealed a total of 117 known driver mutations across 36 genes (per-tumor average=2.5, range 1-6), including 37 base substitutions (32%), 28 indels (24%), 42 amplifications (36%) and 10 homozygous deletions (9%). NGS identified HER2 gene amplification in 6/7 cases scored HER2+ by FISH. The average number of functionally important alterations was surprisingly similar, 2.3 in primaries vs 2.8 in heavily treated MBCs (p=0.32). Remarkably, 25/33 (76%) of primary and 14/17 (82%) of MBCs had at least 1 genomic alteration targetable with an FDA approved drug or novel agent in clinical trials. These included: ERBB2 alterations (n=9), PIK3CA mutations (n=8), NF1 mutations (n=4, candidate for PI3K/MEK inhibitors), AKT1-3 mutations (n=5, PI3K inhibitors), BRCA1/2, (n=6, PARP inhibitors), and CCND2 (n=3)/CDKN2A (n=3) mutations (CDK inhibitors). Numerous other alterations with less apparent therapeutic implications were also observed. Conclusions: Comprehensive NGS profiling in breast cancer needle biopsies showed high frequency of genomic alterations linked to a clinical treatment option or clinical trials of targeted therapies. These results demonstrate it is feasible to use NGS to guide targeted therapy. Prospective testing of the diagnostic/predictive value of this patient selection approach is currently under way.
The role and significance of FoxM1 in invasive breast cancer.
Authors: Yom Cha Kyong , Lee Kyung-Min , Han Wonshik , Kim Sung-Won , Moon Hyeong-Gon , Shin Hee-Chul , Kang Eunyoung , Noh Dong-Young
Published: March 2020

1056

Background: The Forkhead Box protein M1 (FoxM1) is known to regulate a variety of biologic processes in mammalian cells including cell growth and survival, angiogenesis, DNA damage response, chemotherapeutic drug resistance, and cancer cell migration and invasion. We evaluate the role and significance of Fox M1 in primary breast cancer in vitro and analyzed the relation with FoxM1 expression and clinicopathologic features. Methods: Immunohistochemical staining was used for evaluation of cytoplasmic expression of FoxM1 with TMA of invasive breast cancer. In various breast cancer cell lines, we evaluated FoxM1 expression and treated docetaxel/cisplatin in combination with Siomycin A (FoxM1 inhibitor) for BT20 cell line. Results: From Nov 1995 to Jul 2007, in 84 patients with stage 1-3 invasive breast cancer, FoxM1 expression was noted in 58.7%. Median follow-up duration was 85.1 months. Lymphovascular invasion was positively correlated with FoxM1 expression (p=0.040). In multivariate analysis, FoxM1 expression (p=0.005), HR negativity (p=0.002), high histologic grade (p=0.023), hign nuclear grade (p=0.045), lymphovascular invasiveness (p=0.017), and stage 3 cancer (p=0.015) matched poor disease-free survival. In vitro study, FoxM1 was expressed BT474, JIMT-1, BT20, HCC-1937, and MDA-MB-231 cell lines. The inhibition of FoxM1 had synergistic effect on cisplatin treatment, not docetaxel in BT20 cell. Conclusions: FoxM1 expression was noted in triple negative breast cancer cell lines and its inhibition had synergistically cytotoxic effect on BT20 cell line in combination with cisplatin. Although the further in vivo and clinical study should be needed to draw the solid conclusions, FoxM1 could be both a promising target of treatment for triple negative breast cancer and a independent prognostic factor.
Independent validation of prognostic value of 22 micrornas (miRs) in stage I-II squamous cell lung cancer (SqCLC).
Authors: Skrzypski Marcin Tomasz , Czapiewski Piotr , Biernat Wojciech , Goryca Krzysztof , Wyrwicz Lucjan , Jassem Ewa , Pawlowski Ryszard , Rzyman Witold , Jassem Jacek
Published: March 2020

10561

Background: About 50% of NSCLC patients (pts) will develop distant metastases following pulmonary resection. Currently, apart from clinical stage at diagnosis, there are no reliable factors to select high risk pts for adjuvant chemotherapy. We previously demonstrated prognostic value of 22 miRs in frozen tissue samples of early stage SqCLC, and the feasibility of their expression assessment in formalin fixed paraffin embedded (FFPE) samples (Skrzypski et. al. J Clin Oncol 2010;28;15s). In this study, we validated the prognostic value of these miRs in an independent cohort of early SqCLC pts. Methods: FFPE tumor samples were obtained from 132 stage I-II SqCLC pts who underwent radical pulmonary resection, 42% of whom developed distant metastases. Median follow-up of pts who did not develop metastases was 5.6 years (range, 4.1-10.0 years). miRs were isolated from tumor tissue with RecoverAll kit (Ambion). Expression of 22 miRs previously found to be related to the risk of metastases was analyzed by RT-PCR assay (Appliedbiosystems). Raw data were normalized vs. the expression of U6. Individual miRs and the risk score comprising 5 most predictive miRs were correlated with distant metastases-free survival (MFS). Results: Eight miRs were significantly relatedtoMFS. MiR-192 was significantly correlated with MFS (p=0,0007; p=0,02 after correction for multiple comparisons). The 5-miR risk score was an independent prognostic factor (p=1.70E-06) in a multivariate analysis comprising stage (p=1,10E-03) and histological grade (p=0,46). Using the median of the risk score as a cut-off value, the median MFS was 1.73 years in the high risk group, and not reached in the low risk group (HR=2.11). The sensitivity and specificity of the risk score to predict the occurrence of distant metastases were 66% and 64%, respectively. Conclusions: Risk score based on expression of 5 miRs is a strong and independent predictor of distant relapse in operable early SqCLC.
Comparative analysis of myeloid-derived suppressor cell (MDSC) subsets in patients with gastrointestinal (GI) malignancies.
Authors: Duffy Austin G. , Zhao Fei , Fioraventi Suzanne , Greten Tim
Published: March 2020

10566

Background: Immunotherapy represents a potential new treatment option for patients with cancer. Myeloid derived suppressor cells (MDSCs) constitute a heterogenous cell population, which inhibit the host anti-tumor immune response, limiting the effectiveness of both immune and non-immune treatments. MDSC are known to be increased in patients with GI cancer. Characterization of MDSC in humans has been difficult with heterogeneous phenotype classification. Differences in processing are also potentially important. The aim of this study was to perform a comparative analysis of MDSC subsets in patients with GI cancer. Methods: Fluorescence activated cell sorting (FACS) analysis was performed on PBMC and whole blood (WB) from healthy adults (N=44) and patients with GI cancer (N=41). MDSC subsets were enumerated using the following antibodies: anti-CD14; anti-CD15; anti-HLA-DR; anti-CD33; anti-CD11b; 7-AAD. Suppressor function of MDSC subsets was tested using FACS sorted MDSC subpopulations co-cultured with CD3/CD28-stimulated autologous effector cells. Proliferation and IFN-g were measured. Results: The frequencies of Monocytic (Def: CD14+, HLA-DR -/low) and Granulocytic (Def: CD15+,CD14-CD33+CD11b+) MDSC are shown in the table below. Monocytic MDSC were increased in the blood of patients (vs control; P<.0001) and frozen (vs fresh PBMC; P<.05). While CD15+ cells were increased in cancer patients we did not find an increase in granulocytic MDSC. Functional studies will also be presented. Conclusions: Monocytic MDSC are more frequent in patients with advanced GI cancer compared to healthy volunteers. An increase in this population was also found in WB and frozen PBMC compared to fresh PBMC reflecting the need for homogenous preparation in studies evaluating this population in humans. While CD15+CD14- cells were also increased in cancer patients we did not find an increase in CD14-CD15+CD33+CD11b+ cells. [Table: see text]
Retrospective analysis of gene expression profiling and TTF-1 staining in cancer of unknown primary (CUP).
Authors: Chiang Wendy M. , Strauss Gary M. , Nystrom J. Scott
Published: March 2020

10579

Background: CUP involves extensive use of immunohistochemical (IHC) stains because no individual marker is highly both site specific or sensitive. IHC algorithms lack standardization and may even eliminate actual primary sites. Extensive validation studies with gene expression profiling (GEP) has shown it be a new diagnostic technique that further contributes tumor site location in CUP. Thyroid transcription factor-1 (TTF-1) IHC stain is commonly used to identify the pulmonary origin of CUP (particularly adenocarcinoma) and is often used to exclude lung primary in CUP patients. This study evaluates the utility of TTF-1 staining in lung primaries (specifically non-small cell lung carcinoma - NSCLC) of CUP to GEP testing. Methods: This retrospective study contains data obtained from a registry of physicians who received the GEP-based Tissue of Origin (TOO) test (Pathwork Diagnostic, Sunnyvale, CA) between 07/2009 and 12/2009 on CUP cases. Sixty-six physicians contributed 111 TOO test cases. Only cases that had TTF-1 done were included for analysis and these were compared to TOO NSCLC results. Results: Out of 111 cases, there were 73 analyzable TTF-1 results with 12 cases of NSCLC and 61 cases of non-NSCLC by TOO (see TABLE). Assuming that the results of TOO testing accurately indicated the true primary site, the sensitivity and specificity of TTF-1 was 50% and 90%, respectively. The false negative rate was 50%, indicating that half of the identified NSCLC cases in this series had negative TTF-1. On the other hand, 10% of the 61 cases with primaries other than NSCLC (none of whom had thyroid cancer), had positive TTF-1. The “false positive” TTF-1 cases comprised 4 ovarian, 1 breast and 1 colorectal as the site of origin. Conclusions: TTF-1 has limited utility in identifying NSCLC in the setting of CUP. Half of NSCLCs identified by TOO testing had negative TTF-1, and 10% of non-lung primaries were TTF-1 positive. Negative TTF-1 should not be used to exclude NSCLC in the workup of CUP. [Table: see text]
Next-generation sequencing mutational analysis of triple-negative breast cancer patients from matched FFPE and fresh frozen samples.
Authors: Landers Mark , Meredith Rhonda , Lee Jerry , Wang Yipeng , Lee Byung-In , Monforte Joseph
Published: March 2020

1058

Background: TNBC is an aggressive subtype of breast cancer accounting for 10-15% of all cases. TNBC tumors (ER-/PR-/HER-) are more common in patients with BRCA mutations. BRCA mutations leading to homologous DNA repair deficient tumors enhance the efficacy of chemotherapy and PARP inhibitors. BRCA mutations have been identified in 20% of patients without family history. Identification of germline and somatic BRCA mutations in unselected patients could increase the number of patients who benefit from these therapies. Determining BRCA mutational status from FFPE and fresh frozen specimens may enable clinical studies in these patient populations.We describe the development of an NGS BRCA mutational assay compatible with FFPE and fresh frozen samples using tumor/adjacent normal matched tissues. Methods: Matched samples were purchased from Cureline. gDNA was isolated by lysis/column purification (Qiagen) and enriched for BRCA exons/flanking regions (Halogenomics Selector). Fragment libraries were constructed (Ion Torrent frag express) and prepared for sequencing by emPCR (Ion Torrent Template Xpress). Libraries were sequenced for 65 cycles (Ion Torrent PGM) yielding 2-4M reads/sample. Variants were called from tMAP aligned reads by GATK and VarScan. Overlapped exonic variants were filtered by p-value (<0.0001) from VarScan. Results: In the first patient set normalized average depth of BRCA exon coverage was 64X and 72X per 150K reads in FFPE and fresh frozen tissues respectively covering 95-100% of target. hg19 alignment rates varied between 97-99% across all samples. Similar numbers of variants were called in both FFPE (12) vs. fresh frozen (13) with a corresponding mean duplicate removed depth of coverage of 23.3X and 42.4X at the called positions. 10/13 calls in fresh frozen overlapped with those in FFPE. A tumor specific somatic frameshift insertion in BRCA2 was detected in both FFPE and fresh frozen tissues. Conclusions: Results indicate that NGS mediated BRCA mutational analysis demonstrates equivalent utility in both FFPE and fresh frozen tumor samples although more sequencing reads are required to produce equivalent depth of coverage starting from FFPE samples.
A pilot study on the clinical value of 18F-sodium fluoride PET/CT in advanced prostate cancer.
Authors: Kim Joseph W. , Lindenberg Maria Liza , Dahut William L. , Gulley James L. , Madan Ravi A. , Wood Lauren V. , McKinney Yolanda , Choyke Peter L. , Kurdziel Karen A , Apolo Andrea Borghese
Published: March 2020

10589

Background: We evaluated the clinical utility of 18F-sodium fluoride PET/CT bone scan (18F-NaF) in the detection of bone metastases in patients (pts) with prostate cancer in comparison with Technetium-99m MDP bone scan (TcBS). Methods: In a prospective study, from October 2010-December 2011, 30 prostate cancer pts (ages 51-79), 21 with known bone metastases and 9 without known bone metastases, had18F-NaF and a TcBS performed. Abnormal foci of uptake on both TcBS and 18F-NaFwere classified as benign, malignant or indeterminate. Benign lesions included uptake in the joints and linear uptake at the endplates of the vertebral bodies consistent with degenerative changes. Malignant uptake on 18F-NaF scans was confirmed by characteristic osteoblastic features on CT. All TcBS and 18F-NaF were reviewed by an experienced nuclear medicine physician. For the patient-based analysis, scan results were categorized as positive (POS) = any malignant lesion; indeterminate (IND) = not distinctly malignant or benign; negative (NEG) = benign lesions only. Results: In the lesion-based analysis, 21 of 30 (70%) pts had more malignant lesions identified on 18F-NaF than on TcBS. The mean number of additional malignant lesions per patient on 18F-NaF vs TcBS was 4. Eight of the 30 pts had same number of malignant lesions identified in both studies. One of 30 pts had one less malignant lesion identified on 18F-NaF than on TcBS. CT correlation by 18F-NaF PET/CT of this particular lesion did not confirm osteoblastic feature. Malignant lesion distribution on 18F-NaF included: spine (28%), thorax (26%), pelvis (24%), long bones (13%) and skull (10%). In the patient-based analysis, 24 pts (80%) were POS by 18F-NaF, of whom 14 pts were POS, 8 were IND, and 2 were NEG by corresponding TcBS; in the 4 pts with NEG 18F-NaF, zero were POS, 2 were IND and 2 were NEG by corresponding TcBS. Conclusions: 18F-NaF identified more malignant lesions than TcBS. 18F-NaF may also add useful information in the management of advanced prostate cancer pts with and without known bone metastases.
Next-generation sequencing (NGS) to identify actionable genomic changes in common and rare solid tumors: The FMI experience with the initial 50 consecutive patients.
Authors: Palmer Gary A. , Miller Vincent A. , Curran John , Ross Jeffrey S. , Lipson Doron , Yelensky Roman , Stephens Philip , Lancelotta Mary Pat , Cronin Maureen T.
Published: March 2020

10590

Background: Solid tumor (ST) oncology has been transformed by the linkage of genomic changes with targeted therapeutics. Unfortunately, most STs still have no alteration detected with currently available assays. Thus, more informative testing platforms are needed to analyze genomic changes in FFPE biopsy samples often limited by tumor size, heterogeneity and ploidy. Methods: We reviewed the genomic profiles of the first 50 clinical specimens received by our CLIA lab (Foundation Medicine) and analyzed by our NGS assay (Ross J. ASCO 2011). 182 genes known altered in STs and 14 genes known rearranged in STs were included. Genomic alterations were categorized as “actionable” if linked to an approved therapy in the ST under study or another ST (e.g. ALK rearrangement in lung or breast cancer, respectively), a known or suspected contraindication to a given therapy (e.g. KRAS G12D in colorectal cancer) or a clinical trial linked to the alteration (e.g. mTOR inhibition in a tumor with STK11 loss). Results: Fifty tumor specimens were received from 50 patients (median age 54, F/M=30/20) representing 35 sites. Two samples failed analysis and 6 had no detectable genomic alteration. Among 16 primary tumor types, lung (n=15), colorectal (n=7), sarcoma (n=5), breast and esophageal/gastric (n=4 each) were most common. We reported 135 genomic alterations (mean 2.8, range 0-6) unequivocally involved in oncogenesis and 69 actionable alterations. At most, 29 of these (42%) would have been detected by current assays (138% increase with NGS). Thirty seven patients had one or more actionable alterations (range 1-4) detected by NGS (74%, 95% CI 60-85%) versus only 21 (42%, 95% CI 28-57%) in whom changes would have been detected by current assays. Conclusions: Use of a broad, comprehensive NGS assay identifies an unprecedented number of actionable genomic alterations across a variety of STs from routine FFPE samples. This assay can serve as a paradigm for improving access to approved therapies in STs, minimizing use of ineffective therapies and enhancing enrollment in rationally chosen trials. Ongoing and planned trials will study the impact on several efficacy endpoints.
Role of proliferation in response to neoadjuvant chemotherapy in GEICAM/2006-03 and GEICAM/2006-14 breast cancer patients.
Authors: Alba E. , Albanell Joan , Chacon Lopez-Muniz Jose Ignacio , Calvo Lourdes , De la Haba- Rodriguez Juan , Sanchez Rovira Pedro , Plazaola Arrate , Barnadas Agust , Seguí-Palmer Miguel Angel , Arcusa Lanza Angels , Ramos Vazquez Manuel , Rojo Federico , Burgues Octavio , Pajares Bella , Bermejo Begoña , Sanchez-Muñoz Alfonso , Casas Maria Isabel , Caballero Rosalia , Carrasco Eva Maria , Lluch Ana ,   Spanish Breast Cancer Research Group GEICAM
Published: March 2020

10616

Background: Ki67 proliferation biomarker determined by immunohistochemistry (IHC) has been studied as a prognostic and predictive factor in Operable Breast Cancer (OBC). Ki67 modifications after neoadjuvant endocrine therapy have been correlated with long term outcome. However, there is no robust data about its predictive role in Neoadjuvant Chemotherapy (NC). In this study, we investigated Ki67 value as predictor of NC efficacy. Methods: 193 patients (pts) from 2 GEICAM phase II randomized trials (2006-03 and 2006-14) were included: 78 (40%) received epirubicine plus cyclophosphamide followed by docetaxel (EC-D), 41 (21%) EC-D plus carboplatin, and out of the 74 HER2+ pts, 37 (19%) received EC-D plus tratuzumab and 37 (19%) EC-D plus lapatinib. Median age was 49 years. From series, 87% were invasive ductal carcinoma, 58% premenopausal, 50% grade III, 23% luminal , 39% basal and 38% HER2+. Ki67 was centrally assessed by IHC (MIB1 clone) and median score was 40% (range 1-100%). Pathological Complete Response (pCR), defined as absence of invasive cells in breast and lymph nodes, was achieved in 56 pts (29%). Univariate and multivariate logistic regression models were used to study the association of each clinical-pathological variable with pCR. ROC curves were used to determine the most accurate ki67 cut-off for predicting NC response. Results: Ki67≥50% was defined as the most accurate threshold to select patients obtaining benefit from NC. In the univariate analysis, histological grade (p=0.01), treatment (P=0.006), ER (p<0.0001), PR (p<0.0001), HER2 (p=0.01), and Ki67≥50% (p=0.0003) were statistically associated with pCR. A multivariate logistic regression showed that only Ki67≥ 50% (p=0.0003; OR=5.4 CI95% 2.1-13.4), ER (p=0.0001; OR=0.2 CI95% 0.1-0.4), and HER2 status (p<0.0001; OR=8.8 CI95% 3.3-23.6) were predictive for pCR (AUC=0.7812). Conclusions: These results suggest that a high proliferation in breast cancer measured by Ki67 marker is an independent predictive factor for pCR in an unclassified HER2 population of OBC patients treated with NC.
Biomarkers that predict sensitivity to heat shock protein 90 inhibitors (HSP90i).
Authors: Jhaveri Komal L. , Iyengar Neil M. , Corben Adriana , Patil Sujata , Akram Muzaffar , Towers Russell , Sakr Rita , Hudis Clifford , King Tari A. , Rosen Neal , Chandarlapaty Sarat , Modi Shanu
Published: March 2020

10618

Background: HSP90 is an attractive target in tumors as it mediates the maturation and stabilization of client oncoproteins. HSP90i are potentially active in a variety of tumors, but therapeutic benefit is confirmed in only a small subset to date. Predictive biomarkers could identify patients (pts) and/or tumors with sensitivity to HSP90i improving the therapeutic index. We explored potential biomarkers across multiple studies of HSP90i. Methods: Archived pre-treatment tumor specimens from pts treated with any of several HSP90i (17-AAG, 17-DMAG, CNF2024, retaspimycin & ganetespib) on 8 phase I/II trials at MSKCC from 1999 to 2011 were identified. The following antibodies were validated at MSKCC and tumor tissue was tested by immunohistochemistry (IHC) with results defined for each as: ER, PR & AR: >1% pos & <1% neg; HSP90 & HSP70: 0, 1+ neg & 2+, 3+ pos; PTEN: 0, 1+ neg & 2+ pos; HER2: 0, 1+ neg, 2+ equivocal, 3+ pos; EGFR: 0 neg & 1+, 2+, 3+ pos. Clinical response was correlated with IHC using Fisher’s exact test. Results: Of the 164 pts identified, adequate tissue was available for 51, including 11 different solid tumors & 1 CML. Breast (N=31), melanoma & prostate (N=4 each) were most frequent. The mean age at primary diagnosis was 50 yrs (24-81). The median no. of lines of prior chemotherapy for metastatic disease was 2 (0-8). The mean duration of HSP90i therapy was 79 days (0-411). There were 19 responses (5 PR; 14 SD); 14/19 responses strongly correlated with HER2+ status (p = 0.001); 1 pt with ER+/HER2-/EGFR- & 4 with EGFR +/HER2-/ER- disease also responded. A correlation for ER & EGFR with response was not excluded (p=0.07 & p=0.086 respectively). Conclusions: Our findings confirm HER2 as a sensitive client and an effective biomarker for sensitivity to HSP90 inhibitors. ER & EGFR did not meet statistical significance but may warrant further exploration in prospective settings.
Current patterns of chemotherapy and supportive care for early-stage breast cancer (ESBC).
Authors: Lyman Gary H. , Dale David C. , Tomita Dianne , Whittaker Sadie , Crawford Jeffrey
Published: March 2020

1062

Background: ESBC is commonly treated with myelosuppressive chemotherapy, and high relative dose intensity (RDI) correlates with improved overall survival. A retrospective analysis of patients with ESBC treated from 1997–2000 showed that 56% received an RDI < 85% (Lyman et al. JCO. 2003;21:4524-4531). To determine current practice, we evaluated ESBC treatment patterns at 24 US community- and hospital-based oncology practices. Methods: Data were abstracted from medical records of 532 patients with ESBC treated from January 2007–December 2009. Inclusion criteria included surgically resected ESBC (stage I-IIIA); ≥ 18 years old; and completion of at least 1 standard chemotherapy cycle on an every 2 or 3 week schedule. The primary endpoint was RDI over planned cycles. Other endpoints were incidence of dose delays ≥ 7 days, dose reductions ≥ 15% from standard, grade 3/4 neutropenia (SN), febrile neutropenia (FN), FN-related hospitalization, granulocyte-colony stimulating factor (G-CSF) use, and antimicrobial therapy. Descriptive statistics were generated for all endpoints. Results: In this study, mean (range) age was 55 (29–85) years. Relative to previously published results, chemotherapy regimens have shifted from mainly doxorubicin + cyclophosphamide (AC) (previously 35%) to docetaxel + cyclophosphamide (TC; n = 221; 42%) and AC followed by paclitaxel (AC-T; n = 163; 31%). Mean RDI is now higher (93% for both TC and the most common AC-T schedule [dose dense AC-T; n = 84] vs 79% previously); the incidence of dose delays (16% vs 25% previously) and dose reductions (21% vs 37% previously) have decreased; and primary prophylactic use of G-CSF has increased (76% vs 3% previously). In this study, 40% of patients had SN, 3% had FN, 2% had an FN-related hospitalization, and 30% received antimicrobial therapy. These measures were not available in the previously published results. Conclusions: The observed changes between the two studies are noteworthy though inferential comparisons are limited by changes in treatments and other factors. RDI has improved over time, but 16% of patients in this study received an RDI < 85%. Further evaluation is needed to identify factors associated with lower RDI and determine outcomes for these patients.
Using CHFR expression to predict response and survival after first line treatment with carboplatin-paclitaxel in NSCLC.
Authors: Pillai Rathi Narayana , Brodie Seth Aaron , Li Ge , Herman James Gordon , Brock Malcolm , Owonikoko Taofeek Kunle , Ramalingam Suresh S. , Khuri Fadlo Raja , Brandes Johann Christoph
Published: March 2020

10625

Background: The identification of resistance mechanisms for conventional chemotherapy in lung cancer is of fundamental importance not only for personalization of chemotherapy but also for the subsequent development of novel targeted approaches to overcome this resistance. Currently, there is no clinically validated test for the prediction of response to tubulin-targeted agents in NSCLC. Our previous preclinical work identified Checkpoint with Forkhead and Ringfinger domain” (CHFR) as a predictor of taxane cytotoxicity in in vitro models. The current work assessed the translational significance of this finding in a cohort of US veterans treated at a single-institution. Methods: We studied a cohort of patients with advanced NSCLC treated with taxane-containing frontline regimens at the Atlanta VA Medical Center between 2000 and 2009. Archived paraffin-embedded tissue was retrieved and stained for CHFR expression using standard immunohistochemical techniques. Level of protein expression was assessed by light microscopy and scored for intensity of CHFR staining. Intensity of staining was correlated with clinical outcome including objective response and median overall survival using Chi-Square test and Cox proportional models. Results: We analyzed tumor samples from 45 eligible patients with median age 62.6 years, M/F (44/1). In this cohort, high expression of CHFR is strongly associated with adverse outcomes: the risk for progressive disease (PD) after first-line chemotherapy with carboplatin-paclitaxel was 54% in patients with CHFR-high vs. only 18% in those with CHFR-low tumors (HR 3.1; 95% CI 1.09-9.04; p=0.02). Median overall survival was strongly correlated with response to first-line therapy (clinical benefit: 9.24 months; PD: 4.7 months; p<0.001) and with CHFR expression status (CHFR low: 10 months; CHFR high: 5.6 months; p =0.01). Conclusions: CHFR expression is a novel predictive marker of response and overall survival in NSCLC patients treated with taxane-containing chemotherapy.
DNA damage responses in relation to late effects following radiotherapy for early breast cancer.
Authors: Chua Melvin
Published: March 2020

10626

Background: In this study, we assessed if induction of apoptosis in G0 blood lymphocytes following ex vivo irradiation correlated with clinical radiosensitivity and DNA double-strand (DSB) repair in breast radiotherapy (RT) patients. Using small molecule inhibitors, we examined the relationship between DSB repair and radiation-induced apoptosis. Methods: Breast cancer patients with minimal (controls) or marked late RT changes (cases) were selected. DSB were quantified by γH2AX/53BP1 immunostaining 0.5 and 24 h after exposure of lymphocytes to 0.5 and 4 Gy, respectively. Induction of apoptosis was assessed 48 h after 8 Gy using a fluorochrome inhibitor of caspases (FLICA) assay. Apoptotic cells were defined by small cell size and positive FLICA signal, measured using flow cytometry. Small molecule DNA-PK (Nu7441) and ATM (Ku55933) inhibitors were used at concentrations of 1 µM and 10 µM, respectively. Results: Despite similar foci induction at 0.5 h, residual foci levels 24 h after 4 Gy differed significantly between cases and controls (Foci per cell = 12.7 in cases, n = 8 vs 10.3 in controls, n = 8, p = 0.002). In contrast to residual foci levels, comparable levels of apoptosis were observed between cases and controls 48 h after 8 Gy. Mean proportion of apoptotic cells was 37.2% in cases and 34.7% in controls (p = 0.413). Residual foci levels were not correlated with levels of apoptosis in lymphocytes of the same patients (R = -0.059, p = 0.785). To determine if apoptosis is modulated by DSB repair, apoptosis measurements were repeated in lymphocytes treated with Nu7441 to inhibit non-homologous end-joining. We observed that Nu7441-treated cells had higher levels of residual foci and apoptosis compared to mock-treated cells, 48 h after 1 Gy. This effect was wholly dependent on an active ATM function as cells treated simultaneously with Nu7441 and Ku55933 had extremely low levels of apoptosis, comparable to levels observed in cells treated with Ku55933 alone. Conclusions: Higher levels of residual DSB observed among radiosensitive patients suggest a role for DSB repair in the pathogenesis of late radiation-induced effects. Induction of apoptosis appears to be dependent on DSB end-joining following exposure to ionising radiation.
In silico identification of an epithelial core signature in human tumors.
Authors: Goswami Chirayu Pankaj , Cano Oscar D. , Gokmen-Polar Yesim , Badve Sunil S.
Published: March 2020

10628

Background: Gene expression analysis is performed on grossly selected specimens often without any microscopic analysis of tumor content. In studies where histological analyses have been performed, cases having 80% or more tumor content are used for microarray analysis. The variability in amount of epithelial and stromal cells may generate to misleading differential expression analysis and selection for wrong targets for therapeutics. It is also often unclear, whether the genes identified are stromal or epithelial in origin. The goal of this study was to identify genes that define core epithelial phenotype; these genes could provide means of normalization of expression data. Methods: The CABIG GSK microarray (HG-U133_plus_2) data consisting of 950 cell lines from carcinoma (n=562), non-carcinoma (n=385) and normal tissue (n=3) was analyzed to identify epithelial specific genes. 10 carcinomas each from 11 sites (n=110) and an equal number of non-carcinomas were randomly selected. In silico analyses were performed by 1) identifying genes differentially expressed between carcinoma and non-carcinoma samples using a one way ANOVA; 2) identifying gene signature associated with carcinoma using Predictive Analysis of Microarrays (PAM) and 3) a weighted gene coexpression network analysis (WGCNA) was performed to identify co-expression modules. A similar analysis was also performed on tissue samples (E-GEOD-12360) from carcinomas and non-carcinomas. Venn-diagram was generated to identify intersecting set. Results: Comparison of the carcinoma and non-carcinoma samples using ANOVA identified 1455 differential expressed gene probes in cell lines and 540 gene probes in tissues (FDR=1E-10). The cell lines analysis identified 5 modules and a 65-gene signature (43 core and 22 accessory set) that was specific for epithelial cells. In the tissue analysis a 188-gene signature was similarly identified. Cross-comparison identified a smaller 31 gene intersecting set; this was not associated with loss of discriminatory power. Conclusions: A 31 geneset which can be used to determine the epithelial content of heterogeneous tumors, was identified. This study has the potential to significantly impact the use of microarray based gene expression data.