bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Synthetic MicroRNA Designed to Target Glioma-Associated Antigen 1 Transcription Factor Inhibits Division and Induces Late Apoptosis in Pancreatic Tumor Cells
Authors: Tsuda Naotake , Ishiyama Satoshi , Li Yufeng , Ioannides Constantin G. , Abbruzzese James L. , Chang David Z.
Published: April 2023

Abstract
Purpose: To determine whether the synthetic microRNAs (miRNA) could effectively target tumor cells we designed several miRNA complementary to glioma-associated antigen-1 (Gli-1) mRNA and investigated their ability to inhibit tumor cell proliferation. The sonic hedgehog pathway is an early and late mediator of tumorigenesis in epithelial cancers. Activation of sonic hedgehog signaling seems to precede transformation of tissue stem cells to cancerous stem cells, with the Gli-1 transcription factor functioning as a mediator of environmental signals. Inhibiting cancer cell proliferation by targeting the Gli-1 effector pathway is difficult to achieve by chemotherapeutic agents or short interfering RNA.

Experimental Design: We hypothesized that targeting the 3′-untranslated region of Gli-1 mRNA would effectively inhibit tumor cell proliferation. To test this hypothesis, we used synthetic miRNAs of our own design and corresponding duplex/small temporal RNAs by introducing three-nucleotide loops in the 3′-untranslated region Gli-1 sequence of high GU content.

Results: We found that miRNA (Gli-1-miRNA-3548) and its corresponding duplex (Duplex-3548) significantly inhibited proliferation of Gli-1+ ovarian (SK-OV-3) and pancreatic (MiaPaCa-2) tumor cells. The miRNAs mediated delayed cell division and activation of late apoptosis in MiaPaCa-2 cells. This is the first demonstration of inhibition of pancreatic tumor cell division by designed miRNA.

Conclusions: Gli-1 miRNAs should significantly add to the general understanding of the mechanisms of metastasis and contribute toward the design of better treatments for epithelial cancers.
Data from Identification of Matrix Metalloproteinase 11 as a Predictive Tumor Marker in Serum Based on Gene Expression Profiling
Authors: Yang Yan-Hong , Deng Hua , Li Wen-Mei , Zhang Qing-Yun , Hu Xiao-Ting , Xiao Bai , Zhu Hai-Hong , Geng Pai-Li , Lu You-Yong
Published: April 2023

Abstract
Purpose: Prognostic markers discovery is a strategy for early diagnosis and individualization therapy for human cancer. In this study, we focus to integrate different methods to identify specific biomarker and elucidate its clinical significance.

Experimental Design: A powerful tool named Digital Gene Expression Display online was applied to isolate differentially expressed genes correlated with gastric cancer. Matrix metalloproteinase 11 (MMP11) was selected and confirmed at both mRNA and protein level in 10 cell lines, 123 cases of tumor tissues, and 305 cases of gastric cancer serum specimen by semiquantitative PCR, immunohistochemistry staining, and ELISA techniques, respectively.

Results: Our data showed that overexpression of MMP11 at mRNA and protein level was consistently detected in cell lines and primary tumors compared with matched normal tissues. Importantly, serum MMP11 levels were also significantly elevated in gastric cancer patients compared with those of the control subjects (P < 0.001), and the positive expression was well correlated with metastasis in gastric cancer patients (P = 0.009). Furthermore, we have shown that overexpression of MMP11 was associated with the malignant proliferation of AGS cells.

Conclusions: Combination of gene expression profiling and specific clinical resource is a promising approach to validate gene expression patterns associated with malignant phenotype. As a secreted protein, MMP11 may play an important role in carcinogenesis and has potential implication as a biomarker for the diagnosis and prognosis of human cancers including gastric cancer.
Data from Carcinoembryonic Antigen Cell Adhesion Molecule 6 Predicts Breast Cancer Recurrence following Adjuvant Tamoxifen
Authors: Maraqa Loaie , Cummings Michele , Peter Mark B. , Shaaban Abeer M. , Horgan Kieran , Hanby Andrew M. , Speirs Valerie
Published: April 2023

Abstract
Purpose: Tamoxifen remains therapy of choice for premenopausal estrogen receptor α–positive breast cancer. However, resistance and recurrence are serious problems. Our previous work indicated that carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) was significantly up-regulated in tamoxifen-resistant (TAMr) MCF-7 derivatives. The aim of this study was to determine the functional role of CEACAM6 in endocrine-resistant breast cancer and to retrospectively test whether it was predictive of resistance in a large cohort of breast cancers with long-term follow-up.

Experimental Design: siRNA silencing of CEACAM6 was done in TAMr cells and effects on clonogenicity and endocrine sensitivity were determined. CEACAM6 immunohistochemistry was done on a tissue microarray comprising 108 relapsed primary human breast cancers and 243 tamoxifen-sensitive controls.

Results: siRNA-mediated silencing of CEACAM6 reduced both clonogenicity and anchorage-dependent and anchorage-independent growth of TAMr cells. Importantly, CEACAM6 silencing restored sensitivity of TAMr cells to 4-hydroxytamoxifen and proliferative response to 17β-estradiol. Immunohistochemistry showed significantly more CEACAM expression in the relapsed group compared with nonrelapsed controls [35 of 108 (33.3%) and 32 of 243 (13.2%), respectively; odds ratio, 3.16 (95% confidence interval, 1.83-5.47); P < 0.0001]. Additionally, we derived an outcome predictor model based on CEACAM expression that restratified patients in the Nottingham prognostic index intermediate-risk group into either higher-risk or lower-risk group.

Conclusions: Our data support an important role for CEACAM6 in endocrine resistance, which can serve as a powerful predictor of future recurrence.
Data from A Combination of Molecular Markers Accurately Detects Lymph Node Metastasis in Non–Small Cell Lung Cancer Patients
Authors: Xi Liqiang , Coello Michael C. , Litle Virginia R. , Raja Siva , Gooding William E. , Yousem Samuel A. , El-Hefnawy Talal , Landreneau Rodney J. , Luketich James D. , Godfrey Tony E.
Published: April 2023

Abstract
Occult lymph node metastasis (micrometastasis) is a good prognostic indicator in non–small cell lung cancer (NSCLC) and could be used to direct adjuvant chemotherapy in stage I patients. This study was designed to evaluate molecular markers for detection of occult lymph node metastasis in NSCLC, define the best marker or marker combination to distinguish positive from benign lymph nodes, and evaluate these markers in lymph nodes from pathologically node-negative (pN0) NSCLC patients. Potential markers were identified through literature and database searches and all markers were analyzed by quantitative reverse transcription-PCR in a primary screen of six NSCLC specimens and 10 benign nodes. Selected markers were further evaluated on 21 primary NSCLC specimens, 21 positive nodes, and 21 benign nodes, and the best individual markers and combinations were identified. A combination of three markers was further validated on an independent set of 32 benign lymph nodes, 38 histologically positive lymph nodes, and 462 lymph nodes from 68 pN0 NSCLC patients. Forty-two markers were evaluated in the primary screen and eight promising markers were selected for further analysis. A combination of three markers (SFTPB, TACSTD1, and PVA) was identified that provided perfect classification of benign and positive nodes in all sample sets. PVA and SFTPB are particularly powerful in tumors of squamous and adenocarcinoma histologies, respectively, whereas TACSTD1 is a good general marker for NSCLC metastasis. The combination of these genes identified 32 of 462 (7%) lymph nodes from 20 of 68 (29%) patients as potentially positive for occult metastasis. Long-term follow-up will determine the clinical relevance of these findings.
Data from Integrated Genomic and Transcriptomic Analysis of Ductal Carcinoma In situ of the Breast
Authors: Vincent-Salomon Anne , Lucchesi Carlo , Gruel Nadège , Raynal Virginie , Pierron Gaëlle , Goudefroye Rémi , Reyal Fabien , Radvanyi François , Salmon Rémy , Thiery Jean-Paul , Sastre-Garau Xavier , Sigal-Zafrani Brigitte , Fourquet Alain , Delattre Olivier
Published: April 2023

Abstract
Purpose: To gain insight into genomic and trancriptomic subtypes of ductal carcinomas in situ of the breast (DCIS).

Experimental Design: We did a combined phenotypic and genomic analysis of a series of 57 DCIS integrated with gene expression profile analysis for 26 of the 57 cases.

Results: Thirty-two DCIS exhibited a luminal phenotype; 21 were ERBB2 positive, and 4 were ERBB2/estrogen receptor (ER) negative with 1 harboring a bona fide basal-like phenotype. Based on a CGH analysis, genomic types were identified in this series of DCIS with the 1q gain/16q loss combination observed in 3 luminal DCIS, the mixed amplifier pattern including all ERBB2, 12 luminal and 2 ERBB2-/ER- DCIS, and the complex copy number alteration profile encompassing 14 luminal and 1 ERBB2-/ER- DCIS. Eight cases (8 of 57; 14%) presented a TP53 mutation, all being amplifiers. Unsupervised analysis of gene expression profiles of 26 of the 57 DCIS showed that luminal and ERBB2-amplified, ER-negative cases clustered separately. We further investigated the effect of high and low copy number changes on gene expression. Strikingly, amplicons but also low copy number changes especially on 1q, 8q, and 16q in DCIS regulated the expression of a subset of genes in a very similar way to that recently described in invasive ductal carcinomas.

Conclusions: These combined approaches show that the molecular heterogeneity of breast ductal carcinomas exists already in in situ lesions and further indicate that DCIS and invasive ductal carcinomas share genomic alterations with a similar effect on gene expression profile.
Data from Promoter Hypermethylation Identifies Progression Risk in Bladder Cancer
Authors: Yates David R. , Rehman Ishtiaq , Abbod Maysam F. , Meuth Mark , Cross Simon S. , Linkens Derek A. , Hamdy Freddie C. , Catto James W.F.
Published: April 2023

Abstract
Purpose: New methods to accurately predict an individual tumor behavior are urgently required to improve the treatment of cancer. We previously found that promoter hypermethylation can be an accurate predictor of bladder cancer progression, but it is not cancer specific. Here, we investigate a panel of methylated loci in a prospectively collected cohort of bladder tumors to determine whether hypermethylation has a useful role in the management of patients with bladder cancer.

Experimental Design: Quantitative methylation-specific PCR was done at 17 gene promoters, suspected to be associated with tumor progression, in 96 malignant and 30 normal urothelial samples. Statistical analysis and artificial intelligence techniques were used to interrogate the results.

Results: Using log-rank analysis, five loci were associated with progression to more advanced disease (RASSF1a, E-cadherin, TNFSR25, EDNRB, and APC; P < 0.05). Multivariate analysis revealed that the overall degree of methylation was more significantly associated with subsequent progression and death (Cox, P = 0.002) than tumor stage (Cox, P = 0.008). Neuro-fuzzy modeling confirmed that these five loci were those most associated with tumor progression. Epigenetic predictive models developed using artificial intelligence techniques identified the presence and timing of tumor progression with 97% specificity and 75% sensitivity.

Conclusion: Promoter hypermethylation seems a reliable predictor of tumor progression in bladder cancer. It is associated with aggressive tumors and could be used to identify patients with either superficial disease requiring radical treatment or a low progression risk suitable for less intensive surveillance. Multicenter studies are warranted to validate this marker.
Data from T Cell Receptor Genotyping and HOXA/TLX1 Expression Define Three T Lymphoblastic Lymphoma Subsets which Might Affect Clinical Outcome
Authors: Baleydier Frederic , Decouvelaere Anne-Valerie , Bergeron Julie , Gaulard Philippe , Canioni Danielle , Bertrand Yves , Lepretre Stephane , Petit Barbara , Dombret Herve , Beldjord Kheira , Molina Thierry , Asnafi Vahid , Macintyre Elizabeth
Published: April 2023

Abstract
Purpose: T lymphoblastic lymphomas (T-LBL) are rare disorders of immature T cells which predominantly involve the mediastinum. Their oncogenic pathways and prognostic variables are not clear.

Experimental Design: We undertook a retrospective study of 41 cytoplasmic CD3+ T-LBL (nine cases aged <16 years) by assessing stage of maturation arrest based on T cell receptor (TCR) immunogenotyping, immunohistochemistry, and quantification of the oncogenes thought to be important in immature T cell malignancies.

Results: Application of a TCR-based immunogenetic classification allowed the identification of three subcategories: 11 immature IM0/D-LBL showed no TCR or only incomplete TCRD DJ rearrangement and corresponded to cytoplasmic CD3+ precursors of uncertain lineage. Sixteen mature TCRDdel-LBL showed biallelic TCRD deletion and both TCRG and TCRB rearrangement, consistent with TCRαβ lineage restriction. Fourteen intermediate LBL (Int-LBL) showed complete TCRD VDJ and TCRG VJ rearrangement, with TCRB VDJ rearrangement in the majority. All Int-LBL expressed HOX11/TLX1 or HOXA9 transcripts and a proportion of the latter were associated with CALM-AF10 or NUP214-ABL fusion transcripts. IM0/D-LBL were restricted to adults with extrathymic disease and bone marrow involvement, whereas Int-LBL and TCRDdel-LBL were found in children and adults with predominantly thymic disease. In adults, the Int-LBL subgroup was associated with a significantly superior clinical outcome. This subgroup can be identified either by TCR immunogenotyping or HOXA9/TLX1 transcript quantification.

Conclusion: Application of this molecular classification will allow the prospective evaluation of prognostic effects within pediatric and adult protocols.
Data from Global Expression Analysis of Cancer/Testis Genes in Uterine Cancers Reveals a High Incidence of BORIS Expression
Authors: Risinger John Ian , Chandramouli Gadisetti V.R. , Maxwell G. Larry , Custer Mary , Pack Svetlana , Loukinov Dmitri , Aprelikova Olga , Litzi Tracy , Schrump David S. , Murphy Susan K. , Berchuck Andrew , Lobanenkov Victor , Barrett J. Carl
Published: April 2023

Abstract
Purpose: Cancer/testis (CT) genes predominantly expressed in the testis (germ cells) and generally not in other normal tissues are aberrantly expressed in human cancers. This highly restricted expression provides a unique opportunity to use these CT genes for diagnostics, immunotherapeutic, or other targeted therapies. The purpose of this study was to identify those CT genes with the greatest incidence of expression in uterine cancers.

Experimental Design: We queried the expression of known and putative CT gene transcripts (representing 79 gene loci) using whole genome gene expression arrays. Specifically, the global gene expressions of uterine cancers (n = 122) and normal uteri (n = 10) were determined using expression data from the Affymetrix HG-U133A and HG-U133B chips. Additionally, we also examined the brother of the regulator of imprinted sites (BORIS) transcript by reverse transcription-PCR and quantitative PCR because its transcript was not represented on the array.

Results: Global microarray analysis detected many CT genes expressed in various uterine cancers; however, no individual CT gene was expressed in more than 25% of all cancers. The expression of the two most commonly expressed CT genes on the arrays, MAGEA9 (24 of 122 cancers and 0 of 10 normal tissues) and Down syndrome critical region 8 (DSCR8)/MMA1 (16 if 122 cancers and 0 of 10 normal tissues), was confirmed by reverse transcription-PCR methods, validating the array screening approach. In contrast to the relatively low incidence of expression of the other CT genes, BORIS expression was detected in 73 of 95 (77%) endometrial cancers and 24 of 31 (77%) uterine mixed mesodermal tumors.

Conclusions: These data provide the first extensive survey of multiple CT genes in uterine cancers. Importantly, we detected a high frequency of BORIS expression in uterine cancers, suggesting its potential as an immunologic or diagnostic target for these cancers. Given the high incidence of BORIS expression and its possible regulatory role, an examination of BORIS function in the etiology of these cancers is warranted.
Data from Pulmonary Inflammatory Myofibroblastic Tumor Expressing a Novel Fusion, PPFIBP1–ALK: Reappraisal of Anti-ALK Immunohistochemistry as a Tool for Novel ALK Fusion Identification
Authors: Takeuchi Kengo , Soda Manabu , Togashi Yuki , Sugawara Emiko , Hatano Satoko , Asaka Reimi , Okumura Sakae , Nakagawa Ken , Mano Hiroyuki , Ishikawa Yuichi
Published: April 2023

Abstract
Purpose: The anaplastic lymphoma kinase (ALK) inhibitor crizotinib has been used in patients with lung cancer or inflammatory myofibroblastic tumor (IMT), both types harboring ALK fusions. However, detection of some ALK fusions is problematic with conventional anti-ALK immunohistochemistry because of their low expression. By using sensitive immunohistochemistry, therefore, we reassessed “ALK-negative” IMT cases defined with conventional immunohistochemistry (approximately 50% of all examined cases).

Experimental Design: Two cases of ALK-negative IMT defined with conventional anti-ALK immunohistochemistry were further analyzed with sensitive immunohistochemistry [the intercalated antibody-enhanced polymer (iAEP) method].

Results: The two “ALK-negative” IMTs were found positive for anti-ALK immunohistochemistry with the iAEP method. 5′-rapid amplification of cDNA ends identified a novel partner of ALK fusion, protein-tyrosine phosphatase, receptor-type, F polypeptide-interacting protein-binding protein 1 (PPFIBP1) in one case. The presence of PPFIBP1–ALK fusion was confirmed with reverse transcriptase PCR, genomic PCR, and FISH. We confirmed the transforming activities of PPFIBP1–ALK with a focus formation assay and an in vivo tumorigenicity assay by using 3T3 fibroblasts infected with a recombinant retrovirus encoding PPFIBP1–ALK. Surprisingly, the fusion was also detected by FISH in the other case.

Conclusions: Sensitive immunohistochemical methods such as iAEP will broaden the potential value of immunohistochemistry. The current ALK positivity rate in IMT should be reassessed with a more highly sensitive method such as iAEP to accurately identify those patients who might benefit from ALK-inhibitor therapies. Novel ALK fusions are being identified in various tumors in addition to IMT, and thus a reassessment of other “ALK-negative” cancers may be required in the forthcoming era of ALK-inhibitor therapy. Clin Cancer Res; 17(10); 3341–8. ©2011 AACR.
Data from Development of Immunohistochemistry Assays to Assess GALNT14 and FUT3/6 in Clinical Trials of Dulanermin and Drozitumab
Authors: Stern Howard M. , Padilla Mary , Wagner Klaus , Amler Lukas , Ashkenazi Avi
Published: April 2023

Abstract
Purpose: In vitro sensitivity to the proapoptotic receptor agonists dulanermin (rhApo2L/TRAIL) and drozitumab (DR5-agonist antibody) is strongly predicted by the expression of the O-glycosylation enzymes GALNT14 in non–small cell lung cancer (NSCLC) cell lines (among others) and of FUT3/6 in colorectal cancer (CRC) cell lines. We developed immunohistochemistry (IHC) assays that measure GALNT14 and FUT3/6 levels in archival formalin-fixed, paraffin-embedded human tumor tissue to determine marker prevalence in NSCLC and CRC tissue and to enable the future examination of these markers in clinical trials.

Experimental Design: GALNT14 or FUT3/6 ELISA-positive hybridoma clones were screened through IHC on cell pellets with known mRNA levels. The specificity of staining was examined in cell lines, normal tissue, and tumor tissue.

Results: GALNT14 and FUT3/6 IHC exhibited a golgi staining pattern and correlated with GALNT14 and FUT3/6 (but not GALNT2 and FUT4) mRNA expression levels in cell lines and normal tissues, suggesting specificity. GALNT14 and FUT3/6 H-scores were significantly higher in cell lines sensitive to dulanermin (P = 0.01 and P = 0.0004, respectively) and drozitumab (P = 0.03 and P < 0.0001, respectively) versus resistant cell lines. GALNT14 and FUT3/6 H-scores varied widely, with ∼45% of NSCLC samples exhibiting weak to moderate GALNT14 staining (H-score of at least 25) and 70% of CRC samples exhibiting moderate to strong FUT3/6 staining (H-score of at least 125).

Conclusions: GALNT14 and FUT3/6 expression can be assessed in human tumors using sensitive and specific IHC assays. Both assays are being deployed in ongoing clinical trials of dulanermin and drozitumab to assess potential utility for patient selection. Clin Cancer Res; 16(5); 1587–96
Data from Role of ERK-BIM and STAT3-Survivin Signaling Pathways in ALK Inhibitor–Induced Apoptosis in EML4-ALK–Positive Lung Cancer
Authors: Takezawa Ken , Okamoto Isamu , Nishio Kazuto , Jänne Pasi A. , Nakagawa Kazuhiko
Published: April 2023

Abstract
Purpose:EML4-ALK (echinoderm microtubule-associated protein–like 4 anaplastic lymphoma kinase) was recently identified as a transforming fusion gene in non–small cell lung cancer. The purpose of the present study was to characterize the mechanism of malignant transformation by EML4-ALK.

Experimental Design: We established NIH 3T3 cells that stably express variant 1 or 3 of EML4-ALK and examined the signaling molecules that function downstream of EML4-ALK.

Results: Forced expression of EML4-ALK induced marked activation of extracellular signal–regulated kinase (ERK) and STAT3, but not that of AKT. Inhibition of ERK or STAT3 signaling resulted in substantial attenuation of the proliferation of cells expressing either variant of EML4-ALK, suggesting that these signaling pathways function downstream of EML4-ALK in lung cancer cells. The specific ALK inhibitor TAE684 induced apoptosis that was accompanied both by upregulation of BIM, a proapoptotic member of the Bcl-2 family, and by downregulation of survivin, a member of the inhibitor of apoptosis protein (IAP) family, in EML4-ALK–expressing NIH 3T3 cells as well as in H3122 human lung cancer cells harboring endogenous EML4-ALK. Depletion of BIM and overexpression of survivin each inhibited TAE684-induced apoptosis, suggesting that both upregulation of BIM and downregulation of survivin contribute to TAE684-induced apoptosis in EML4-ALK–positive lung cancer cells. Furthermore, BIM and survivin expression was found to be independently regulated by ERK and STAT3 signaling pathways, respectively.

Conclusions: ALK inhibitor–induced apoptosis is mediated both by BIM upregulation resulting from inhibition of ERK signaling as well as by survivin downregulation resulting from inhibition of STAT3 signaling in EML4-ALK–positive lung cancer cells. Clin Cancer Res; 17(8); 2140–8. ©2011 AACR.
Data from Inherited Determinants of Ovarian Cancer Survival
Authors: Goode Ellen L. , Maurer Matthew J. , Sellers Thomas A. , Phelan Catherine M. , Kalli Kimberly R. , Fridley Brooke L. , Vierkant Robert A. , Armasu Sebastian M. , White Kristin L. , Keeney Gary L. , Cliby William A. , Rider David N. , Kelemen Linda E. , Jones Monica B. , Peethambaram Prema P. , Lancaster Johnathan M. , Olson Janet E. , Schildkraut Joellen M. , Cunningham Julie M. , Hartmann Lynn C.
Published: April 2023

Abstract
Purpose: Due to variation of outcome among cases, we sought to examine whether overall survival in ovarian cancer was associated with common inherited variants in 227 candidate genes from ovarian cancer–related pathways including angiogenesis, inflammation, detoxification, glycosylation, one-carbon transfer, apoptosis, cell cycle regulation, and cellular senescence.

Experimental Design: Blood samples were obtained from 325 women with invasive epithelial ovarian cancer diagnosed at the Mayo Clinic from 1999 to 2006. During a median follow-up of 3.8 years (range, 0.1-8.6 years), 157 deaths were observed. Germline DNA was analyzed at 1,416 single nucleotide polymorphisms (SNP). For all patients, and for 203 with serous subtype, we assessed the overall significance of each gene and pathway, and estimated risk of death via hazard ratios (HR) and 95% confidence intervals (CI), adjusting for known prognostic factors.

Results: Variation within angiogenesis was most strongly associated with survival time overall (P = 0.03) and among patients with serous cancer (P = 0.05), particularly for EIF2B5 rs4912474 (all patients HR, 0.69; 95% CI, 0.54-0.89; P = 0.004), VEGFC rs17697305 (serous subtype HR, 2.29; 95% CI, 1.34-3.92; P = 0.003), and four SNPs in VHL. Variation within the inflammation pathway was borderline significant (all patients, P = 0.09), and SNPs in CCR3, IL1B, IL18, CCL2, and ALOX5 which correlated with survival time are worthy of follow-up.

Conclusion: An extensive multiple-pathway assessment found evidence that inherited differences may play a role in outcome of ovarian cancer patients, particularly in genes within the angiogenesis and inflammation pathways. Our work supports efforts to target such mediators for therapeutic gain. Clin Cancer Res; 16(3); 995–1007
Data from Development of an Integrated Genomic Classifier for a Novel Agent in Colorectal Cancer: Approach to Individualized Therapy in Early Development
Authors: Pitts Todd M. , Tan Aik Choon , Kulikowski Gillian N. , Tentler John J. , Brown Amy M. , Flanigan Sara A. , Leong Stephen , Coldren Christopher D. , Hirsch Fred R. , Varella-Garcia Marileila , Korch Christopher , Eckhardt S. Gail
Published: April 2023

Abstract
Background: A plethora of agents is in early stages of development for colorectal cancer (CRC), including those that target the insulin-like growth factor I receptor (IGFIR) pathway. In the current environment of numerous cancer targets, it is imperative that patient selection strategies be developed with the intent of preliminary testing in the latter stages of phase I trials. The goal of this study was to develop and characterize predictive biomarkers for an IGFIR tyrosine kinase inhibitor, OSI-906, that could be applied in CRC-specific studies of this agent.

Methods: Twenty-seven CRC cell lines were exposed to OSI-906 and classified according to IC50 value as sensitive (≤1.5 μmol/L) or resistant (>5 μmol/L). Cell lines were subjected to immunoblotting and immunohistochemistry for effector proteins, IGFIR copy number by fluorescence in situ hybridization, KRAS/BRAF/phosphoinositide 3-kinase mutation status, and baseline gene array analysis. The most sensitive and resistant cell lines were used for gene array and pathway analyses, along with shRNA knockdown of highly ranked genes. The resulting integrated genomic classifier was then tested against eight human CRC explants in vivo.

Results: Baseline gene array data from cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which, in combination with IGFIR fluorescence in situ hybridization and KRAS mutational status, was able to predict with 100% accuracy a test set of patient-derived CRC xenografts.

Conclusions: These results indicate that an integrated approach to the development of individualized therapy is feasible and should be applied early in the development of novel agents, ideally in conjunction with late-stage phase I trials. Clin Cancer Res; 16(12); 3193–204. ©2010 AACR.
Data from Bevacizumab plus Fotemustine as First-line Treatment in Metastatic Melanoma Patients: Clinical Activity and Modulation of Angiogenesis and Lymphangiogenesis Factors
Authors: Del Vecchio Michele , Mortarini Roberta , Canova Stefania , Di Guardo Lorenza , Pimpinelli Nicola , Sertoli Mario R. , Bedognetti Davide , Queirolo Paola , Morosini Paola , Perrone Tania , Bajetta Emilio , Anichini Andrea
Published: April 2023

Abstract
Purpose: To assess the clinical and biological activity of the association of bevacizumab and fotemustine as first-line treatment in advanced melanoma patients.

Experimental Design: Previously untreated, metastatic melanoma patients (n = 20) received bevacizumab (at 15 mg/kg every 3 weeks) and fotemustine (100 mg/m2 by intravenous administration on days 1, 8, and 15, repeated after 4 weeks) in a multicenter, single-arm, open-label, phase II study. Primary endpoint was the best overall response rate; other endpoints were toxicity, time to progression (TTP), and overall survival (OS). Serum cytokines, angiogenesis, and lymphangiogenesis factors were monitored by multiplex arrays and by in vitro angiogenesis assays. Effects of fotemustine on melanoma cells, in vitro, on vascular endothelial growth factor (VEGF)-C release and apoptosis were assessed by ELISA and flow cytometry, respectively.

Results: One complete response, 2 partial responses (PR), and 10 patients with stable disease were observed. TTP and OS were 8.3 and 20.5 months, respectively. Fourteen patients experienced adverse events of toxicity grade 3–4. Serum VEGF-A levels in evaluated patients (n = 15) and overall serum proangiogenic activity were significantly inhibited. A significant reduction in VEGF-C levels was found in several post-versus pretherapy serum samples. In vitro, fotemustine inhibited VEGF-C release by melanoma cells without inducing significant cell death. Serum levels of interleukin (IL)-10 and IL-12p70 showed the highest levels in sera of PR patients, compared with patients with stable or progressive disease whereas IL-23 showed the opposite pattern.

Conclusions: The combination of bevacizumab plus fotemustine has clinical activity in advanced melanoma and promotes systemic modulation of angiogenesis and lymphangiogenesis factors. Clin Cancer Res; 16(23); 5862–72. ©2010 AACR.
Data from Prevention of Chemotherapy-Induced Anemia and Thrombocytopenia by Constant Administration of Stem Cell Factor
Authors: Bartucci Monica , Dattilo Rosanna , Martinetti Daniela , Todaro Matilde , Zapparelli Giuseppina , Di Virgilio Antonio , Biffoni Mauro , De Maria Ruggero , Zeuner Ann
Published: April 2023

Abstract
Purpose: Chemotherapy-induced apoptosis of immature hematopoietic cells is a major cause of anemia and thrombocytopenia in cancer patients. Although hematopoietic growth factors such as erythropoietin and colony-stimulating factors cannot prevent the occurrence of drug-induced myelosuppression, stem cell factor (SCF) has been previously shown to protect immature erythroid and megakaryocytic cells in vitro from drug-induced apoptosis. However, the effect of SCF in vivo as a single myeloprotective agent has never been elucidated.

Experimental Design: The ability of SCF to prevent the occurrence of chemotherapy-induced anemia and thrombocytopenia was tested in a mouse model of cisplatin-induced myelosuppression. To highlight the importance of maintaining a continuous antiapoptotic signal in immature hematopoietic cells, we compared two treatment schedules: in the first schedule, SCF administration was interrupted during chemotherapy treatment and resumed thereafter, whereas in the second schedule, SCF was administered without interruption for 7 days, including the day of chemotherapy treatment.

Results: The administration of SCF to cisplatin-treated mice could preserve bone marrow integrity, inhibit apoptosis of erythroid and megakaryocytic precursors, prevent chemotherapy-induced anemia, and rapidly restore normal platelet production. Treatment with SCF increased the frequency of Bcl-2/Bcl-XL–positive bone marrow erythroid cells and sustained Akt activation in megakaryocytes. Myeloprotection was observed only when SCF was administered concomitantly with cisplatin and kept constantly present during the days following chemotherapy treatment.

Conclusions: SCF treatment can prevent the occurrence of chemotherapy-induced anemia and thrombocytopenia in mice, indicating a potential use of this cytokine in the supportive therapy of cancer patients. Clin Cancer Res; 17(19); 6185–91. ©2011 AACR.
Data from Immunobiological Characterization of Cancer Stem Cells Isolated from Glioblastoma Patients
Authors: Di Tomaso Tiziano , Mazzoleni Stefania , Wang Ena , Sovena Gloria , Clavenna Daniela , Franzin Alberto , Mortini Pietro , Ferrone Soldano , Doglioni Claudio , Marincola Francesco M. , Galli Rossella , Parmiani Giorgio , Maccalli Cristina
Published: April 2023

Abstract
Purpose: Cancer stem cells (CSC) have been isolated from human tumors, including glioblastoma multiforme (GBM). The aims of this study were the immunobiological characterization of GBM CSCs and the assessment of whether these cells represent suitable targets for immunotherapy.

Experimental Design: GBM CSC lines and their fetal bovine serum (FBS)–cultured non-CSC pair lines were generated and examined by flow cytometry for expression of known tumor antigens, MHC-I and MHC-II molecules, antigen-processing machinery components, and NKG2D ligands. In addition, immunogenicity and immunosuppression of such cell lines for autologous or allogeneic T lymphocytes were tested by cytokine secretion (ELISPOT) or proliferation (carboxyfluorescein diacetate succinimidyl ester) assays, respectively.

Results: Both GBM CSC and FBS lines were weakly positive and negative for MHC-I, MHC-II, and NKG2D ligand molecules, respectively. Antigen-processing machinery molecules were also defective in both cell types. Upregulation of most molecules was induced by IFNs or 5-Aza deoxycytidine, although more efficiently in FBS than in CSCs. Patient T-cell responses, mediated by both TH1 and the TH2 subsets, against autologous CSC could be induced in vitro. In addition, CSC but not their paired FBS tumor lines inhibited T-cell proliferation of healthy donors. Notably, a differential gene signature that was confirmed at the protein levels for some immunologic-related molecules was also found between CSC and FBS lines.

Conclusions: These results indicate lower immunogenicity and higher suppressive activity of GBM CSC compared with FBS lines. The immunogenicity, however, could be rescued by immune modulation leading to anti-GBM T cell–mediated immune response. Clin Cancer Res; 16(3); 800–13
Data from HER2-Affitoxin: A Potent Therapeutic Agent for the Treatment of HER2-Overexpressing Tumors
Authors: Zielinski Rafal , Lyakhov Ilya , Hassan Moinuddin , Kuban Monika , Shafer-Weaver Kimberly , Gandjbakhche Amir , Capala Jacek
Published: April 2023

Abstract
Purpose: Cancers overexpressing the HER2/neu gene are usually more aggressive and are associated with poor prognosis. Although trastuzumab has significantly improved the outcome, many tumors do not respond or acquire resistance to current therapies. To provide an alternative HER2-targeted therapy, we have developed and characterized a novel recombinant protein combining an HER2-specific Affibody and modified Pseudomonas aeruginosa exotoxin A (PE 38), which, after binding to HER2, is internalized and delivered to the cytosol of the tumor cell, where it blocks protein synthesis by ADP ribosylation of eEF-2.

Experimental Design: The effect of the Affitoxin on cell viability was assessed using CellTiter-Glo (Promega). To assess HER2-specific efficacy, athymic nude mice bearing BT-474 breast cancer, SK-OV-3 ovarian cancer, and NCI-N87 gastric carcinoma xenografts were treated with the Affitoxin (HER2- or Tag-specific), which was injected every third day. Affitoxin immunogenicity in female BALB/c mice was investigated using standard antibody production and splenocyte proliferation assays.

Results:In vitro experiments proved that HER2-Affitoxin is a potent agent that eliminates HER2-overexpressing cells at low picomolar concentrations. Therapeutic efficacy studies showed complete eradication of relatively large BT-474 tumors and significant effects on SK-OV-3 and NCI-N87 tumors. HER2-Affitoxin cleared quickly from circulation (T1/2 < 10 minutes) and was well tolerated by mice at doses of 0.5 mg/kg and below. Immunogenicity studies indicated that HER2-Affitoxin induced antibody development after the third injected dose.

Conclusions: Our findings showed that HER2-Affitoxin is an effective anticancer agent and a potential candidate for clinical studies. Clin Cancer Res; 17(15); 5071–81. ©2011 AACR.
Data from Synergistic Effect between Erlotinib and MEK Inhibitors in KRAS Wild-Type Human Pancreatic Cancer Cells
Authors: Diep Caroline H. , Munoz Ruben M. , Choudhary Ashish , Von Hoff Daniel D. , Han Haiyong
Published: April 2023

Abstract
Purpose: The combination of erlotinib and gemcitabine has shown a small but statistically significant survival advantage when compared with gemcitabine alone in patients with advanced pancreatic cancer. However, the overall survival rate with the erlotinib and gemcitabine combination is still low. In this study, we sought to identify gene targets that, when inhibited, would enhance the activity of epidermal growth factor receptor (EGFR)-targeted therapies in pancreatic cancer cells.

Experimental Design: A high-throughput RNA interference (RNAi) screen was carried out to identify candidate genes. Selected gene hits were further confirmed and mechanisms of action were further investigated using various assays.

Results: Six gene hits from siRNA screening were confirmed to significantly sensitize BxPC-3 pancreatic cancer cells to erlotinib. One of the hits, mitogen-activated protein kinase (MAPK) 1, was selected for further mechanistic studies. Combination treatments of erlotinib and two MAP kinase kinase (MEK) inhibitors, RDEA119 and AZD6244, showed significant synergistic effect for both combinations (RDEA119–erlotinib and AZD6244–erlotinib) compared with the corresponding single drug treatments in pancreatic cancer cell lines with wild-type KRAS (BxPC-3 and Hs 700T) but not in cell lines with mutant KRAS (MIA PaCa-2 and PANC-1). The enhanced antitumor activity of the combination treatment was further verified in the BxPC-3 and MIA PaCa-2 mouse xenograft model. Examination of the MAPK signaling pathway by Western blotting indicated effective inhibition of the EGFR signaling by the drug combination in KRAS wild-type cells but not in KRAS mutant cells.

Conclusions: Overall, our results suggest that combination therapy of an EGFR and MEK inhibitors may have enhanced efficacy in patients with pancreatic cancer. Clin Cancer Res; 17(9); 2744–56. ©2011 AACR.
Data from Pazopanib Reveals a Role for Tumor Cell B-Raf in the Prevention of HER2+ Breast Cancer Brain Metastasis
Authors: Gril Brunilde , Palmieri Diane , Qian Yong , Smart DeeDee , Ileva Lilia , Liewehr David J. , Steinberg Seth M. , Steeg Patricia S.
Published: April 2023

Abstract
Purpose: Brain metastases of breast cancer contribute significantly to patient morbidity and mortality. We have tested pazopanib, a recently approved antiangiogenic drug that targets VEGFR1, VEGFR2, VEGFR3, PDGFRβ, PDGFRα, and c-kit, for prevention of experimental brain metastases and mechanism of action.

Experimental Design:In vitro assays included B-Raf enzymatic assays, Western blots, and angiogenesis assays. For in vivo assays, HER2 transfectants of the brain seeking sublines of MDA-MB-231 cells (231-BR-HER2) and MCF7 cells (MCF7-HER2-BR3, derived herein) were injected into the left cardiac ventricle of mice and treated with vehicle or pazopanib beginning on day 3 postinjection. Brain metastases were counted histologically, imaged, and immunostained.

Results: Treatment with 100 mg/kg of pazopanib resulted in a 73% decline in large 231-BR-HER2 metastases (P < 0.0001) and a 39% decline in micrometastases (P = 0.004). In vitro, pazopanib was directly antiproliferative to 231-BR-HER2 breast cancer cells and inhibited MEK and ERK activation in vitro despite B-Raf and Ras mutations. Enzymatic assays demonstrated that pazopanib directly inhibited the wild type and exon 11 oncogenic mutant, but not the V600E mutant forms of B-Raf. Activation of the B-Raf targets pERK1/2 and pMEK1/2 was decreased in pazopanib-treated brain metastases whereas blood vessel density was unaltered. In the MCF7-HER2-BR3 experimental brain metastasis model, pazopanib reduced overall brain metastasis volume upon magnetic resonance imaging (MRI) by 55% (P = 0.067), without affecting brain metastasis vascular density.

Conclusions: The data identify a new activity for pazopanib directly on tumor cells as a pan-Raf inhibitor and suggest its potential for prevention of brain metastatic colonization of HER2+ breast cancer. Clin Cancer Res; 17(1); 142–53. ©2010 AACR.
Data from Nrf2 and Keap1 Abnormalities in Non–Small Cell Lung Carcinoma and Association with Clinicopathologic Features
Authors: Solis Luisa M. , Behrens Carmen , Dong Wenli , Suraokar Milind , Ozburn Natalie C. , Moran Cesar A. , Corvalan Alejandro H. , Biswal Shyam , Swisher Stephen G. , Bekele B. Nebiyou , Minna John D. , Stewart David J. , Wistuba Ignacio I.
Published: April 2023

Abstract
Purpose: To understand the role of nuclear factor erythroid-2–related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) in non–small cell lung cancer (NSCLC), we studied their expression in a large series of tumors with annotated clinicopathologic data, including response to platinum-based adjuvant chemotherapy.

Experimental Design: We determined the immunohistochemical expression of nuclear Nrf2 and cytoplasmic Keap1 in 304 NSCLCs and its association with patients' clinicopathologic characteristics, and in 89 tumors from patients who received neoadjuvant (n = 26) or adjuvant platinum-based chemotherapy (n = 63). We evaluated NFE2L2 and KEAP1 mutations in 31 tumor specimens.

Results: We detected nuclear Nrf2 expression in 26% of NSCLCs; it was significantly more common in squamous cell carcinomas (38%) than in adenocarcinomas (18%; P < 0.0001). Low or absent Keap1 expression was detected in 56% of NSCLCs; it was significantly more common in adenocarcinomas (62%) than in squamous cell carcinomas (46%; P = 0.0057). In NSCLC, mutations of NFE2L2 and KEAP1 were very uncommon (2 of 29 and 1 of 31 cases, respectively). In multivariate analysis, Nrf2 expression was associated with worse overall survival [P = 0.0139; hazard ratio (HR), 1.75] in NSCLC patients, and low or absent Keap1 expression was associated with worse overall survival (P = 0.0181; HR, 2.09) in squamous cell carcinoma. In univariate analysis, nuclear Nrf2 expression was associated with worse recurrence-free survival in squamous cell carcinoma patients who received adjuvant treatment (P = 0.0410; HR, 3.37).

Conclusions: Increased expression of Nrf2 and decreased expression of Keap1 are common abnormalities in NSCLC and are associated with a poor outcome. Nuclear expression of Nrf2 in malignant lung cancer cells may play a role in resistance to platinum-based treatment in squamous cell carcinoma. Clin Cancer Res; 16(14); 3743–53. ©2010 AACR.
Data from A Novel HSP90 Inhibitor Delays Castrate-Resistant Prostate Cancer without Altering Serum PSA Levels and Inhibits Osteoclastogenesis
Authors: Lamoureux Francois , Thomas Christian , Yin Min-Jean , Kuruma Hidetoshi , Fazli Ladan , Gleave Martin E. , Zoubeidi Amina
Published: April 2023

Abstract
Purpose: Prostate cancer responds initially to antiandrogen therapies; however, progression to castration-resistant disease frequently occurs. Therefore, there is an urgent need for novel therapeutic agents that can prevent the emergence of castrate-resistant prostate cancer (CRPC). HSP90 is a molecular chaperone involved in the stability of many client proteins including Akt and androgen receptor (AR). 17-Allylamino-17-demethoxy-geldanamycin (17-AAG) has been reported to inhibit tumor growth in various cancers; however, it induces tumor progression in the bone microenvironment.

Methods: Cell growth, apoptosis, and AR transactivation were examined by crystal violet assay, flow cytometric, and luciferase assays, respectively. The consequence of HSP90 therapy in vivo was evaluated in LNCaP xenograft model. The consequence of PF-04928473 therapy on bone metastasis was studied using an osteoclastogenesis in vitro assay.

Results: PF-04928473 inhibits cell growth in a panel of prostate cancer cells, induces cell-cycle arrest at sub-G1, and leads to apoptosis and increased caspase-3 activity. These biological events were accompanied by decreased activation of Akt and Erk as well as decreased expression of Her2, and decreased AR expression and activation in vitro. In contrast to 17-AAG, PF-04928473 abrogates RANKL-induced osteoclast differentiation by affecting NF-κB activation and Src phosphorylation. Finally, PF-04929113 inhibited tumor growth and prolonged survival compared with controls. Surprisingly, PF-04929113 did not reduce serum prostate-specific antigen (PSA) levels in vivo; in parallel, these decrease in tumor volume.

Conclusion: These data identify significant anticancer activity of PF-04929113 in CRPC but suggest that serum PSA may not prove useful as pharmacodynamic tool for this drug. Clin Cancer Res; 17(8); 2301–13. ©2011 AACR.
Data from Pancreatic Tumor Suppression by Benzyl Isothiocyanate Is Associated with Inhibition of PI3K/AKT/FOXO Pathway
Authors: Boreddy Srinivas Reddy , Pramanik Kartick C. , Srivastava Sanjay K.
Published: April 2023

Abstract
Purpose: Our previous studies have shown that benzyl isothiocyanate (BITC) suppress pancreatic cancer growth by inducing apoptosis but the molecular mechanism was unclear. In this study we hypothesized the involvement of PI3K/AKT/FOXO pathway in BITC-induced apoptosis.

Experimental Design: Mice were implanted BxPC-3 tumor xenografts and orally gavaged with 12 μmol BITC. Plasma and tumor BITC concentration was estimated by liquid chromatography/tandem mass spectrometry. BxPC-3 and PanC-1 cells were used to elucidate PI3K/AKT/FOXO pathway. Electrophoretic mobility shift assay (EMSA), DNA binding activity, immunofluorescence, and gene transfection were used to delineate the mechanism.

Results: BITC-treated mice showed 43% less tumor growth as compared with control mice and correlated well with the therapeutic concentrations of 6.5 μmol/L BITC achieved in plasma and 7.5 μmol/g BITC in tumor tissue. Western blot analyses and immunohistochemistry revealed that tumors from BITC-treated mice showed reduced phosphorylation of PI3K, AKT, PDK1, mTOR, FOXO1, and FOXO3a and increased apoptosis. Complementing our in vivo results, we made similar observations in a dose- and time-dependent manner in BITC-treated BxPC-3 and Panc-1 cells. Binding of FOXO1 with 14-3-3 proteins was also reduced drastically by BITC treatment indicating nuclear retention of FOXO1 and this observation was further confirmed with EMSA, immunofluorescence, DNA binding, and upregulation of FOXO-responsive proteins Bim, p27, and p21 in BxPC-3 cells. Overexpression of AKT by transient transfection significantly blocked the modulation of FOXO proteins and protected the cells from BITC-mediated apoptosis and growth suppression.

Conclusions: Our results provide convincing evidence on the involvement of PI3K/AKT/FOXO pathway in BITC-mediated pancreatic tumor growth suppression. Clin Cancer Res; 17(7); 1784–95. ©2011 AACR.