Publications by authors named "Ahmed Chenna"

19 Publications

  • Page 1 of 1

Molecular and Clinical Activity of CDX-3379, an Anti-ErbB3 Monoclonal Antibody, in Head and Neck Squamous Cell Carcinoma Patients.

Clin Cancer Res 2019 10 15;25(19):5752-5758. Epub 2019 Jul 15.

Department of Medicine, Division of Hematology/Oncology, University of Arizona Cancer Center, Tucson, Arizona.

Purpose: ErbB3 and its ligand neuregulin-1 (NRG1) are widely expressed in head and neck squamous cell carcinoma (HNSCC) and associated with tumor progression. A "window-of-opportunity" study (NCT02473731) was conducted to evaluate the pharmacodynamic effects of CDX-3379, an anti-ErbB3 mAb, in patients with HNSCC.

Patients And Methods: Twelve patients with newly diagnosed, operable HNSCC received two infusions of CDX-3379 (1,000 mg) at a 2-week interval prior to tumor resection. The primary study objective was to achieve ≥50% reduction in tumor ErbB3 signaling (phosphorylation of ErbB3; pErbB3) in ≥30% of patients. Other potential tumor biomarkers, pharmacokinetics, safety, and tumor measurements were also assessed.

Results: pErbB3 was detectable in all tumors prior to treatment and decreased for 10 of 12 (83%) patients following CDX-3379 dosing, with ≥50% reduction in 7 of 12 (58%; = 0.04; 95% confidence interval, 27.7%-84.8%). Target trough CDX-3379 serum levels were achieved in all patients. CDX-3379 treatment-related toxicity was grade 1-2 and included diarrhea, fatigue, and acneiform dermatitis. Five of 12 (42%) patients had shrinkage in tumor burden, including a marked clinical response in a patient with human papillomavirus-negative oral cavity HNSCC. All patients with tumor shrinkage had tumors that expressed both NRG1 and ErbB3 and demonstrated reduced pErbB3 with CDX-3379 treatment.

Conclusions: This study demonstrates that CDX-3379 can inhibit tumor ErbB3 phosphorylation in HNSCC. CDX-3379 was well tolerated and associated with measurable tumor regression. A phase II study (NCT03254927) has been initiated to evaluate CDX-3379 in combination with cetuximab for patients with advanced HNSCC.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6820348PMC
October 2019

p95HER2-T cell bispecific antibody for breast cancer treatment.

Sci Transl Med 2018 10;10(461)

Preclinical Research Program, Vall d'Hebron Institute of Oncology (VHIO), 08035 Barcelona, Spain.

T cell bispecific antibodies (TCBs) are engineered molecules that include, within a single entity, binding sites to the T cell receptor and to tumor-associated or tumor-specific antigens. The receptor tyrosine kinase HER2 is a tumor-associated antigen in ~25% of breast cancers. TCBs targeting HER2 may result in severe toxicities, likely due to the expression of HER2 in normal epithelia. About 40% of HER2-positive tumors express p95HER2, a carboxyl-terminal fragment of HER2. Using specific antibodies, here, we show that p95HER2 is not expressed in normal tissues. We describe the development of p95HER2-TCB and show that it has a potent antitumor effect on p95HER2-expressing breast primary cancers and brain lesions. In contrast with a TCB targeting HER2, p95HER2-TCB has no effect on nontransformed cells that do not overexpress HER2. These data pave the way for the safe treatment of a subgroup of HER2-positive tumors by targeting a tumor-specific antigen.
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http://dx.doi.org/10.1126/scitranslmed.aat1445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498439PMC
October 2018

p95HER2 Methionine 611 Carboxy-Terminal Fragment Is Predictive of Trastuzumab Adjuvant Treatment Benefit in the FinHer Trial.

Clin Cancer Res 2018 07 13;24(13):3046-3052. Epub 2018 Mar 13.

Department of Oncology, Helsinki University Hospital & Helsinki University, Helsinki, Finland.

Expression of p95HER2 (p95), a truncated form of the HER2 receptor, which lacks the trastuzumab binding site but retains kinase activity, has been reported as a prognostic biomarker for poor outcomes in patients with trastuzumab-treated HER2-positive metastatic breast cancer. The impact of p95 expression on trastuzumab treatment efficacy in early HER2-positive breast cancer is less clear. In the current study, p95 was tested as a predictive marker of trastuzumab treatment benefit in the HER2-positive subset of the FinHer adjuvant phase III trial. In the FinHer trial, 232 patients with HER2-positive early breast cancer were randomized to receive chemotherapy plus 9 weeks of trastuzumab or no trastuzumab treatment. Quantitative p95 protein expression was measured in formalin-fixed paraffin-embedded samples using the p95 VeraTag assay (Monogram Biosciences), specific for the M611 form of p95. Quantitative HER2 protein expression was measured using the HERmark assay (Monogram Biosciences). Distant disease-free survival (DDFS) was used as the primary outcome measure. In the arm receiving chemotherapy only, increasing log(p95) correlated with shorter DDFS (HR, 2.0; = 0.02). In the arm receiving chemotherapy plus trastuzumab ( = 95), increasing log(p95) was not correlated with a shorter DDFS. In a combined analysis of both treatment arms, high breast tumor p95 content was significantly correlated with trastuzumab treatment benefit in multivariate models (interaction = 0.01). A high p95HER2/HER2 ratio identified patients with metastatic breast cancer with poor outcomes on trastuzumab-based therapies. Further investigation of the p95HER2/HER2 ratio as a potential prognostic or predictive biomarker for HER2-targeted therapy is warranted. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-3250DOI Listing
July 2018

High p95HER2/HER2 Ratio Associated With Poor Outcome in Trastuzumab-Treated HER2-Positive Metastatic Breast Cancer NCCTG N0337 and NCCTG 98-32-52 (Alliance).

Clin Cancer Res 2018 07 12;24(13):3053-3058. Epub 2018 Mar 12.

Center for Breast Health, Mayo Clinic, Jacksonville, Florida.

p95HER2 is a truncated form of HER2 that confers resistance to trastuzumab , but clinical results have been conflicting to date. Given that p95HER2 levels correlate with total HER2 expression levels, which confer better outcomes, we sought to evaluate the p95HER2/HER2 ratio in the North Central Cancer Treatment Group N0337 and N98-32-52 trials. The HERmark assay and VeraTag technology (Monogram Biosciences) were used to measure total HER2 and p95HER2 expression levels in 91 patient samples. In the multivariate model, increasing total HER2 level was significantly associated with longer (OS; HR, 0.33; = 0.002) and decreasing p95HER2 level was significantly associated with longer OS (HR, 4.2; = 0.01). Total HER2 expression level was significantly associated with longer progression-free survival (PFS) (HR, 0.57; = 0.04), whereas p95HER2 level was not (HR, 1.7; = 0.25). However, there was a positive association between p95HER2 and total HER2 expression levels ( = 0.48; < 0.001). Consistent with our hypothesis, the ratio of p95HER2/HER2 was significantly associated with worsening PFS (HR, 1.7; = 0.04) and OS (HR, 2.8; = 0.002). Patients with the highest tertile of p95HER2/HER2 values had significantly less favorable PFS (HR, 1.8; = 0.06) and OS (HR, 2.3; = 0.02). A high p95HER2/HER2 ratio identified patients with metastatic breast cancer with poor outcomes on trastuzumab-based therapies. Further investigation of the p95HER2/HER2 ratio as a potential prognostic or predictive biomarker for HER2-targeted therapy is warranted. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-1864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314664PMC
July 2018

Adverse Health Effects of Thirdhand Smoke: From Cell to Animal Models.

Int J Mol Sci 2017 Apr 28;18(5). Epub 2017 Apr 28.

Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.

The newly identified smoke hazard, thirdhand smoke (THS), has gained public attention in recent years but its health impact and biological effects are largely unknown. THS may be defined by "the four Rs": tobacco chemicals that remain, react, re-emit, and/or are resuspended long after active smoking has ceased. This review summarizes recent research progress in the effects of THS on genotoxicity, metabolism and early life development using cellular and animal models. We first reported that THS generated in laboratory systems caused significant DNA damage in human cell lines. Our finding that THS significantly induces oxidative base lesions has been confirmed in skin wounds of mice models exposed to THS. THS also induced metabolomic changes in human reproductive cell lines. Furthermore, we demonstrated that early exposure to THS not only negatively impacts body weight in both male and female mice, but also induces persistent changes to immunological parameters in peripheral blood in these mice. These results indicate that THS is genotoxic at realistic experimental doses and that there may be a window of susceptibility for some forms of cellular damage induced by THS.
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http://dx.doi.org/10.3390/ijms18050932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5454845PMC
April 2017

Prolonged Response to Trastuzumab in a Patient With HER2-Nonamplified Breast Cancer With Elevated HER2 Dimerization Harboring an ERBB2 S310F Mutation.

J Natl Compr Canc Netw 2015 Sep;13(9):1066-70

From Mayo Clinic, Jacksonville, Florida; Monogram Biosciences/LabCorp, South San Francisco, California; Foundation Medicine, Cambridge, Massachusetts; and University of Maryland Greenebaum Cancer Center, Baltimore, Maryland.

In the current genomic era, increasing evidence demonstrates that approximately 2% of HER2-negative breast cancers, by current standard testings, harbor activating mutations of ERBB2. However, whether patients with HER2-negative breast cancer with activating mutations of ERBB2 also experience response to anti-HER2 therapies remains unclear. This case report describes a patient with HER2-nonamplified heavily pretreated breast cancer who experienced prolonged response to trastuzumab in combination with pertuzumab and fulvestrant. Further molecular analysis demonstrated that her tumors had an elevated HER2 dimerization that corresponded to ERBB2 S310F mutation. Located in the extracellular domain of the HER2 protein, this mutation was reported to promote noncovalent dimerization that results in the activation of the downstream signaling pathways. This case highlights the fact that HER2-targeted therapy may be valuable in patients harboring an ERBB2 S310F mutation.
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http://dx.doi.org/10.6004/jnccn.2015.0132DOI Listing
September 2015

Increased Expression of HER2, HER3, and HER2:HER3 Heterodimers in HPV-Positive HNSCC Using a Novel Proximity-Based Assay: Implications for Targeted Therapies.

Clin Cancer Res 2015 Oct 2;21(20):4597-606. Epub 2015 Jul 2.

University of California San Francisco, San Francisco, California.

Purpose: In other cancer types, HPV infection has been reported to coincide with overexpression of HER2 (ERBB2) and HER3 (ERBB3); however, the association between HER2 or HER3 expression and dimer formation in HNSCC has not been reported. Overexpression of HER2 and HER3 may contribute to resistance to EGFR inhibitors, including cetuximab, although the contribution of HPV in modulating cetuximab response remains unknown. Determination of heterodimerization of HER receptors is challenging and has not been reported in HNSCC. The present study aimed to determine the expression of HER proteins in HPV(+) versus HPV(-) HNSCC tumors using a proximity-based protein expression assay (VeraTag), and to determine the efficacy of HER-targeting agents in HPV(+) and HPV(-) HNSCC cell lines.

Experimental Design: Expression of total HER1, HER2, and HER3, p95HER2, p-HER3, HER1:HER1 homodimers, HER2:HER3 heterodimers, and the HER3-PI3K complex in 88 HNSCC was determined using VeraTag, including 33 baseline tumors from individuals treated in a trial including cetuximab. Inhibition of cell growth and protein activation with cetuximab and afatinib was compared in HPV(+) and HPV(-) cetuximab-resistant cell lines.

Results: Expression of total HER2, total HER3, HER2:HER3 heterodimers, and the HER3:PI3K complex were significantly elevated in HPV(+) HNSCC. Total EGFR was significantly increased in HPV(-) HNSCC where VeraTag assay results correlated with IHC. Afatinib significantly inhibited cell growth when compared with cetuximab in the HPV(+) and HPV(-) cetuximab-resistant HNSCC cell lines.

Conclusions: These findings suggest that agents targeting multiple HER proteins may be effective in the setting of HPV(+) HNSCC and/or cetuximab resistance.
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http://dx.doi.org/10.1158/1078-0432.CCR-14-3338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4609280PMC
October 2015

Quantitative HER2 and p95HER2 levels in primary breast cancers and matched brain metastases.

Neuro Oncol 2015 Sep 13;17(9):1241-9. Epub 2015 Feb 13.

Military Institute of Medicine, Warsaw, Poland (R.D.); Monogram Biosciences, Inc, South San Francisco, California (J.S., A.C., W.H., J.M.W., J.W., M.H., A.P., Y.L.); Medical University of Lublin, Lublin, Poland (T.T., B.J.); Institute of Oncology, Warsaw, Poland (T.M.); Białystok Oncology Center, Białystok, Poland (B.C.-A.); Opole Oncology Center, Opole, Poland (B.R.); Interior Affairs Hospital, Olsztyn, Poland (R.S.); Regional Oncology Center, Łódź, Poland (E.K.-W.); Oncology Center, Warsaw, Poland (M.C.); Medical University of Gdańsk, Gdańsk, Poland (A.K., W.B., J.J.).

Background: Patients with advanced breast cancer positive for human epidermal growth factor receptor 2 (HER2) are at high risk for brain metastasis (BM). The prevalence and significance of expression of HER2 and its truncated form p95HER2 (p95) in BM is unknown.

Methods: Seventy-five pairs of formalin-fixed paraffin-embedded samples from matched primary breast cancers (PBCs) and BM were assayed for quantitative p95 and HER2-total (H2T) protein expression using the p95 VeraTag and HERmark assays, respectively.

Results: There was a net increase in p95 and H2T expression in BM relative to the matched PBC (median 1.5-fold, P = .0007 and 2.1-fold, P < .0001, respectively). Cases with H2T-positive tumors were more likely to have the largest (≥5-fold) increase in p95 (odds ratio = 6.3, P = .018). P95 positivity in PBC correlated with progression-free survival (hazard ratio [HR] = 2.2, P = .013), trended with shorter time to BM (HR = 1.8, P = .070), and correlated with overall survival (HR = 2.1, P = .042). P95 positivity in BM correlated with time to BM (HR = 2.0, P = .016) but did not correlate with overall survival from the time of BM diagnosis (HR = 1.2, P = .61).

Conclusions: This is the first study of quantitative p95 and HER2 expression in matched PBC and BM. BM of breast cancer shows significant increases in expression of both biomarkers compared with matched PBC. These data provide a rationale for future correlative studies on p95 and HER2 levels in BM.
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http://dx.doi.org/10.1093/neuonc/nov012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4588754PMC
September 2015

HER2 over-expressing high grade endometrial cancer expresses high levels of p95HER2 variant.

Gynecol Oncol 2015 Apr 17;137(1):160-6. Epub 2015 Jan 17.

Vincent Center for Reproductive Biology, Vincent Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, MA 02114, United States; Gynecologic Oncology Division, Vincent Department of Obstetrics & Gynecology, Massachusetts General Hospital, Boston, MA 02114, United States; Harvard Medical School, Boston, MA 02115, United States.

Background: Subsets of high grade endometrial cancer (EnCa) over-express HER2 (ERBB2), yet clinical trials have failed to demonstrate any anti-tumor activity utilizing trastuzumab, an approved platform for HER2 positive breast cancer (BrCa). A truncated p95HER2 variant lacking the trastuzumab binding site may confer resistance. The objective of this investigation was to characterize the expression of the p95HER2 truncated variant in EnCa.

Materials And Methods: With institutional approval, 86 high grade EnCa tumors were identified with tumor specimens from surgeries performed between 2000 and 2011. Clinical data were collected and all specimens underwent tumor genotyping, HER2 immunohistochemistry (IHC, HercepTest®), HER2 fluorescent in situ hybridization (FISH), along with total HER2 (H2T) and p95HER2 assessment with VeraTag® testing. Regression models were used to compare a cohort of 86 breast tumors selected for equivalent HER2 protein expression.

Results: We identified 44 high grade endometrioid and 42 uterine serous carcinomas (USC). IHC identified high HER2 expression (2+ or 3+) in 59% of the tumors. HER2 gene amplification was observed in 16 tumors (12 USC, 4 endometrioid). Both HER2 gene amplification and protein expression correlated with H2T values. High p95HER2 expression above 2.8RF/mm2 was observed in 53% (n=54) with significant correlation with H2T levels. When matched to a cohort of 107 breast tumors based on HercepTest HER2 expression, high grade EnCa presented with higher p95 levels (p<0.001).

Conclusions: These data demonstrate that compared to BrCa, high grade EnCa expresses higher levels of p95HER2 possibly providing rationale for the trastuzumab resistance observed in EnCa.
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http://dx.doi.org/10.1016/j.ygyno.2015.01.533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380649PMC
April 2015

High HER2 expression correlates with response to the combination of lapatinib and trastuzumab.

Clin Cancer Res 2015 Feb 2;21(3):569-76. Epub 2014 Dec 2.

Human Oncology and Pathogenesis Program (HOPP), Memorial Sloan Kettering Cancer Center, New York, New York. Breast Medicine Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.

Purpose: Expression of p95HER2 has been associated with resistance to trastuzumab-based therapy in patients with metastatic breast cancer. Conversely, high levels of HER2 have been linked with increased clinical benefit from anti-HER2 therapy. In this work, we aimed to investigate whether the levels of p95HER2 and HER2 can predict response to anti-HER2 therapy in patients with breast cancer.

Experimental Design: We measured p95HER2 and HER2 by VeraTag and HERmark, respectively, in primary tumors of patients enrolled in the neoadjuvant phase III study NeoALTTO and correlated these variables with pathologic complete response (pCR) and progression-free survival (PFS) following lapatinib (L), trastuzumab (T), or the combination of both agents (L+T).

Results: A positive correlation between p95HER2 and HER2 levels was found in the 274 cases (60%) in which quantification of both markers was possible. High levels of these markers were predictive for pCR, especially in the hormone receptor (HR)-positive subset of patients. High HER2 expression was associated with increased pCR rate upon L+T irrespective of the HR status. To examine whether the levels of either p95HER2 or HER2 could predict for PFS in patients treated with lapatinib, trastuzumab or L+T, we fit to the PFS data in Cox models containing log2(p95HER2) or log2(HER2). Both variables correlated with longer PFS.

Conclusions: Increasing HER2 protein expression correlated with increased benefit of adding lapatinib to trastuzumab. HER2 expression is a stronger predictor of pCR and PFS than p95HER2 for response to lapatinib, trastuzumab and, more significantly, L+T.
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http://dx.doi.org/10.1158/1078-0432.CCR-14-1824DOI Listing
February 2015

Quantitative measurements of tumoral p95HER2 protein expression in metastatic breast cancer patients treated with trastuzumab: independent validation of the p95HER2 clinical cutoff.

Clin Cancer Res 2014 May 25;20(10):2805-13. Epub 2014 Mar 25.

Authors' Affiliations: Military Institute of Medicine, Warsaw; Lublin Oncology Center, Lublin; Białystok Oncology Center, Białystok; Greater Poland Cancer Center, Poznań; West Pomeranian Oncology Center, Szczecin; Opole Oncology Center, Opole; Warmia and Masuria Oncology Center, Olsztyn; Bydgoszcz Oncology Center, Bydgoszcz; Beskidy Oncology Center, Bielsko-Biała; Medical University of Gdańsk, Gdańsk, Poland; and Monogram Biosciences, Inc., South San Francisco, California

Purpose: P95HER2 (p95) is a truncated form of the HER2, which lacks the trastuzumab-binding site and contains a hyperactive kinase domain. Previously, an optimal clinical cutoff of p95 expression for progression-free survival (PFS) and overall survival (OS) was defined using a quantitative VeraTag assay (Monogram Biosciences) in a training set of trastuzumab-treated metastatic breast cancer (MBC) patients.

Experimental Design: In the current study, the predictive value of the p95 VeraTag assay cutoff established in the training set was retrospectively validated for PFS and OS in an independent series of 240 trastuzumab-treated MBC patients from multiple institutions.

Results: In the subset of 190 tumors assessed as HER2-total (H2T)-positive using the quantitative HERmark assay (Monogram Biosciences), p95 VeraTag values above the predefined cutoff correlated with shorter PFS (HR = 1.43; P = 0.039) and shorter OS (HR = 1.94; P = 0.0055) where both outcomes were stratified by hormone receptor status and tumor grade. High p95 expression correlated with shorter PFS (HR = 2.41; P = 0.0003) and OS (HR = 2.57; P = 0.0025) in the hormone receptor-positive subgroup of patients (N = 78), but not in the hormone receptor-negative group. In contrast with the quantitative p95 VeraTag measurements, p95 immunohistochemical expression using the same antibody was not significantly correlated with outcomes.

Conclusions: The consistency in the p95 VeraTag cutoff across different cohorts of patients with MBC treated with trastuzumab justifies additional studies using blinded analyses in larger series of patients.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-2782DOI Listing
May 2014

HER3, p95HER2, and HER2 protein expression levels define multiple subtypes of HER2-positive metastatic breast cancer.

Breast Cancer Res Treat 2013 Aug;141(1):43-53

Breast Oncology Research, Division of Hematology/Oncology, Department of Medicine, Penn State Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USA.

Trastuzumab is effective in the treatment of HER2/neu over-expressing breast cancer, but not all patients benefit from it. In vitro data suggest a role for HER3 in the initiation of signaling activity involving the AKT–mTOR pathway leading to trastuzumab insensitivity. We sought to investigate the potential of HER3 alone and in the context of p95HER2 (p95), a trastuzumab resistance marker, as biomarkers of trastuzumab escape. Using the VeraTag® assay platform, we developed a dual antibody proximity-based assay for the precise quantitation of HER3 total protein (H3T) from formalin-fixed paraffin-embedded (FFPE) breast tumors. We then measured H3T in 89 patients with metastatic breast cancer treated with trastuzumab-based therapy, and correlated the results with progression-free survival and overall survival using Kaplan–Meier and decision tree analyses that also included HER2 total (H2T) and p95 expression levels. Within the sub-population of patients that over-expressed HER2, high levels of HER3 and/or p95 protein expression were significantly associated with poor clinical outcomes on trastuzumab-based therapy. Based on quantitative H3T, p95, and H2T measurements, multiple subtypes of HER2-positive breast cancer were identified that differ in their outcome following trastuzumab therapy. These data suggest that HER3 and p95 are informative biomarkers of clinical outcomes on trastuzumab therapy, and that multiple subtypes of HER2-positive breast cancer may be defined by quantitative measurements of H3T, p95, and H2T.
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http://dx.doi.org/10.1007/s10549-013-2665-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3758835PMC
August 2013

Thirdhand smoke causes DNA damage in human cells.

Mutagenesis 2013 Jul 5;28(4):381-91. Epub 2013 Mar 5.

Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.

Exposure to thirdhand smoke (THS) is a newly described health risk. Evidence supports its widespread presence in indoor environments. However, its genotoxic potential, a critical aspect in risk assessment, is virtually untested. An important characteristic of THS is its ability to undergo chemical transformations during aging periods, as demonstrated in a recent study showing that sorbed nicotine reacts with the indoor pollutant nitrous acid (HONO) to form tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-4-(3-pyridyl)butanal (NNA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The goal of this study was to assess the genotoxicity of THS in human cell lines using two in vitro assays. THS was generated in laboratory systems that simulated short (acute)- and long (chronic)-term exposures. Analysis by liquid chromatography-tandem mass spectrometry quantified TSNAs and common tobacco alkaloids in extracts of THS that had sorbed onto cellulose substrates. Exposure of human HepG2 cells to either acute or chronic THS for 24h resulted in significant increases in DNA strand breaks in the alkaline Comet assay. Cell cultures exposed to NNA alone showed significantly higher levels of DNA damage in the same assay. NNA is absent in freshly emitted secondhand smoke, but it is the main TSNA formed in THS when nicotine reacts with HONO long after smoking takes place. The long amplicon-quantitative PCR assay quantified significantly higher levels of oxidative DNA damage in hypoxanthine phosphoribosyltransferase 1 (HPRT) and polymerase β (POLB) genes of cultured human cells exposed to chronic THS for 24h compared with untreated cells, suggesting that THS exposure is related to increased oxidative stress and could be an important contributing factor in THS-mediated toxicity. The findings of this study demonstrate for the first time that exposure to THS is genotoxic in human cell lines.
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http://dx.doi.org/10.1093/mutage/get013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681537PMC
July 2013

Profiling the HER3/PI3K pathway in breast tumors using proximity-directed assays identifies correlations between protein complexes and phosphoproteins.

PLoS One 2011 Jan 28;6(1):e16443. Epub 2011 Jan 28.

Department of Oncology, Monogram Biosciences, South San Francisco, California, United States of America.

Background: The identification of patients for targeted antineoplastic therapies requires accurate measurement of therapeutic targets and associated signaling complexes. HER3 signaling through heterodimerization is an important growth-promoting mechanism in several tumor types and may be a principal resistance mechanism by which EGFR and HER2 expressing tumors elude targeted therapies. Current methods that can study these interactions are inadequate for formalin-fixed, paraffin-embedded (FFPE) tumor samples.

Methodology And Principal Findings: Herein, we describe a panel of proximity-directed assays capable of measuring protein-interactions and phosphorylation in FFPE samples in the HER3/PI3K/Akt pathway and examine the capability of these assays to inform on the functional state of the pathway. We used FFPE breast cancer cell line and tumor models for this study. In breast cancer cell lines we observe both ligand-dependent and independent activation of the pathway and strong correlations between measured activation of key analytes. When selected cell lines are treated with HER2 inhibitors, we not only observe the expected molecular effects based on mechanism of action knowledge, but also novel effects of HER2 inhibition on key targets in the HER receptor pathway. Significantly, in a xenograft model of delayed tumor fixation, HER3 phosphorylation is unstable, while alternate measures of pathway activation, such as formation of the HER3PI3K complex is preserved. Measurements in breast tumor samples showed correlations between HER3 phosphorylation and receptor interactions, obviating the need to use phosphorylation as a surrogate for HER3 activation.

Significance: This assay system is capable of quantitatively measuring therapeutically relevant responses and enables molecular profiling of receptor networks in both preclinical and tumor models.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016443PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3030586PMC
January 2011

Benzene-derived N2-(4-hydroxyphenyl)-deoxyguanosine adduct: UvrABC incision and its conformation in DNA.

Toxicol Lett 2010 Mar 16;193(1):26-32. Epub 2009 Dec 16.

Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132, USA.

Benzene, a ubiquitous human carcinogen, forms DNA adducts through its metabolites such as p-benzoquinone (p-BQ) and hydroquinone (HQ). N(2)-(4-Hydroxyphenyl)-2'-deoxyguanosine (N(2)-4-HOPh-dG) is the principal adduct identified in vivo by (32)P-postlabeling in cells or animals treated with p-BQ or HQ. To study its effect on repair specificity and replication fidelity, we recently synthesized defined oligonucleotides containing a site-specific adduct using phosphoramidite chemistry. We here report the repair of this adduct by Escherichia coli UvrABC complex, which performs the initial damage recognition and incision steps in the nucleotide excision repair (NER) pathway. We first showed that the p-BQ-treated plasmid was efficiently cleaved by the complex, indicating the formation of DNA lesions that are substrates for NER. Using a 40-mer substrate, we found that UvrABC incises the DNA strand containing N(2)-4-HOPh-dG in a dose- and time-dependent manner. The specificity of such repair was also compared with that of DNA glycosylases and damage-specific endonucleases of E. coli, both of which were found to have no detectable activity toward N(2)-4-HOPh-dG. To understand why this adduct is specifically recognized and processed by UvrABC, molecular modeling studies were performed. Analysis of molecular dynamics trajectories showed that stable G:C-like hydrogen bonding patterns of all three Watson-Crick hydrogen bonds are present within the N(2)-4-HOPh-G:C base pair, with the hydroxyphenyl ring at an almost planar position. In addition, N(2)-4-HOPh-dG has a tendency to form more stable stacking interactions than a normal G in B-type DNA. These conformational properties may be critical in differential recognition of this adduct by specific repair enzymes.
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http://dx.doi.org/10.1016/j.toxlet.2009.12.005DOI Listing
March 2010

A novel proximity assay for the detection of proteins and protein complexes: quantitation of HER1 and HER2 total protein expression and homodimerization in formalin-fixed, paraffin-embedded cell lines and breast cancer tissue.

Diagn Mol Pathol 2009 Mar;18(1):11-21

Departments of Research and Development, Clinical Research, and Operations, Monogram Biosciences Inc., 345 Oyster Point Boulevard, South San Francisco, CA 94080, USA.

The availability of drugs targeting the EGFR/HER/erbB signaling pathway has created a need for diagnostics that accurately predict treatment responses. We have developed and characterized a novel assay to provide sensitive and quantitative measures of HER proteins and homodimers in formalin-fixed, paraffin-embedded (FFPE) cell lines and breast tumor tissues, to test these variables. In the VeraTag assay, HER proteins and homodimers are detected through the release of fluorescent tags conjugated to specific HER antibodies, requiring proximity to a second HER antibody. HER2 protein quantification was normalized to tumor area, and compared to receptor numbers in 12 human tumor cell lines determined by fluorescence-activated cell sorting (FACS), and with HER immunohistochemistry (IHC) test categories and histoscores in cell lines and 170 breast tumors. HER1 and HER2 expression levels determined by the VeraTag assay are proportional to receptor number over more than a 2 log10 range, and HER homodimer levels are consistent with crosslinking and immunoprecipitation results. VeraTag HER2 measurements of breast tumor tissue and cell lines correlate with standard IHC test categories (P<0.001). VeraTag HER2 levels also agree with IHC histoscores at lower HER2 protein levels, but are continuous and overlapping between IHC test categories, extending the dynamic range 5-fold to 10-fold at higher HER2 levels. The VeraTag assay specifically and reproducibly measures HER1 and HER2 protein and homodimers in FFPE tissues. The continuous measure of HER2 protein levels over a broad dynamic range, and the novel HER2 homodimer measure, are presently being assessed as predictive markers for responses to targeted HER2 therapy.
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http://dx.doi.org/10.1097/PDM.0b013e31818cbdb2DOI Listing
March 2009

Synthesis of the fully protected phosphoramidite of the benzene-DNA adduct, N2-(4-Hydroxyphenyl)-2'-deoxyguanosine and incorporation of the later into DNA oligomers.

Nucleosides Nucleotides Nucleic Acids 2008 Aug;27(8):979-91

Monogram Biosciences Inc, South San Francisco, California, USA.

N(2)- (4-Hydroxyphenyl)-2'-deoxyguanosine-5'-O-DMT-3'-phosphoramidite has been synthesized and used to incorporate the N(2)-(4-hydroxyphenyl)-2'-dG (N(2)-4-HOPh-dG) into DNA, using solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium (Xantphos-chelated) catalyzed N(2)-arylation (Buchwald-Hartwig reaction) of a fully protected 2'-deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield and the adduct was converted to the required 5'-O-DMT-3'-O-phosphoramidite by standard methods. The latter was used to synthesize oligodeoxynucleotides in which the N(2)-4-HOPh-dG adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC. Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers.
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http://dx.doi.org/10.1080/15257770802258034DOI Listing
August 2008

Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis.

Nucleic Acids Res 2004 Sep 8;32(16):e126. Epub 2004 Sep 8.

ACLARA BioSciences, Inc., 1288 Pear Avenue, Mountain View, CA 94043, USA.

We describe a novel multiplexing technology using a library of small fluorescent molecules, termed eTag molecules, to code and quantify mRNA targets. eTag molecules, which have the same fluorometric property, but distinct charge-to-mass ratios possess pre-defined electrophoretic characteristics and can be resolved using capillary electrophoresis. Coupled with primary Invader mRNA assay, eTag molecules were applied to simultaneously quantify up to 44 mRNA targets. This multiplexing approach was validated by examining a panel of inflammation responsive genes in human umbilical vein endothelial cells stimulated with inflammatory cytokine interleukin 1beta. The laser-induced fluorescence detection and electrokinetic sample injection process in capillary electrophoresis allows sensitive quantification of thousands of copies of mRNA molecules in a reaction. The assay is precise, as evaluated by measuring qualified Z' factor, a dimensionless and simple characteristic for applications in high-throughput screening using mRNA assays. Our data demonstrate the synergy between the multiplexing capability of eTag molecules by sensitive capillary electrophoresis detection and the isothermal linear amplification characteristics of the Invader assay. eTag multiplex mRNA assay presents a unique platform for sensitive, high sample throughput and multiplex gene expression analysis.
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http://dx.doi.org/10.1093/nar/gnh119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC519123PMC
September 2004

Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea.

Mutat Res 2003 Oct;531(1-2):191-203

Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.

1,N(6)-Ethanoadenine (EA) is an exocyclic adduct formed from DNA reaction with the antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). To understand the role of this adduct in the mechanism of mutagenicity or carcinogenicity by BCNU, an oligonucleotide with a site-specific EA was synthesized using phosphoramidite chemistry. We now report the in vitro miscoding properties of EA in translesion DNA synthesis catalyzed by mammalian DNA polymerases (pols) alpha, beta, eta and iota. These data were also compared with those obtained for the structurally related exocyclic adduct, 1,N(6)-ethenoadenine (epsilonA). Using a primer extension assay, both pols alpha and beta were primarily blocked by EA or epsilonA with very minor extension. Pol eta, a member of the Y family of polymerases, was capable of catalyzing a significant amount of bypass across both adducts. Pol eta incorporated all four nucleotides opposite EA and epsilonA, but with differential preferences and mainly in an error-prone manner. Human pol iota, a paralog of human pol eta, was blocked by both adducts with a very small amount of synthesis past epsilonA. It incorporated C and, to a much lesser extent, T, opposite either adduct. In addition, the presence of an A adduct, e.g. epsilonA, could affect the specificity of pol iota toward the template T immediately 3' to the adduct. In conclusion, the four polymerases assayed on templates containing an EA or epsilonA showed differential bypass capacity and nucleotide incorporation specificity, with the two adducts not completely identical in influencing these properties. Although there was a measurable extent of error-free nucleotide incorporation, all these polymerases primarily misincorporated opposite EA, indicating that the adduct, similar to epsilonA, is a miscoding lesion.
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http://dx.doi.org/10.1016/j.mrfmmm.2003.07.006DOI Listing
October 2003