Identifying Mcl-1 protein dependencies using dimerization-specific antibody biomarker for predicting response to targeted apoptosis inducing therapies.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 7038-7038
Author(s):  
Michael H. Cardone ◽  
Sae Rin Jean ◽  
Bahriye Karakas ◽  
Sonia Kumar ◽  
Max Narovlyansky ◽  
...  
Keyword(s):  
Leukemia Cell ◽  
Predictive Biomarker ◽  
Response To Therapy ◽  
Cancer Therapies ◽  
Cancer Treatments ◽  
Protein Levels ◽  
Formalin Fixed Paraffin ◽  
Mimetic Peptides ◽  
Formalin Fixed Paraffin Embedded ◽  
Bh3 Mimetic

7038 Background: The anti-apoptotic Bcl-2 family proteins facilitate pro-survival and resistance to anti-cancer therapies. Measuring the function of these proteins has shown utility in predicting response to therapy. A method that expands the principle of BH3 profiling to solid tumors is presented. Measuring the occurrence of heterodimers of Myeloid Leukemia Cell Differentiation Protein (Mcl-1), and pro-apoptotic binding protein Bim is an indicator of cancer cell apoptotic priming state. The readout of Mcl-1 containing complex-specific biomarkers can identify survival dependencies in cancer cells potentially providing clinical utility in guiding cancer treatments. Methods: Engineered immunogens that recapitulate conformation-specific epitopes induced during binding of the Mcl-1/Bim protein complex were used to generate. monoclonal antibodies. One Heterodimer Specific Mcl-1 Bim (HsMcB) was chosen. The selective binding was confirmed using ELISA, fluorescence polarization, immunofluorescence microscopy and flow cytometry in Bim or Mcl-1 knockdown cells, and by immunohistochemistry in formalin-fixed paraffin-embedded patient tissue. Correlation of HsMcB measurements to BH3 profiling readouts from the Mcl-1 restricted Noxa peptide was explored. Results: HsMcB signal depends on both Mcl-1 and Bim protein levels. The ratio of the HsMcB to unbound protein (Mcl-1) signal ([HsMcB]/[Mcl-1]) was measured. Disruption of the complexes by BH3 mimetics targeted to Mcl-1 and depletion of Mcl-1 level using CDK9 inhibitors diminished the [HsMcB]/[Mcl-1] readout. We see correlations between these readouts and BH3 mimetic peptides readouts from the Mcl-1 specific Noxa and MS1 BH3 mimetic peptides in AML patient samples that have been treated with Mcl-1 inhibitors. Conclusions: Mcl-1 dependence is a predictive biomarker for venetoclax resistance and for response to Mcl-1 targeted therapies. Flow cytometric and IHC based measurements of a heterodimer complex offer a direct and simpler approach that harbors potential for use in clinical settings. Additional antibodies targeting Mcl-1/Bak and Mcl-1 Noxa complexes are being tested.

scholarly journals Molecular Markers for Prostate Cancer in Formalin-Fixed Paraffin-Embedded Tissues

2013 ◽  
Vol 2013 ◽  
pp. 1-15 ◽  
Author(s):  
Tamara Sequeiros ◽  
Marta García ◽  
Melania Montes ◽  
Mireia Oliván ◽  
Marina Rigau ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Molecular Markers ◽  
Tumor Grade ◽  
Developed Countries ◽  
Response To Therapy ◽  
Tissue Samples ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

Prostate cancer (PCa) is the most frequently diagnosed type of cancer in developed countries. The decisive method of diagnosis is based on the results of biopsies, morphologically evaluated to determine the presence or absence of cancer. Although this approach leads to a confident diagnosis in most cases, it can be improved by using the molecular markers present in the tissue. Both miRNAs and proteins are considered excellent candidates for biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues, due to their stability over long periods of time. In the last few years, a concerted effort has been made to develop the necessary tools for their reliable measurement in these types of samples. Furthermore, the use of these kinds of markers may also help in establishing tumor grade and aggressiveness, as well as predicting the possible outcomes in each particular case for the different treatments available. This would aid clinicians in the decision-making process. In this review, we attempt to summarize and discuss the potential use of microRNA and protein profiles in FFPE tissue samples as markers to better predict PCa diagnosis, progression, and response to therapy.

scholarly journals Mass Spectrometry Imaging Reveals Neutrophil Defensins as Additional Biomarkers for Anti-PD-(L)1 Immunotherapy Response in NSCLC Patients

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 863 ◽  
Author(s):  
Eline Berghmans ◽  
Julie Jacobs ◽  
Christophe Deben ◽  
Christophe Hermans ◽  
Glenn Broeckx ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Immune Response ◽  
Cancer Cells ◽  
Mass Spectrometry Imaging ◽  
Predictive Biomarker ◽  
Protein Biomarkers ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Nsclc Patients

(1) Background: Therapeutic blocking of the interaction between programmed death-1 (PD-1) with its ligand PD-L1, an immune checkpoint, is a promising approach to restore the antitumor immune response. Improved clinical outcomes have been shown in different human cancers, including non-small cell lung cancer (NSCLC). Unfortunately, still a high number of NSCLC patients are treated with immunotherapy without obtaining any clinical benefit, due to the limitations of PD-L1 protein expression as the currently sole predictive biomarker for clinical use; (2) Methods: In this study, we applied mass spectrometry imaging (MSI) to discover new protein biomarkers, and to assess the possible correlation between candidate biomarkers and a positive immunotherapy response by matrix-assisted laser desorption/ionization (MALDI) MSI in 25 formalin-fixed paraffin-embedded (FFPE) pretreatment tumor biopsies (Biobank@UZA); (3) Results: Using MALDI MSI, we revealed that the addition of neutrophil defensin 1, 2 and 3 as pretreatment biomarkers may more accurately predict the outcome of immunotherapy treatment in NSCLC. These results were verified and confirmed with immunohistochemical analyses. In addition, we provide in-vitro evidence of the immune stimulatory effect of neutrophil defensins towards cancer cells; and (4) Conclusions: With proteomic approaches, we have discovered neutrophil defensins as additional prospective biomarkers for an anti-PD-(L)1 immunotherapy response. Thereby, we also demonstrated that the neutrophil defensins contribute in the activation of the immune response towards cancer cells, which could provide a new lead towards an anticancer therapy.

Thymidylate synthase (TS) gene expression in patients with ALK positive (+) non-small cell lung cancer (NSCLC): Implications for therapy.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 7582-7582 ◽  
Author(s):  
David R. Gandara ◽  
Eric Huang ◽  
Sonal Desai ◽  
Philip C. Mack ◽  
Laurel Beckett ◽  
...  
Keyword(s):  
Gene Expression ◽  
Thymidylate Synthase ◽  
Predictive Biomarker ◽  
Rt Pcr ◽  
Kras Mutations ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Report Analysis ◽  
Increased Sensitivity ◽  
Alk Positive

7582 Background: ALK+ NSCLC represents a molecular target-defined patient population highly responsive to the ALK inhibitor crizotinib. Previous reports have also suggested increased sensitivity of ALK+ NSCLC to the chemotherapeutic agent pemetrexed. Thymidylate synthase (TS) is a candidate predictive biomarker for pemetrexed activity. Here we report analysis of the Response Genetics Inc. (RGI) database for this association and implications for therapy. Methods: ALK fusion was identified by a novel RT-PCR assay (Danenberg et al: ASCO 2010). For TS, RNA from microdissected formalin-fixed paraffin-embedded tumors was analyzed as previously described, reported as the ratio of gene expression to β-actin. For reference, a TS level <2.33 is the cutpoint for sensitivity. Results: TS levels were available from 63 ALK+ patients and 1,698 ALK- control lung adenocarcinoma patients. All ALK+ patients had adenocarcinomas without EGFR or KRAS mutations. Median age: 59.0 (range 33-88), gender (male/female) 32/31 (51%/49%). Median TS RNA level in ALK+ patients was 2.02, range (0.55-19.44), and in ALK- patients was 3.32 (0.36-53.51), p<0.0001 (Mann-Whitney test). The majority of ALK+ patients (N=43, 68%) had a TS level <2.33 cutpoint, compared to only 32% of ALK- patients (N=551, p<0.0001). Conclusions: This analysis demonstrates relatively low TS gene expression in ALK+ patient tumors as determined by RT-PCR. These data provide a mechanism of action supportive of pemetrexed sensitivity for ALK+ NSCLC. [Table: see text]

scholarly journals Developing a routine lab test for absolute quantification of Her2 in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tissues using Quantitative Dot Blot (QDB) method

2020 ◽  
Author(s):  
Guohua Yu ◽  
Wenfeng Zhang ◽  
Yunyun Zhang ◽  
Jiahong Lv ◽  
Shishou Wu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Clinical Practice ◽  
Daily Clinical Practice ◽  
Dot Blot ◽  
Her2 Protein ◽  
Protein Levels ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Abstract Background: Developing immunoassay for Her2 assessment suitable for Formalin Fixed Paraffin Embedded (FFPE) samples promises improved objectivity, consistency and accuracy in daily clinical practice. Yet, this effort is hindered by lacking available technique. The feasibility of Quantitative Dot Blot (QDB) method to meet this demand was evaluated in this study. Methods: QDB-based Immunoassay was developed for quantitative measurement of Her2 protein levels in this retrospective study using clinically validated antibody for immunohistochemistry (IHC antibody). Total protein lysates were extracted from 2X15 um FFPE slices collected from 332 breast cancer patients sequentially and non-selectively from local hospital. The absolutely quantitated Her2 levels in these samples were compared with the results from methods of Immunohistochemistry (IHC) and Fluorescence in situ hybridization (FISH) analyses using Receiver Operative Characteristics (ROC) analysis. Results: Her2 levels measured with two IHC antibodies of EP3 and 4B5 were strongly correlated (r=0.963, p=0.0000). We also achieved area under the curve (AUC) at 0.9753±0.01026 (95% CI: 0.9551 to 0.9954, p<0.0001, n=322) using provided results from IHC and FISH analyses, with 99.6% concordance with IHC and 88.1% with FISH at optimized cutoff of 0.261 nmole/g. The concordance rate with FISH analysis was improved to 94.1% when disagreed samples were re-analyzed by third party independently. Conclusions: We introduced the first immunoassay for absolute quantitation of protein biomarker levels in FFPE samples. This method fits well with daily clinical practice with its objectivity, simplicity and consistency, especially for local hospitals and hospitals in developing countries. The quantitatively measured Her2 protein levels in FFPE samples also allow statistically analysis at population level to meet the need of precision medicine. .

Comparison of three methods to detect mutations in the PI3K oncogene in FFPE cancer samples

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e22212-e22212
Author(s):  
J. Rodgers ◽  
C. Murray ◽  
N. Leaves
Keyword(s):  
Breast Cancer ◽  
Cell Signalling ◽  
Test Sample ◽  
Response To Therapy ◽  
Breast Cancer Patients ◽  
Formalin Fixed Paraffin ◽  
Test Kit ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Pathology Review

e22212 Background: PI3Kinase is an important target for new oncology therapies, since it is central to several cell signalling pathways associated with proliferation and survival. Many pharmaceutical companies have PI3K inhibitors in their drug development pipelines all of which are in early phase trials. Activating mutations in exons 9 and 20 of the PI3KCA oncogene have been observed in a number of important cancer types including: gastrointestinal, breast, liver, lung and genito-urinary cancers. The presence of such mutations may prove useful as companion diagnostic biomarkers for prediction of response to therapy. Methods: Source BioScience (Nottingham, UK) tested three different methods for the detection of PI3K mutations in archival tumour samples, in their CPA and GLP accredited laboratories. Eighty formalin-fixed paraffin-embedded (‘FFPE') samples from histologically confirmed breast cancer samples were tested by the new DxS P13K Mutation Test Kit using real-time PCR, by pyrosequencing (Qiagen), and by conventional bidirectional capillary sequencing. Tumour content was evaluated by pathology review. Results: The results of the comparison showed that the DxS test using rtPCR technology was easy to perform, convenient and robust. The DxS test had a high degree of sensitivity provided there was more than 20% tumour content in the test sample as determined by histology and pathology review. Conclusions: In conclusion, the DxS test is a valuable tool for detecting PI3K mutations in FFPE samples from breast cancer patients. No significant financial relationships to disclose.

Immunohistochemical staining for programmed cell-death ligand 1 (PD-L1) in malignant thymoma and thymic carcinoma.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e20003-e20003
Author(s):  
Alexei Shimanovsky ◽  
Richard Cartun ◽  
Mary Fiel-Gan ◽  
Daniza Mandich ◽  
Jonathan Earle ◽  
...  
Keyword(s):  
Thymic Carcinoma ◽  
Tumor Grade ◽  
Predictive Biomarker ◽  
Moderate Intensity ◽  
Malignant Thymoma ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Automated Platform ◽  
Formalin Fixed ◽  
Immunohistochemical Testing

e20003 Background: Recent development of anti-PD-1/L1 antibodies has demonstrated activity in various neoplasms. Thymic malignancies (TMS) are rare and treatment in advanced disease is limited. To evaluate the potential impact of anti-PD-1/L1 therapy in TMS, we examined the expression of PD-L1 in previously resected thymoma (TM) and thymic carcinoma (TC). Methods: We examined resected specimens from patients at Hartford Hospital with TM and TC between 2000 and 2014. Expression of PD-L1 was evaluated on formalin-fixed paraffin-embedded tissue. Immunohistochemical testing was done using four different clones of PD-L1 antibodies on the Leica Bond Max automated platform. The four clones include: E1L3N (Cell Signaling Technology), 28-8 (Epitomics) and SP142 (Spring Bioscience), and CAL10 (BioCare). PD-L1 expression was evaluated based on the percentage of tumor cells positive and their intensity graded as negative, weak (1+), moderate (2+), and strong (+3). The scoring was performed by three pathologists and was blinded for clinicopathologic data and antibody clones. Results: We evaluated a total of 29 patients, including 26 patients with TM and 3 with TC. Among the 29 available specimens, 12 had completed PD-L1 expression assessment at the time of submission. PD-L1 expression is present in 75-100% of the evaluated patients. All had positive PD-L1 staining by SP142 and CAL10. Three patients showed strong intensity by CAL10, and one by SP142. E1L3N and 28-8 had positive PD-L1 expression in 9 and 8 patients respectively with weak/moderate intensity. SP142 and CLA10 demonstrated the strongest concordance (R2 = 0.91) but there was significant variation between antibodies (R2 = 0.31-0.91). No correlation was detected between tumor grade and PD-L1 expression. There were focal areas that lacked expression in all of the evaluated specimens. Conclusions: There is increased expression of PD-L1 in TMS. The level of PD-L1 expression varies between the four PD-L1 antibodies. Increased PD-L1 expression provides evidence for the use of PD-L1 inhibitors in TMS. The variable staining highlights the heterogeneity of TMS and challenges in developing predictive biomarker in this cancer.

scholarly journals PD-L1 in Cytological Samples: A Review and a Practical Approach

2021 ◽  
Vol 8 ◽  
Author(s):  
Eva Tejerina ◽  
Laura García Tobar ◽  
José I. Echeveste ◽  
Carlos E. de Andrea ◽  
Elena Vigliar ◽  
...  
Keyword(s):  
Predictive Biomarkers ◽  
Cost Effective ◽  
Standard Of Care ◽  
Observer Agreement ◽  
Cancer Treatments ◽  
Formalin Fixed Paraffin ◽  
Programmed Death ◽  
Wide Range ◽  
Formalin Fixed Paraffin Embedded ◽  
Programmed Death 1

With a growing number of predictive biomarkers needed to manage patients with non-small cell lung cancer (NSCLC), there has been a paradigm shift in care and handling of diagnostic samples. Among the various testing methods, immunohistochemistry (IHC) is the most cost- effective and widely available. Furthermore, over the past decade immunotherapy has emerged as one of the most promising cancer treatments. In this scenario IHC is the most used testing method available for PDL-1/PD1 immunotherapy. Several monoclonal antibodies targeting programmed death 1 (PD-1)/programmed death ligand-1 (PD-L1) pathways have been integrated into standard-of-care treatments of a wide range of cancer types, once provided evidence of PD-L1 expression in tumor cells by immunohistochemistry (IHC). Since currently available PD-L1 assays have been developed on formalin-fixed paraffin embedded (FFPE) histological specimens, a growing body of research is being dedicated to confirm the feasibility of applying PDL-1 assays also to cytological samples. Albeit promising results have been reported, several important issues still need to be addressed. Among these are the type of cytological samples, pre-analytical issues, cyto-histological correlation, and inter-observer agreement. This review briefly summarizes the knowledge of the role of cytopathology in the analysis of PD-L1 by immunocytochemistry (ICC) and future directions of cytopathology in the immunotherapy setting.

Measurements of Mcl-1 protein dependencies using dimerization-specific antibodies as predictive biomarkers for BH-3 mimetic class therapies in AML.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e19505-e19505
Author(s):  
Michael H. Cardone ◽  
Andrew Kinloch ◽  
Mine Canakci ◽  
Jingping Ge
Keyword(s):  
Cell Lines ◽  
Protein Complexes ◽  
Treatment Options ◽  
Predictive Biomarkers ◽  
Predictive Biomarker ◽  
Response To Treatment ◽  
Clinical Settings ◽  
Cancer Therapies ◽  
Protein Levels ◽  
Combination Treatments

e19505 Background: The anti-apoptotic Bcl-2 family proteins facilitate pro-survival and resistance to anti-cancer therapies. Measuring the function of these proteins has shown utility in predicting response to treatment. A biomarker platform that assesses the functionality of these proteins by measuring BH3 mediated signaling potential in individual patients’ cancers, could provide a potential guiding platform for treating AML. In AML venetoclax is becoming widely prescribed and highly effective in combination with other drugs. Recent data indicate that Mcl-1 dependence is a resistance factor to venetoclax and that methods for identifying this could provide guidance for combination treatments. We are developing a technology to directly measure the occurrence of heterodimers of Mcl-1, or Bcl-xL, bound to pro-apoptotic BH3-only protein Bim. These measurements are combined in algorithms developed to indicate the cancer cell apoptotic priming state and offer great potential for identifying best AML treatment options. Methods: Monoclonal antibodies against conformation-specific epitopes induced in Mcl-1/Bim and Bcl-xL/Bim protein complexes were made. Selective bonding of Heterodimer Specific Mcl-1 Bim (HSMCB) and Heterodimer Specific Bcl-xL Bim (HSBXB) mabs were confirmed using ELISA, fluorescence polarization, immunofluorescence microscopy and flow, and by immunohistochemistry in genetically defined cell lines and in AML patient biopsied samples. Results: We show that HSMCB signal depends on both Mcl-1 and Bim protein levels while HSBXB requires Bc-xL and Bim levels. The relative signals of HSMCB to unbound Mcl-1 signal ([HsMcB]/[Mcl-1]) provide a biomarker for Mcl-1 dependence. We carefully established binding metrics that correlate to drug response in cell lines. The pharmacological disruption of these complexes is monitored by the ratiometric readouts that serve as predictive biomarkers for BH-3 mimetic drugs in AML cells. Conclusions: Mcl-1 dependence is a predictive biomarker for venetoclax resistance and for response to Mcl-1 targeted therapies. Flow cytometric and IHC based measurements of a heterodimer complex offer a direct and simpler approach that harbors potential for use in clinical settings. Additional antibodies targeting Mcl-1/Bak and Bcl-2/Bim complexes are being tested.

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