Range Of HER2 Expression Research Articles

Abstract 2377: HER2-ADC efficacy is rooted in its induction of immunogenic cell death with FcGR activation and a systemic bystander effect

Abstract Background: T-DXd is an antibody-drug conjugate (ADC) targeting HER2 that has demonstrated superior clinical efficacy over previous HER2-targeting agent Trastuzumab emtansine (T-DM1). Notably, T-Dxd is clinically effective in breast cancers (BC) with a range of HER2 expression. While its purported mechanism of action (MOA) involves its cleavable linker leading to bystander killing, it remains unclear how this potential MOA accounts for efficacy in HER2-low cancers or the role FCGR-mediated signaling and immune induction plays in its clinical efficacy. Method: In vitro studies utilized co-culture, internalization, FCGR activation, ADCP, flow cytometry, and gene expression by QRT-PCR and ELISA assays. Assessments of tumor antigen-specific T cell stimulation involved JEDI T cells. In vivo studies were conducted using in vivo implantation of HER2+ and HER2- cells, PK assessments of T-Dxd/DM1 and therapeutic effects determined using an endogenous HER2 transgenic mouse model that measured HER2-specific adaptive immune responses, in combination with immune checkpoint blockade anti-CD47. Results: T-Dxd elicited a systemic bystander effect that was only partially dependent on HER2 expression, mediated by specific cathepsins in the extracellular TME. Notably, T-Dxd cytotoxicity induced immunogenic cell death (ICD), while also retaining the ability to engage FCGRs to promote ADCP and innate immune activation. ICD was mediated by secretion of specific Dxd driven DAMPs (HMGB1 and eATP), resulting in TLR4/STING activation. ICD and FCGR activation along with antigen ADCP together elicited myeloid cell activation (CD80, CD40) and antigen presentation, resulting in effective expansion of tumor antigen-specific T cells responses, demonstrated by co-culture assays using EGFP/HER2+ BC cells and CD8+ JEDI T cells. T cell activation required TLR4 and STING pathways while in vivo, T-Dxd anti-tumor responses associated with augmented HER2-specific T and B cell responses. However, T-Dxd also induced CD47 to limit phagocytosis. But, the use of CD47/SIRPa blocking antibodies dramatically enhanced CD8+ T cell expansion in vitro and anti-tumor efficacy in vivo upon T-Dxd treatment. Conclusion: Our study demonstrates that T-Dxd is cleavable in HER2 negative BC in vivo, which permits more widespread Dxd activity against HER2 low BC. Critically, Dxd elicits ICD, resulting in innate immune stimulation of myeloid cells, which when combined with FCGR activation and ADCP, leads to a striking induction of tumor-specific adaptive immunity. This combination was unique to T-Dxd, which may underlie its clinical superior efficacy. However, T-Dxd activity also elicits suppressive CD47 expression which could be negated using CD47/SIRPA blockade. We found that this combination dramatically enhanced T-Dxd resulted therapeutic efficacy, thus supporting clinical exploration of these combinations. Citation Format: Li-Chung Tsao, Xingru Ma, John S. Wang, Timothy N. Trotter, Tao Wang, Jun-Ping Wei, Gang-jun Lei, Jason McBane, Ping Fan, Ivan Spasojevic, Zachary C. Hartman. HER2-ADC efficacy is rooted in its induction of immunogenic cell death with FcGR activation and a systemic bystander effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2377.

Abstract P6-04-17: High Intra- and Inter-block concordance of HER2 immunohistochemistry (IHC) scores across breast cancer samples and impact of decalcification procedures

Abstract Background & Objective Trastuzumab deruxtecan is effective in HER2-positive (IHC3+ or ISH-positive) and HER2-low (IHC1+; IHC2+/ISH-negative) breast cancer. We assessed consistency of HER2-positive and HER2-low categorization by IHC across and between breast cancer samples with a range of HER2 expression. The impact of decalcification procedures on HER2 IHC staining was also assessed to determine whether HER2 expression is likely to be compromised in decalcified samples from metastatic bone disease. Methods Three non-consecutive tumor sections (approximately 100 μm apart) from 50 commercially obtained breast cancer resections (2 blocks/resection) and 20 biopsies (sections ~40 μm apart) representing a range of HER2 IHC expression, and with known ISH status, were stained using the VENTANA anti-HER2/neu 4B5 IHC assay, scored per ASCO-CAP (2018) guidelines and intra-block/inter-block concordance reported for HER2-positive and HER2-low categorization. To investigate the impact of decalcification, four resection samples were formalin-fixed, divided into four pieces, of which three were pre-treated with either 10% formic acid, Decal STAT or Richard Allan Scientific< sup>TM Decalcifying Solution before processing and staining with the 4B5 IHC assay. Results A single pathologist reviewed all samples. Intra-block agreement for HER2-low (proportion of blocks in which 3 of 3 sections had identical status) was 89.5% for biopsies (17/19) and 80.6% for resection samples (n=79/98). Inter-block agreement (proportion of resections in which two independent blocks had the same majority status for HER2-low) was 91.7%. Intra and inter-block agreement for HER2-positive was >90%. Exposure to Decal STAT or Richard Allan Scientific< sup>TM Decalcifying Solution caused a significant reduction in HER2 IHC1+ and IHC2+ staining in breast cancer samples. 10% formic acid had no apparent impact on HER2 IHC staining in all four samples tested. Conclusion There was high HER2-positive and HER2-low agreement within breast cancer biopsies and resection blocks and highly consistent HER2-positive and HER2-low categorization between different blocks from tumor resections. Our results suggest HER2 staining heterogeneity does not impact determination of HER2-positive and HER2-low status in the majority of cases. Accurate assessment of HER2 IHC1+ and IHC2+ status was observed to be compromised by Decal STAT and Richard Allan decalcification solutions. Decalcification of bone metastasis samples for HER2-positive and HER2-low status determination may be possible using formic acid, but this would require further validation. Citation Format: Asimina Tsirka, Charo Garrido, Marietta Scott, Paul Scorer, Anne-Marie Boothman, Tsuyoshi Matsuo, Craig Barker. High Intra- and Inter-block concordance of HER2 immunohistochemistry (IHC) scores across breast cancer samples and impact of decalcification procedures [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-17.

Abstract 5128: Quantitative mass spectrometry of HER2 protein levels reveals high variability within HER2 IHC grades

Abstract About 15% of breast cancers are HER2 over-expressing (HER2 IHC 3+ or IHC 2+/ISH+)), but another 45% have low levels of HER2 (HER2 IHC 2+/ISH- or IHC 1+), and these patients are not currently approved for treatment with trastuzumab. Recently, a new HER2 ADC, DS-8201 showed anti-tumor activity, not only in patients with HER2 over-expressing breast cancer but also in HER2 low expressing tumors in whom to date, there are no effective anti-HER2 therapies indicated. FDA-approved HER2 in vitro diagnostic tests have recognized several limitations including effects of pre-analytical variable (fixation affects antibody sensitivity), limited dynamic range of chromogen-based IHC, and subjectivity in interpretation of the HER2 score. Additionally, the cut-off values (percentage of cells to be positive) defining HER2 positive have been changing over time. Therefore, more accurate, sensitive, precise and objective assays to better identify patients who may benefit from anti-Her2 treatment therapies (e.g DS-8201) are needed. To address this gap, we evaluated upcoming technologies targeted MS and QRT-PCR and aim to compare expression with current diagnostic HER2 tests in FFPE samples Using selected reaction monitoring mass spectrometry (SRM-MS), we quantified proteins from formalin-fixed, paraffin-embedded tissue samples that had been classified as HER2 0, 1+, 2+ or 3+ by IHC (n=107). HER2 protein concentration measured by SRM-MS was compared between patients in different HER2 IHC classifications using an ANOVA, adjusting for multiple comparisons. HER2 concentration (measured by SRM-MS) was progressively increased according to HER2 IHC grouping (i.e. lowest concentration in HER2 0 samples, highest in HER2 3+ samples). HER2 levels were significantly elevated in 2+ vs. 0 (2.2-fold increase, p < 0.05) samples, and trended higher in 2+ vs. 1+ (1.6-fold increase, p = 0.07) and in 1+ vs. 0 (1.4-fold increase, p = 0.17) samples. About 73% of samples scored as IHC0 had detectable Her2 by SRM-MS (from 168 to 623 amol/µg). Among HER2 IHC 0 samples, ~15% (7/47) had HER2 concentrations above the median levels for the 1+ group. Similarly, 19% (3/16) 1+ samples had HER2 levels above the median for the 2+ group. About 20% of samples co-expressed either ERBB1 and/or ERBB3. Simultaneously from FFPE sections we quantified protein level of payload response and resistance markers (MDR, MRP1, Topo1 and SLFN11). We used an objective multiplex non-antibody-based method to quantify multiple targets from FFPE tissue. SRM-MS revealed a range of HER2 expression over 100 orders of magnitude and identify markers of payload response or resistance in the same assay. The differences seen in payload markers expression could affect therapeutic efficacy and may suggest differing responses to Her2-targeted ADC, depending on tumor biology. Multiplexed quantitative MS could be used to accurately predict which patients will derive the most benefit from Her2-ADC therapy based on the specific biology of their tumor. These studies are ongoing. Citation Format: Fabiola Cecchi, Mark Gustavson, Danielle Carroll, Sriram Sridhar, Steven Coats, Anuja Bhalkikar, Sheeno Thyparambil, Wei-Li Liao, Todd Hembrough. Quantitative mass spectrometry of HER2 protein levels reveals high variability within HER2 IHC grades [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5128.

Abstract 4163: Clinical proteomics for patient stratification - a GCLP quantitative mass spectrometry workflow for multiplexed measurement of protein biomarkers in FFPE tissues

Abstract a) The advent of precision oncology has led a shift towards biomarker-driven clinical trial designs and molecular profiling of individual patients. Identification of patients with the target biomarker profile may be useful in guiding patient selection as an enrichment strategy for clinical trials. Targeted multiple reaction monitoring mass spectrometry (MRM-MS) analysis for multiplexed quantitation of biomarker proteins in FFPE tissue provides direct, more accurate and precise quantification over current ‘gold standard' immunohistochemistry (IHC) methods. However, MRM-MS has not yet been broadly applied to clinical trials. In this study, we demonstrate the systematic development, optimization and analytical validation of quantitative, multiplexed MRM-MS assays for robust biomarker quantification in clinical FFPE tissues, including sample analysis under GCLP. Results from an MRM panel targeting 8 clinically relevant biomarker proteins will also be shown, including the measured HER2 levels in FFPE breast tumors classified by IHC as 0, 1+, 2+ or 3+. b) MRM-MS biomarker panels were developed and optimized for multiplexed quantitation of ≤12 proteins, in which unique peptides derived from each target protein were monitored as a surrogate measure of protein levels. Tumor regions from FFPE tissue sections were dissected using laser capture or macrodissection, solubilized, digested with trypsin to generate peptides for analysis, spiked with fixed levels of stable isotope labeled (SIL) peptide standards, and analyzed by MRM-MS. Analytical validation was performed per NCI CPTAC guidelines, including response curves, assay repeatability, selectivity, stability, and reproducibility of endogenous detection. Clinical performance was assessed using commercially sourced FFPE-tumor tissues, including a cohort of breast tumor tissues with a wide range of HER2 expression. c) Assay performance results were protein/peptide dependent, with sensitivity in the low pg/μg total protein range. For HER2, assay linearity was demonstrated over 2.5 to 3 orders of magnitude, with a precision and accuracy of <15% over 3 independent runs. In sample analysis, the MRM-MS was sufficiently sensitive to detect HER2 in 1 μg total protein from FFPE breast tumor classified by IHC as negative (0). d) GCLP-compliant quantitative multiplexed large-scale clinical analysis of protein biomarkers by MRM-MS in FFPE tissue is feasible and enables precise and accurate quantitation of proteins when IHC methods are unsuitable or unavailable. Data can be used for patient stratification, optimization of treatment outcomes, drug resistance prediction, and to support clinical development of novel therapies. Citation Format: Maxim Isabelle, Michael Schirm, Gwenael Pottiez, Rudolf Guilbaud, Lorella Di Donato. Clinical proteomics for patient stratification - a GCLP quantitative mass spectrometry workflow for multiplexed measurement of protein biomarkers in FFPE tissues [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4163.

Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer

An on-demand, closed RT-qPCR, the GeneXpert (GX) system, has the potential to provide biomarker information in low-resourced settings and elsewhere. We used this system with a research use only version of the Breast Cancer STRAT4 cartridge that measures the mRNA expression levels of ERBB2, ESR1, PGR, and MKi67. Here we evaluated the impact of non-macrodissected (non m-d) versus macrodissected (m-d) samples using STRAT4 on formalin-fixed, paraffin-embedded (FFPE) core needle biopsies. Two cohorts were assessed: (1) 60 FFPE infiltrating ductal carcinoma (IDCA) cases and (2) 20 FFPE IDCA cases with ductal carcinoma in situ (DCIS) with a range of HER2 expression as determined by clinical immunohistochemistry and fluorescence in situ hybridization (IHC/FISH). We observed about half of the core needle biopsy area as invasive tumor in both IDCA (mean = 51.5%) and IDCA with DCIS (mean = 53.5%) cohorts, but also found the mRNA levels were independent of tumor area. We found excellent agreement of the mRNA transcript level between the paired samples, m-d versus non m-d, for ERBB2, ESR1, PGR, and MKi67 for both the IDCA and IDCA with DCIS cohorts. No significant difference (P > 0.99) was observed when we compared the mRNA transcript level between the paired samples m-d versus non m-d. In addition, we noted a significant concordance (P < 0.001) between RT-qPCR and IHC/FISH for HER2-positivity, ER-positivity, and PR-positivity, independent of specimen dissection. These data suggest that mRNA expression for ERBB2, ESR, and PGR is sufficiently low in surrounding tissue cells such that macrodissection is not required for assessment of key breast cancer mRNA markers and is independent of the amount of input tumor. This approach may be valuable in settings lacking pathology expertise or using specimen types, such as fine-needle aspirates, where it may be challenging to separate non-tumor from tumor tissue.The authors compared mRNA expression in a closed system real-time quantitative reverse transcription PCR assay for key breast cancer markers in non-micro-dissected tissue versus micro-dissected tissue in both invasive and ductal carcinoma in situ breast cancer cohorts. They found that microdissection of whole sections is not required for concordance with immunohistochemistry and fluorescence in situ hybridization.

Abstract P2-03-02: Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer

Abstract Background: An on-demand, closed system RT-qPCR (the GeneXpert system, Cepheid, Sunnyvale, CA) has the potential to provide biomarker information in low resourced settings. The system consists of an inexpensive, single-use, disposable, macrofluidic cartridge and an instrument that automates RT-qPCR. Here we use it with a research use only cartridge (STRAT4) that measures the mRNA expression levels of ESR1, PGR, ERBB2, and MKi67 using a single 5uM thick FFPE tissue section from an excisional or core biopsy specimen containing invasive carcinoma of the breast. The assay, results are expressed as a delta cycle threshold (dCt) value, defined as the Ct of a control gene (CYFIP1) minus the Ct of the target gene (ESR1, PGR, ERBB2, or MKi67). We determine whether the dCt result for each marker is equivalent using the entire non-macrodissected section (non m-d) to the dCt results obtained following tumor macro-dissection (m-d) to eliminate non-tumor elements from the assay. Methods: We evaluated the impact of m-d versus non m-d using STRAT4 on a cohort of 62 formalin-fixed paraffin-embedded (FFPE) tumor core needle biopsy specimens with a range of HER2 expression determined by clinical immunohistochemistry and fluorescence in situ hybridization (IHC/FISH). Concordance (sensitivity and specificity) of the STRAT4 ESR1 and HER2 mRNA versus ER and HER2 IHC/FISH measurements were also assessed. Results: We observed excellent agreement of the resulting dCt between the paired samples, m-d versus non m-d, for ESR1 (R2=0.92), PGR (R2=0.90), ERBB2 (R2=0.94) and MKi67 (R2=0.90). No significant difference (P value &amp;gt; 0.99) was observed when we compared the dCt between the paired samples m-d versus non m-d. In addition, using the predefined STRAT4 dCt cutoff for ESR and ERBB2 positivity, we found a significant concordance between RT-qPCR and IHC/FISH for ESR-positivity for the paired samples, m-d (P value &amp;lt; 0.001; sensitivity = 0.98; specificity = 1; PPV = 1; NPV = 0.95) versus non m-d (P value &amp;lt; 0.001; sensitivity = 0.98; specificity = 1; PPV = 1; NPV = 0.95) and HER2-positivity for the paired samples, m-d (P value &amp;lt; 0.001; sensitivity = 0.85; specificity = 0.98; PPV = 0.92; NPV = 0.96) versus non m-d (P value &amp;lt; 0.001; sensitivity = 0.71; specificity = 0.98; PPV = 0.90; NPV = 0.92), respectively. Conclusion: These data suggest that mRNA for ESR and ERBB2 is sufficiently low in surrounding tissues that m-d of whole sections is not required for accurate assessment of key breast cancer mRNA markers in a closed system RT-qPCR assay. The simplicity of the assay workflow may be particularly valuable in low resourced settings where routine access to pathology expertise and to high quality IHC/FISH is challenging. Citation Format: Gupta S, Carvajal-Hausdorf DE, Wasserman BE, Ho K, Weidler J, Wong W, Rhees B, Bates M, Rimm DL. Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-03-02.

Abstract 3810: Validation of novel high-plex protein spatial profiling quantitation based on NanoString's Digital Spatial Profiling (DSP) technology with quantitative fluorescence (QIF)

Abstract Background: Protein expression in formalin-fixed, paraffin-embedded (FFPE) tissue is routinely measured by Immunohistochemistry (IHC) on only one protein, or with quantitative fluorescence (QIF) on a handful of proteins on a single section. NanoString’s Digital Spatial Profiling (DSP)* technology can detect and quantify proteins at significantly higher multiplex (currently 30 plex, potential for up to 800 plex) with spatial resolution using molecular digital color-coded “barcodes”, within specific regions of interest on FFPE tissue. Here, we compare Nanostring DSP technology to automated QIF (AQUA), for accurate and reproducible measurement of protein expression on a continuous scale. Additionally, using the multiplexing potential of Nanostring technology, we did a pilot study to assess cold ischemic time as a variable to monitor tissue quality by assessment of epitope degradation in Non-Small Cell Lung Cancer (NSCLC). Methods: The DSP technology uses a cocktail of primary antibodies conjugated to indexing DNA oligos with a UV Photocleavable linker that can be counted on the nCounter™ platform. Regions of interest (ROI) on the tissue are selected with fluorescently labeled antibodies, and oligos from that region are released via UV mediated linker cleavage. Free oligos are captured via microcapillary fluidics into a microtiter plate and then quantitated on the nCounter. The comparator for this technology was the AQUA method of QIF. We examined a breast cancer tissue microarray with a range of HER2 expression, and a NSCLC time to fixation standardization array with timepoints from 20 min to 48 hrs. Results: Multiple markers were assessed and a high correlation was found between Nanostring DSP counts and QIF scores (R²&amp;gt;0.7), when the measurements were performed in the same region of interest (defined by cytokeratin expression). The dynamic range of DSP exceeded the quantification range of QIF (nearly 4 logs vs about 2 logs). When the 28 protein markers’ expression was compared at different fixation timepoints, most were found to be stable over different cold ischemic timepoints. Two markers, including phospho-ERK (Extracellular Signal-Regulated Kinases) and phospho-GSK (Glycogen Synthase Kinase) showed epitope loss as a function of delay to fixation (R²=0.0064 and R²=0.05 respectively). Conclusion: The Nanostring DSP is a reproducible method with the capability of highly multiplexed measurements of protein expression on a field of view averaged basis. It shows high concordance with the AQUA method of QIF, an extensively validated technique for protein quantification. For the first time, the high-plex capacity of DSP allows inclusion of markers that are sensitive to time to fixation as an intrinsic control for tissue quality. *FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Maria I. Toki, Chris Merritt, Giang Ong, Joseph M. Beechem, Daniel E. Carvajal-Hausdorf, Yalai Bai, David Rimm. Validation of novel high-plex protein spatial profiling quantitation based on NanoString's Digital Spatial Profiling (DSP) technology with quantitative fluorescence (QIF) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3810. doi:10.1158/1538-7445.AM2017-3810

Abstract LB-061: HER2-targeted PEGylated liposomal doxorubicin (MM-302) efficiently targets the HER2 intermediate cell population in vitro and in vivo

Abstract Introduction: MM-302 is an antibody-liposomal drug conjugate designed specifically to target doxorubicin to HER2-overexpressing tumor cells. MM-302 is currently being evaluated in a Phase II trial in HER2 positive metastatic breast cancer (NCT02213744). HER2-positive breast cancer accounts for about 15-20% of breast cancer cases and is defined as IHC 3+ or 2+ and HER2 FISH amplified. A substantial percentage (∼30%) of breast cancer patients show positive HER2 IHC (1+/2+) without HER2 gene amplification (“HER2 intermediate”). This population is not eligible for treatment with currently approved HER2-targeted therapies. The purpose of this study is to investigate the in vitro and in vivo delivery/activity of MM-302 in the HER2 intermediate population. Methods: In vitro binding and viability studies were performed with MM-302, PEGylated liposomal doxorubicin (PLD) and T-DM1 with a panel of cell lines representing a range of HER2 expression. HER2-mediated cellular delivery of MM-302 was investigated in vivo in different HER2 expressing tumor models using a novel PEG immunofluorescent assay herein described. Frozen tumor tissues were stained for PEG, HER2 and cytokeratin, side-by-side with two cell standard arrays: A PEG array, obtained by cell incubation with increasing concentrations of MM-302, and a HER2 array, built with a panel of cell lines at different HER2 expression (from ∼50,000 to over 1,000,000 HER2). Image analysis and subsequent regression of the PEG and HER2 fluorescent intensities from the respective standards allowed for the quantification of the number of liposomes in individual tumor cells at distinct HER2 receptor numbers. Tumor cell apoptosis following MM-302 cellular delivery was measured by immunofluorescence followed by image analysis. Results: MM-302 efficiently bound to, and induced, tumor cell death across a panel of cell lines, with no significant distinction between cell lines expressing 300-400,000 HER2 or above 1,000,000 HER2 (IHC 3+). Conversely, T-DM1, used as control, significantly bound to and induced cell death only to cells above 1,000,000 HER2 (IHC 3+). In vivo evaluation of HER2-mediated cellular delivery via PEG immunofluorescent staining showed that MM-302 can be equally efficiently internalized in tumor cells above 1,000,000 HER2 (IHC 3+) and in the HER2 intermediate expression range. Preliminary analysis on post-treatment patient biopsies collected during a MM-302 Phase I study (NCT01304797) support clinical translation of these preclinical observations. Conclusions: Treatment with MM-302 results in efficient HER2 binding and liposome cellular delivery across a panel of HER2 models that extend beyond the traditional HER2 positive definition. This study suggests that MM-302 may be a promising candidate for the treatment of patients with intermediate HER2 expression who represent a significant unmet medical need. Citation Format: Elena Geretti, Christopher Espelin, Bambang Adiwijaya, Nancy Dumont, Silvia Coma, Zachary Koncki, Minh Pham, Gabriela Garcia, Troy Bloom, Victoria Rimkunas, Joe Reynolds, Karen Campbell, Victor Moyo, Istvan Molnar, Patricia LoRusso, Ian Krop, Kathy Miller, Cynthia Ma, Pamela Munster, Thomas Wickham. HER2-targeted PEGylated liposomal doxorubicin (MM-302) efficiently targets the HER2 intermediate cell population in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-061.

Abstract 3398: Development of a mass spectrometry based antibody-drug conjugate biomarker panel

Abstract Background: Antibody-Drug Conjugates (ADCs) are poised to become an extremely important class of therapeutics in oncology. By conjugating cytotoxic payloads to antibodies that target proteins found primarily on cancer cells, ADCs represent a novel mechanism for directing extremely toxic small molecules specifically to tumor cells. Due to the unique mechanism of ADCs, patient selection should involve screening not only for the presence of the antibody target, but also screening for the presence of any markers of resistance or response to the payload. Several proteins, such as multi-drug effluxers and tubulin-beta 3, have been implicated in resistance to small molecule cytotoxins and microtubule inhibitor drugs. OncoPlex Diagnostics has built a multiplexed ADC biomarker panel that simultaneously quantifies the levels of the antibody target and putative resistance markers for several known payloads, such as maytansinoids, auristatins and taxanes, as well as response markers for the topoisomerase inhibitor payloads SN-38 and doxorubicin. Methods: Liquid Tissue-Selected Reaction Monitoring (LT-SRM) is a multiplexed, quantitative method that uses mass spectrometry to quantify proteins based on a unique sequence of amino acids, and thus does not have the same limitations as traditional antibody-based, semi-quantitative protein detection methods, such as immunohistochemistry. We developed a LT-SRM assay to quantify protein levels of EGFR, FRalpha, Her2, CD30 and Mesothelin (antibody targets) and MCL1, MDR, MRP1, tubulin-beta3, Topo1 and Topo2a (payload response and resistance markers) simultaneously from FFPE biopsies. Calibration curves for all the proteins in the ADC panel are linear over 5-orders of magnitude, with limits of detection for each analyte between 25 and 400 amol/ug of tissue. Results: Analysis of FFPE tumor tissues show a broad range of expression for the ADC proteins, with some tissues showing no detectable levels of some payload markers. Clinical analysis of FRalpha showed a range of expression from &amp;lt;LOD to 13500 amol/ug. An examination of Her2 positive tissue showed a wide range of Her2 expression (over 20-orders of magnitude), as well as a wide range of expression for the payload markers. The differences seen in the payload markers suggest differing responses to Her2-targeted ADCs, independent of Her2 levels, and indicate that different patient populations might respond better to different payloads, depending on tumor biology. Conclusions: The OncoPlexDx ADC panel can determine of a cutoff for expression levels of the antibody-target protein necessary for ADC response as well as identify markers of payload response or resistance to further understand how these markers affect therapeutic efficacy. This panel can be used to predict which patients will derive the most benefit from ADC therapy based on the specific biology of their tumor. Citation Format: Adele Blackler, Wei-Li Liao, Sheeno Thyparambil, Eunkyung An, Fabiola Cecchi, Marlene Darfler, Todd Hembrough, Jon Burrows. Development of a mass spectrometry based antibody-drug conjugate biomarker panel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3398. doi:10.1158/1538-7445.AM2015-3398

Abstract P1-07-19: Mass Spectrometry Based Quantitative Analysis of the HER Family receptors in FFPE Breast Cancer Tissue

Abstract The human EGF receptor family (HER's) consists of two clinically validated drug targets (EGFR and HER2), a third (HER3) currently under investigation for its possible role in the acquisition of multidrug resistance and a fourth (HER4), the role of which is still matter of debate. Drugs inhibiting EGFR or HER2 show significant antitumor activity in the clinic, however, acquisition of resistance is a hallmark of these and most targeted therapies. In the case of EGFR and HER2, one of the emerging resistance mechanisms is the co-expression of HER3. Indeed, recent reports show that inhibition of the PI3K pathway leads to upregulation of HER3, and subsequent resistance. Clinical analysis of protein levels in formalin fixed paraffin embedded (FFPE) tissues is limited to immunohistochemistry (IHC), which is semi-quantitative and requires significant amounts of tissue. Moreover, the vast majority of research groups consider specific HER3 staining by IHC particularly challenging. However, accurate measurement of these targets is critical both for properly defining treatment groups and predicting patterns of resistance. In order to address these issues, we used trypsin digestion mapping and stable isotope labeled peptides to develop a panel of quantitative mass spectrometric (MS) assays to measure the levels of EGFR, HER2, HER3 and other clinically relevant targets in FFPE breast cancer tissue. These quantitative MS assays were then multiplexed to analyze 1μg of tumor protein. In this study, we multiplexed HER family analysis on 31 HER2 positive breast cancers. Tumor tissue was microdissected from FFPE sections, and subjected to quantitative MS analysis of EGFR, HER2, HER3 as well as IGF1R and cMET. Quantitation of HER2 showed a broad range of HER2 expression in these tissues. The highest expresser measured 26 fmol/ug tumor tissue, representing amplification and massive protein over expression. In contrast, five tissues showed low levels of HER2 expression, below 1 fmol/ug, similar to HER2 non-amplified cell lines. This suggests that MS quantitation can identify patients with low expression of HER2 who are unlikely to respond to trastuzumab therapy. As a matter of fact, 3 of these 5 low expressing patients had outcome data and showed no response to trastuzumab treatment. In 28 of 31 patient tissue samples, HER3 showed low levels of expression (100–300 amol/ug tumor tissue) similar to HER3 expression in cell lines, and comparable to low expressing EGFR and HER2 cell lines. The remaining 3 patients had no detectable HER3. This study demonstrates the feasibility of measuring HER3 in multiplex in FFPE breast cancer tissue. Based on the low but widespread expression of HER3 in this cohort, it may be most useful to assess HER3 expression after initial treatment as a marker of potential resistance to targeted therapies. Taken together, these data demonstrate that a sensitive and quantitative assay to measure oncoproteins in FFPE clinical samples may help stratify patients with variable expression of these targets. Our quantitation of oncogene expression from clinical samples uses a small amount of tissue, is clinically applicable and alleviates the problem of scoring either positive or negative for the expression of a given protein. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-19.