An enormous amount of research effort has been devoted to biomarker discovery and validation. With the completion of the human genome, proteomics is now playing an increasing role in this search for ...new and better biomarkers. Here, what leads to successful biomarker development is reviewed and how these features may be applied in the context of proteomic biomarker research is considered. The “fit‐for‐purpose” approach to biomarker development suggests that untargeted proteomic approaches may be better suited for early stages of biomarker discovery, while targeted approaches are preferred for validation and implementation. A systematic screening of published biomarker articles using MS‐based proteomics reveals that while both targeted and untargeted technologies are used in proteomic biomarker development, most researchers do not combine these approaches. i) The reasons for this discrepancy, (ii) how proteomic technologies can overcome technical challenges that seem to limit their translation into the clinic, and (iii) how MS can improve, complement, or replace existing clinically important assays in the future are discussed.
Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to ...be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a 'green' xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm
showed a high correlation between replicates (r
= 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.
Cancer remains a major challenge for modern medicine. It has become evident that cancer is a complex disease involving many coexisting genomic alterations; the molecular consequences of detected ...genetic and transcriptomic alterations are yet poorly understood. Risk and patient stratification based on the genetic phenotype alone has proven to have limited efficacy on the individual treatment outcome. Multi-omics approaches combine various high-throughput analytical techniques to characterize the genome, transcriptome, proteome and metabolome, as well as other cellular components of individual tumors simultaneously. Multi-omics has emerged as a promising tool for providing a more comprehensive and holistic view of cancer biology, which may lead to the identification of novel therapeutic targets and biomarkers. The aim of this doctoral thesis is to investigate the potential of multi-omics approaches for the identification of therapeutic vulnerabilities in hard-to-treat malignancies.This thesis presents a systematic review of the literature on multi-omics approaches in clinical research, with a focus on identifying common challenges and opportunities in the field. We present two original research articles on the application of multi-omics for molecular characterization of malignancies. We established an optimized and automatable sample preparation pipeline for mass spectrometry-based quantitative proteomics from limited volume clinical specimens, such as formalin-fixed, paraffin embedded (FFPE) tissue micro array cores (~0.4 mm³ core volume). This accelerated method enables molecular profiling of individual tumors within 24 h, from sample receival to analysis, and does not require special training or equipment, promoting a translation into the clinic. We used this method for a proteomic ‘landscaping’ of non-invasive breast ductal carcinoma compared to invasive breast ductal carcinoma (IDC). Ductal carcinoma in situ (DCIS) is the most common type (80%) of non-invasive breast lesions. The lack of validated prognostic markers, limited patient numbers and tissue quality significantly impact diagnosis, risk stratification, as well as patient enrolment and results of clinical studies. Our study validated 22 putative biomarkers from independent genetic studies and reveals more than 380 differentially expressed proteins and metabolic vulnerabilities, that can inform new therapeutic strategies for DCIS and IDC. Due to the readily druggable nature of proteins, this study is of high interest for clinical research and the pharmaceutical industry.Overall, this thesis demonstrates the potential of multi-omics approaches to uncover new therapeutic vulnerabilities in hard-to-treat malignancies. The findings of this research will contribute to the development of personalized and targeted therapies for cancer patients, which can ultimately improve patient outcomes and reduce the burden of cancer on society
To prevent doping practices in sports, the World Anti-Doping Agency implemented the Athlete Biological Passport (ABP) program, monitoring biological variables over time to indirectly reveal the ...effects of doping rather than detect the doping substance or the method itself. In the context of this program, a highly multiplexed mass spectrometry-based proteomics assay for 319 peptides corresponding to 250 proteins was developed, including proteins associated with blood-doping practices. “Baseline” expression profiles of these potential biomarkers in capillary blood (dried blood spots (DBS)) were established using multiple reaction monitoring (MRM). Combining DBS microsampling with highly multiplexed MRM assays is the best-suited technology to enhance the effectiveness of the ABP program, as it represents a cost-effective and robust alternative analytical method with high specificity and selectivity of targets in the attomole range. DBS data were collected from 10 healthy athlete volunteers over a period of 140 days (28 time points per participant). These comprehensive findings provide a personalized targeted blood proteome “fingerprint” showcasing that the targeted proteome is unique to an individual and likely comparable to a DNA fingerprint. The results can serve as a baseline for future studies investigating doping-related perturbations.
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Background: Immuno-oncology revolutionized lung cancer treatment, but predictive biomarkers of response to checkpoint inhibitors (CPI) are still lacking. Only 50% of PD-L1 positive tumors by ...immunohistochemistry (IHC) respond to anti-PD-L1 treatment. Analytical variability and post-translational modifications (PTM) of the PD-1 signaling associated proteins (e.g.: glycosylation), can explain some of this discrepancy. Plus, the tumor immune microenvironment (TME) is complex and PD-L1 IHC alone is a flawed surrogate its status. Mass spectrometry based technologies have the potential to overcome these challenges by integrating sensitivity, specificity and absolute quantitation of proteins and PTMs in a standardized fashion. Here, we demonstrate the advantages of using anti-peptide antibodies to purify surrogate peptides followed by liquid-chromatography (LC) and multiple reaction monitoring (MRM), herby termed as iMRM, to gain new insight into the TME. Methods: To determine the concentration of PD-L1, PD-1, PD-L2, NT5E, LCK and ZAP70, we used unique and well detectable proteolytic peptides as surrogates. Our previously described protocol (ASCO 2022, #377181) allows robust quantitation of 13 peptides and monitors the glycosylation status of PD-L1, PD-L2, and PD-1. NSCLC samples were either fresh frozen (n = 42) or FFPE (n = 77) and sometimes both (n = 17). PD-L1 quantitation by iMRM was compared to PD-L1 IHC 22C3. Results: This multiplexed iMRM assay successfully quantified the PD-1/PD-L1 axis proteins in 96% of NSCLC patients (61/63). PD-L1 glycosylation levels ranged from 82 to 100% (n = 69, median = 100%, SD = 3.7%). Only PD-1 was significantly different between the fresh frozen (mean = 11±6 amol/µg total protein) and FFPE group (mean = 5±2 amol/µg total protein, ρ = 0.0001), all other peptides showed comparable levels. In our 17 matched FF/FFPE samples, PD-L1 moderately correlated (R = 0.45, ρ = 0.045) and the other peptides did not correlate (R < 0.03). Intra/intertumoral heterogeneity, differences in cellularity and tumor origin likely contributed to this discrepancy. IMRM results correlated moderately (R = 0.56, ρ < 0.01) with PD-L1 IHC. Most of these patients were not CPI-treated, but survival data was available. A trend was noted between the concentration of certain targets and patient survival. Linear regression was used to establish a cut-off for each peptide from which an immunoscore was calculated. The immunoscore could predict long-term survival (accuracy 75.4%) irrespective of tumor staging, grade or subtype and survival was significantly associated with the immunoscore (log-rank ρ = 0.005). Conclusions: Our iMRM workflow provides a new understanding of the TME in NSCLC through the PD-1/PD-L1 axis with an easy-to-read immunoscore. A set of 60 tumors from CPI-treated patients is currently being processed to validate the clinical utility of the assay in relation with CPI-response.
The tumor suppressor PTEN is the main negative regulator of PI3K/AKT/mTOR signaling and is commonly found downregulated in breast cancer (BC). Conflicting data from conventional immunoassays such as ...immunohistochemistry (IHC) has sparked controversy about PTEN’s role as a prognostic and predictive biomarker in BC, which can be largely attributed to the lack of specificity, sensitivity, and interlaboratory standardization. Here, we present a fully standardized, highly sensitive, robust microflow immuno-MRM (iMRM) assay that enables precise quantitation of PTEN concentrations in cells and fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissues, down to 0.1 fmol/10 μg of extracted protein, with high interday and intraday precision (CV 6.3%). PTEN protein levels in BC PDX samples that were determined by iMRM correlate well with semiquantitative IHC and WB data. iMRM, however, allowed the precise quantitation of PTENeven in samples that were deemed to be PTEN negative by IHC or western blot (WB)while requiring substantially less tumor tissue than WB. This is particularly relevant because the extent of PTEN downregulation in tumors has been shown to correlate with severity. Our standardized and robust workflow includes an 11 min microflow LC-MRM analysis on a triple-quadrupole MS and thus provides a much needed tool for the study of PTEN as a potential biomarker for BC.
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Background: More accurate predictive biomarkers of response to checkpoint inhibitors (CPIs) still is a major unmet need in oncology. PD-L1 immunohistochemistry (IHC) limitations include its ...analytical variability and the post-translational modifications of PD-1 signaling-associated proteins like glycosylation. Moreover, PD-L1 IHC is an imperfect surrogate of the tumor immune microenvironment, and immunoscoring is important but difficult to assess in a clinical setting. Proteomic based technologies can overcome these challenges, but the low concentration of these proteins and the presence of high background noise in formalin-fixed paraffin embedded (FFPE) tumors were limiting obstacles. In this study, we evaluate the benefit of a new approach we used with anti-peptide antibodies to purify surrogate peptides, while liquid chromatography (LC) was coupled to multiple reaction monitoring mass spectrometry (iMRM) to improve specificity and precision of protein quantitation. Methods: To determine the concentration of PD-L1, PD-1, PD-L2, NT5E, LCK and ZAP70, we used unique and well detectable proteolytic peptides as surrogates. In a refined protocol, we optimized protein extraction and digestion, peptide immuno-enrichment, LC and MRM parameters to maximize recovery, increase target-specific signal and reduce noise. Plus, we assessed the glycosylation status of PD-L1, PD-L2, and PD-1. The entire workflow was fully validated using 31 NSCLC FFPE tumors. PD-L1 quantitation by iMRM was compared to PD-L1 IHC clone 22C3. Results: On average, 71±29 µg (n = 52) of protein could be extracted from each 1–3 mm
3
NSCLC tumor FFPE core. The optimized iMRM method allowed the quantitation of PD-L1 and PD-1 down to 21 amol on-column. Inter- and intra-day repeatability were well below FDA guidelines (coefficients of variation CV < 20%) with average CVs of 5.2±4.0% (intra-day) and 4.5±2.6% (inter-day). Sample storage had no significant effect on peptide quantitation. The final multiplexed iMRM assay enables quantitation all targets and glycosylation states for > 40 samples in only 3 days (including external calibration and quality controls) and was used to quantify the PD-1/PD-L1 axis proteins successfully in all 31 NSCLC FFPE tumors. PD-L1 expression ranged from 2 amol/μg to 61 amol/μg of total protein. As expected, iMRM results correlated moderately (R = 0.56, ρ < 0.001) with PD-L1 IHC. PD-L1 glycosylation status ranged from 99.9±0.2%, and therefore did not explain the discrepancies between IHC and iMRM for these samples. Conclusions: Herein a robust iMRM workflow was developed for the quantitation of the PD-1/PD-L1 axis in FFPE. This proof-of-concept supports that MS-based assay can provide otherwise unavailable data (e.g., PD-L1 concentration, glycosylation status). CPI treated patient tumors are being currently processed to validate the predictive value of the assay.
Abstract only
e21040
Background: Improving the predictive biomarkers arena of checkpoint inhibitors (CPIs) beyond PD-L1 immunohistochemistry (IHC) is one of the most important unmet need in NSCLC. Up ...to 50% of patients that show positive PD-L1 expression by IHC do not respond to anti-PD-L1 treatments, and patients with low/undetectable PD-L1 have significantly improved survival with CPI. Moreover, PD-L1 IHC is heterogeneous, can be affected by tissue fixation time and post-translational modifications like glycosylation. Also, multiple studies indicated that PD-L1 expression alone does not reliably reflect the immune status of the tumor, thus requiring the measurement of other members of the PD1 signalling pathway. Methods: To address these issues, we developed a multiplexed targeted mass spectrometry-based (MS) assay for the quantitation of protein members of the PD-1/PD-L1 axis in formalin fixed paraffin-embedded (FFPE) tissue.The effect of fixation time on protein recovery was determined using differentially fixed H1915 cells. Liquid chromatography (LC) coupled to MRM was used to develop a targeted assay for PD-L1, PD-1, PD-L2, NT5E, LCK and ZAP70. Results: After 30 minutes fixation 33.6 µg of protein were extracted per mg of FFPE H1915 cells, while protein recovery after 7 days was 55.8 µg/mg, with greater variability (18% and 28% CV, p-value = ns). The optimized LC-MRM method allows the quantitation of PD-L1 and PD-1 down to 23 amol on-column. We evaluated the utility of our MRM assays using the H1915 FFPE cells (PD-L1 3+ by IHC) and determined an endogenous concentration of PD-L1 and NT5E as being 33.2±0.1 amol/µg of total protein and 4.8±0.5 fmol/µg respectively. Noteworthy, a known glycosylation site of PD-L1 was quantified at 10.9±0.3 amol/µg of total protein (30% of total). As increases sensitivity was required, anti-peptide antibodies were generated against the 15 best peptides. We then evaluated this LC-MRM method using MDA-MB-436 cells with lower levels of PD-L1 protein assessed by IHC. Determination of endogenous PD-L1 concentration (3.0 amol/µg of total protein) was achieved using anti-peptide immuno-enrichment followed by MRM. Conclusions: We developed a fixation time independent extraction technique for FFPE and optimized a highly sensitive LC-MRM method that allows the absolute quantification of our targets. This proteomic workflow allows absolute quantification of the PD-1/PD-L1 axis from FFPE tissue using immuno-enrichment and a multiplexed LC-MRM method.
Abstract Capivasertib is a potent selective inhibitor of AKT. It was recently FDA-approved in combination with fulvestrant to treat HR+, HER2-negative breast cancers with certain genetic ...alteration(s) activating the PI3K pathway. In Phase I trials, heavily pre-treated patients with tumours selected for activating PI3K pathway mutations treated with capivasertib monotherapy demonstrated objective response rates of <30%. We investigated the proteomic profile associated with capivasertib response in genetically pre-selected patients and cancer cell lines. We analyzed samples from 16 PIK3CA-mutated patient tumours collected prior to capivasertib monotherapy in the Phase I trial. PI3K pathway proteins were precisely quantified with immuno-MALDI-MS. Global proteomic profiles were also obtained. Patients were classified according to response to capivasertib monotherapy: “clinical benefit (CB)” (≥12 weeks without progression, n=7) or “no clinical benefit (NCB)” (progression in <12 weeks, n=9). Proteins that differed between the patient groups were subsequently quantified in AKT1- or PIK3CA-altered breast cancer cell lines with varying capivasertib sensitivity. The measured concentrations of AKT1 and AKT2 varied among the PIK3CA-mutated tumours but did not differ between the CB and NCB groups. However, analysis of the global proteome data showed that translational activity was higher in tumours of the NCB vs. CB group. When reproducibly quantified by validated LC-MRM-MS assays, the same proteins of interest similarly distinguished between capivasertib-sensitive vs. -resistant cell lines. The results provide further evidence that increased mTORC1-driven translation functions as a mechanism of resistance to capivasertib monotherapy. Protein concentrations may offer additional insights for patient selection for capivasertib, even among genetically pre-selected patients.
Hotspot testing for activating
mutations is used in precision oncology to select colorectal cancer (CRC) patients who are eligible for anti-EGFR treatment. However, even for
tumors anti-EGFR response ...rates are <30%, while mutated-
does not entirely rule out response, indicating the need for improved patient stratification. We performed proteogenomic phenotyping of
and
CRC liver metastases (mCRC). Among >9000 proteins we detected considerable expression changes including numerous proteins involved in progression and resistance in CRC. We identified peptides representing a number of predicted somatic mutations, including KRAS
. For eight of these, we developed a multiplexed parallel reaction monitoring (PRM) mass spectrometry assay to precisely quantify the mutated and canonical protein variants. This allowed phenotyping of eight mCRC tumors and six paired healthy tissues, by determining mutation rates on the protein level. Total KRAS expression varied between tumors (0.47-1.01 fmol/µg total protein) and healthy tissues (0.13-0.64 fmol/µg). In
-mCRC, G12V-mutation levels were 42-100%, while one patient had only 10% KRAS
but 90% KRAS
. This might represent a missed therapeutic opportunity: based on hotspot sequencing, the patient was excluded from anti-EGFR treatment and instead received chemotherapy, while PRM-based tumor-phenotyping indicates the patient might have benefitted from anti-EGFR therapy.
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