TROP2 Is Associated with Primary Resistance to Immune Checkpoint Inhibition in Patients with Advanced Non-Small Cell Lung Cancer.

PURPOSE: Mechanisms of primary resistance to inhibitors of the programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) signaling axis in non-small cell lung cancer (NSCLC) are still poorly understood. While some studies suggest the involvement of trophoblast cell surface antigen 2 (TROP2) in modulating tumor cell resistance to therapeutic drugs, its specific role in the context of PD-1/PD-L1 axis blockade is not definitively established. EXPERIMENTAL DESIGN: We performed high-throughput analysis of transcriptomic data from 891 NSCLC tumors from patients treated with either the PD-L1 inhibitor atezolizumab or chemotherapy in two large randomized clinical trials. To confirm our results at the protein level, we complemented this transcriptional approach by performing a multiplex immunofluorescence analysis of tumor tissue samples as well as a proteomic profiling of plasma. RESULTS: We observed a significant association of TROP2 overexpression with worse progression-free survival and overall survival on PD-L1 blockade, independent of other prognostic factors. Importantly, we found increased TROP2 expression to be predictive of survival in patients treated with atezolizumab but not chemotherapy. TROP2 overexpression was associated with decreased T-cell infiltration. We confirmed these results at the proteomic level both on tumor tissue and in plasma. CONCLUSIONS: Our results suggest an important contribution of TROP2 expression to the primary resistance to PD-L1 blockade in NSCLC. TROP2-biomarker-based strategy may be relevant in selecting patients with NSCLC who are more likely to benefit from a combination of immunotherapy and an anti-TROP2 agent.

Upregulation of Indoleamine 2,3-Dioxygenase 1 in Tumor Cells and Tertiary Lymphoid Structures is a Hallmark of Inflamed Non-Small Cell Lung Cancer.

PURPOSE: Overexpression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) has been reported in several tumor types, including non-small cell lung cancer (NSCLC), and has been shown to promote tumor-immune evasion and inhibit T-cell activation through increased tryptophan degradation and the production of several immunosuppressive metabolites collectively known as kynurenines. However, it remains unclear whether IDO1 expression by tumor cells is detrimental specifically in the context of programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) axis blockade. EXPERIMENTAL DESIGN: We analyzed the transcriptome of 891 NSCLC tumor samples from patients enrolled in two large randomized clinical trials investigating the safety and activity of atezolizumab, a humanized IgG1 mAb that targets PD-L1, versus docetaxel in patients with advanced NSCLC. We complemented these transcriptomics results at the protein level by using multiplex immunofluorescence and at the functional level with in vitro experiments. RESULTS: The increased expression of the tryptophan-catabolizing enzyme IDO1 was significantly associated with improved objective response, progression-free survival, and overall survival in patients treated with PD-L1 inhibitors, but not in those treated with chemotherapy. Strikingly, inflamed tumors had higher levels of IDO1, and IDO1 was also expressed in tertiary lymphoid structures (TLS) by mature follicular dendritic cells. L-kynurenine impaired the differentiation of antibody-producing B cells induced by follicular helper T (Tfh)/B-cell interactions, a hallmark process within TLS. CONCLUSIONS: IDO1 pathway in NSCLC is driven by the immune system rather than by tumor cells. Targeting IDO1 in combination with anti-PD-1/PD-L1 might be beneficial only in patients with inflamed tumors and particularly in those bearing TLS.

Standardized Pathology Screening of Mature Tertiary Lymphoid Structures in Cancers.

Mature tertiary lymphoid structures (mTLSs) are organized lymphoid structures containing B lymphocytes admixed to CD23+ follicular dendritic cells. Their presence has been linked to improved survival and sensitivity to immune checkpoint inhibitors in several cancers, emerging as a promising pancancer biomarker. However, the requirements for any biomarker are clear methodology, proven feasibility, and reliability. In 357 patients' samples, we studied tertiary lymphoid structures (TLSs) parameters using multiplex immunofluorescence (mIF), hematoxylin-eosin-saffron (HES) staining, double CD20/CD23 staining, and single CD23 immunohistochemistry. The cohort included carcinomas (n = 211) and sarcomas (n = 146), gathering biopsies (n = 170), and surgical specimens (n = 187). mTLSs were defined as TLSs containing either a visible germinal center on HES staining or CD23+ follicular dendritic cells. Focusing on 40 TLSs assessed using mIF, double CD20/CD23 staining was less sensitive than mIF to assess maturity in 27.5% (n = 11/40) but was rescued by single CD23 staining in 90.9% (n = 10/11). In 97 patients, several samples (n = 240) were reviewed to characterize TLS distribution. The likelihood of finding TLSs in surgical material was 6.1 higher than in biopsy and 2.0 higher in primary samples than in metastasis after adjustment with a type of sample. Interrater agreement rates over 4 examiners were 0.65 (Fleiss kappa, 95% CI [0.46, 0.90]) for the presence of TLS and 0.90 for maturity (95% CI [0.83, 0.99]). In this study, we propose a standardized method to screen mTLSs in cancer samples using HES staining and immunohistochemistry that can be applied to all specimens.

Proliferative Tumor-Infiltrating Lymphocytes' Abundance within the Microenvironment Impacts Clinical Outcome in Cutaneous B-Cell Lymphomas.

Primary cutaneous large B-cell lymphoma, leg-type (PCLBCL-LT) is the most aggressive primary cutaneous B-cell lymphoma (PCBCL). Tumor microenvironment has a crucial role in tumor development, and tumor-infiltrating lymphocytes (TILs) can be targeted by immunotherapies. We characterized TILs in 20 PCBCLs to identify the tumor microenvironment features associated with clinical outcomes. We developed a seven‒multiplex immunofluorescence panel using Opal staining and image analysis using HALO software. In PCLBCL-LT, TILs were sparsely intermingled within tumor infiltrate in contrast to those in indolent PCBCL where TILs were scattered around tumor nodule edges with variable tumor infiltration. In PCLBCL-LT, TILs were composed of CD8 and CD4, whereas CD4 was predominant in indolent PCBCL. Proliferative TILs (CD3+Ki-67+ cells) were more abundant in PCLBCL-LT (P = 0.0036) than in indolent PCBCL. In PCLBCL-LT, proliferative TILs' abundance tended to be associated with better progression-free survival. These data were confirmed in a second independent cohort of 23 cases showing that proliferative TILs were more abundant in PCLBCL-LT (P = 0.0205) and that in PCLBCL-LT, high CD3+Ki-67+ cell density was associated with better progression-free survival (P = 0.002). These distinct TILs composition and distribution among PCBCL suggest that proliferative T lymphocytes represent a good prognostic factor in PCLBCL-LT and that stimulating their functions may represent a therapeutic approach.

Spatial transcriptomics of macrophage infiltration in non-small cell lung cancer reveals determinants of sensitivity and resistance to anti-PD1/PD-L1 antibodies.

BACKGROUND: Tumor-associated macrophages (TAMs) having immunosuppressive properties are one of the most abundant immune cells in the tumor microenvironment (TME). Preclinical studies have highlighted the potential role of TAMs in resistance to immune checkpoint blockers (ICBs). Here, we investigated the predictive value of TAM infiltration in patients with non-small cell lung cancer (NSCLC) treated with ICBs and characterized their transcriptomic profiles. METHODS: Tumor samples were collected from 152 patients with NSCLC before ICB treatment onset. After immunohistochemical staining and image analysis, the correlation between CD163+ cell infiltration and survival was analyzed. Spatial transcriptomic analyses were performed using the NanoString GeoMx Immune Pathways assay to compare the gene expression profile of tumors with high or low levels of CD163+ cell infiltration and to identify determinants of response to ICBs in tumors with high CD163+ infiltration. RESULTS: Low intratumoral CD163+ cell infiltration was associated with longer progression-free survival (PFS; HR 0.61, 95% CI 0.40 to 0.94, p=0.023) and overall survival (OS; HR 0.48, 95% CI 0.28 to 0.80, p=0.004) under ICB treatment. Spatial transcriptomic profiles of 16 tumors revealed the upregulation of ITGAM, CD27, and CCL5 in tumors with high CD163+ cell infiltration. Moreover, in tumors with high macrophage infiltration, the upregulation of genes associated with the interferon-γ signaling pathway and the M1 phenotype was associated with better responses under immunotherapy. Surprisingly, we found also a significantly higher expression of CSF1R in the tumors of responders. Analysis of three independent data sets confirmed that high CSF1R expression was associated with an increased durable clinical benefit rate (47% vs 6%, p=0.004), PFS (median 10.89 months vs 1.67 months, p=0.001), and OS (median 23.11 months vs 2.66 months, p<0.001) under ICB treatment. CONCLUSIONS: Enrichment of TAMs in the TME of NSCLC is associated with resistance to immunotherapy regardless of the programmed death ligand 1 status and is driven by upregulation of CD27, ITGAM, and CCL5 gene expression within the tumor compartment. Our transcriptomic analyses identify new potential targets to alter TAM recruitment/polarization and highlight the complexity of the CSF1R pathway, which may not be a suitable target to improve ICB efficacy.

Altered inhibitory function in hippocampal CA2 contributes in social memory deficits in Alzheimer's mouse model.

Parvalbumin (PV)-expressing interneurons which are often associated with the specific extracellular matrix perineuronal net (PNN) play a critical role in the alteration of brain activity and memory performance in Alzheimer's disease (AD). The integrity of these neurons is crucial for normal functioning of the hippocampal subfield CA2, and hence, social memory formation. Here, we find that social memory deficits of mouse models of AD are associated with decreased presence of PNN around PV cells and long-term synaptic plasticity in area CA2. Furthermore, single local injection of the growth factor neuregulin-1 (NRG1) is sufficient to restore both PV/PNN levels and social memory performance of these mice. Thus, the PV/PNN disruption in area CA2 could play a causal role in social memory deficits of AD mice, and activating PV cell pro-maturation pathways may be sufficient to restore social memory.

Regorafenib-avelumab combination in patients with biliary tract cancer (REGOMUNE): a single-arm, open-label, phase II trial.

BACKGROUND: Regorafenib has shown substantial clinical activity in patients with advanced biliary tract cancers (BTCs). Preclinical data suggested that this drug modulates antitumour immunity and is synergistic with immune checkpoint inhibition. PATIENTS AND METHODS: This is a single-arm, multicentric phase II trial. Regorafenib was given 3 weeks/4, 160 mg quaque die (once a day) (QD); avelumab 10 mg/kg IV was given every two weeks, beginning at C1D15 until progression or unacceptable toxicity. The primary end-point was the confirmed objective response rate under treatment, as per Response Evaluation Criteria in Solid Tumours 1.1. The secondary end-points included the following: 1-year non-progression rate; progression-free survival (PFS) and overall survival; safety and biomarkers studies performed on sequential tumour samples obtained at baseline and at cycle 2 day 1. RESULTS: Thirty-four patients were enrolled in four centres. Twenty-nine patients were assessable for efficacy after central radiological review. The best response was partial response for four patients (13.8%), stable disease for 11 patients (37.9%) and progressive disease for 14 patients (48.3%). The median PFS and overall survival were 2.5 months (95% confidence interval [CI] [1.9-5.5]) and 11.9 months (95%CI [6.2-NA]) respectively. The most common grade 3 or 4 clinical adverse events related to treatment were hypertension (17.6%), fatigue (14.7%) and maculopapular rash (11.8%). High baseline levels of programmed cell death ligand 1 and of indoleamine 2, 3-dioxygénase expression were associated with improved outcomes. CONCLUSIONS: Regorafenib combined with avelumab has antitumour activity in a subset of heavily pretreated biliary tract cancer population. Further investigations are needed in patients selected based on tumour microenvironment features. CLINICAL TRIAL REGISTRATION: NCT03475953.

Mature tertiary lymphoid structures predict immune checkpoint inhibitor efficacy in solid tumors independently of PD-L1 expression.

Only a minority of patients derive long-term clinical benefit from anti-PD1/PD-L1 monoclonal antibodies. The presence of tertiary lymphoid structures (TLS) has been associated with improved survival in several tumor types. Here, using a large-scale retrospective analysis of three independent cohorts of cancer patients treated with anti-PD1/PD-L1 antibodies, we showed that the presence of mature TLS was associated with improved objective response rate, progression-free survival, and overall survival independently of PD-L1 expression status and CD8+ T-cell density. These results pave the way for using TLS detection to select patients who are more likely to benefit from immune checkpoint blockade.

IDO Targeting in Sarcoma: Biological and Clinical Implications.

Sarcomas are heterogeneous malignant mesenchymal neoplasms with limited sensitivity to immunotherapy. We recently demonstrated an increase in Kynurenine Pathway (KP) activity in the plasma of sarcoma patients treated with pembrolizumab. While the KP has already been described to favor immune escape through the degradation of L-Tryptophan and production of metabolites including L-Kynurenine, Indoleamine 2,3 dioxygenase (IDO1), a first rate-limiting enzyme of the KP, still represents an attractive therapeutic target, and its blockade had not yet been investigated in sarcomas. Using immunohistochemistry, IDO1 and CD8, expression profiles were addressed within 203 cases of human sarcomas. At a preclinical level, we investigated the modulation of the KP upon PDL1 blockade in a syngeneic model of sarcoma through mRNA quantification of key KP enzymes within the tumor. Furthermore, in order to evaluate the possible anti-tumor effect of IDO blockade in combination with PDL1 blockade, an innovative IDO inhibitor (GDC-0919) was used. Its effect was first assessed on Kynurenine to Tryptophan ratio at plasmatic level and also within the tumor. Following GDC-0919 treatment, alone or in combination with anti-PDL1 antibody, tumor growth, immune cell infiltration, and gene expression profiling were measured. IDO1 expression was observed in 39.1% of human sarcoma cases and was significantly higher in tumors with high CD8 infiltration. In the pre-clinical setting, blockade of PDL1 led to a strong anti-tumor effect and was associated with an intratumoral inflammatory cytokines signature driven by Ifng but also with a modulation of the KP enzymes including Ido1 and Ido2. IDO1 inhibition using GDC-0919 resulted in (i) a significant decrease of plasmatic Kynurenine to Tryptophan ratio and in (ii) a decrease of tumoral Kynurenine. However, GDC-0919 used alone or combined with anti-PDL1, did not show anti-tumoral activity and did not affect the tumor immune cell infiltrate. In order to elucidate the mechanism(s) underlying the lack of effect of GDC-0919, we analyzed the gene expression profile of intratumoral biopsies. Interestingly, we have found that GDC-0919 induced a downregulation of the expression of pvr and granzymes, and an upregulation of inhba and Dtx4 suggesting a potential role of the IDO pathway in the control of NK function.

Maturation of PNN and ErbB4 Signaling in Area CA2 during Adolescence Underlies the Emergence of PV Interneuron Plasticity and Social Memory.

Adolescence is a vulnerable period characterized by major cognitive changes. The mechanisms underlying the emergence of new cognitive functions are poorly understood. We find that a long-term depression of inhibitory transmission (iLTD) from parvalbumin-expressing (PV+) interneurons in the hippocampal area Cornu Ammonis 2 (CA2) is absent in young mice but emerges at the end of adolescence. We demonstrate that the maturation of both the perineuronal net (PNN) and signaling through ErbB4 is required for this plasticity. Furthermore, we demonstrate that social recognition memory displays the same age dependence as iLTD and is impaired by targeted degradation of the PNN or iLTD blockade in area CA2. Our data reveal an unusual developmental rule for plasticity at the PV+ interneuron transmission in area CA2 and indicate that this plasticity is involved in the emergence of higher cognitive function, such as social memory formation, in late adolescence.

Early disruption of parvalbumin expression and perineuronal nets in the hippocampus of the Tg2576 mouse model of Alzheimer's disease can be rescued by enriched environment.

Recent findings show that parvalbumin (PV) interneuron function is impaired in Alzheimer's disease (AD), and that this impairment in PV function can be linked to network dysfunction and memory deficits. PV cells are often associated with a specific extracellular matrix, the perineuronal net (PNN). PNNs are believed to protect PV cell integrity, and whether the amyloidopathy affects PNNs remains unclear. Here, we evaluated the number of PV cells with and without PNNs in the hippocampus of the Tg2576 mouse model of AD at different stages of the disease. We show a deficit of PV+ and/or PV+/PNN+ cells in the areas CA1, CA2, and CA3 in Tg2576 as young as 3 months of age. Importantly, transient exposure to an enriched environment, which has proven long-lasting beneficial effects on memory in AD subjects, rescues the PV/PNN cell number deficits. We conclude that cognitive improvements induced by enriched environment in AD mouse models could be supported by a remodeling of hippocampal PV cell network and their PNNs.

Activity of trabectedin and the PARP inhibitor rucaparib in soft-tissue sarcomas.

BACKGROUND: Trabectedin has recently been approved in the USA and in Europe for advanced soft-tissue sarcoma patients who have been treated with anthracycline-based chemotherapy without success. The mechanism of action of trabectedin depends on the status of both the nucleotide excision repair (NER) and homologous recombination (HR) DNA repair pathways. Trabectedin results in DNA double-strand breaks. We hypothesized that PARP-1 inhibition is able to perpetuate trabectedin-induced DNA damage. METHODS: We explored the effects of combining a PARP inhibitor (rucaparib) and trabectedin in a large panel of soft-tissue sarcoma (STS) cell lines and in a mouse model of dedifferentiated liposarcoma. RESULTS: The combination of rucaparib and trabectedin in vitro was synergistic, inhibited cell proliferation, induced apoptosis, and accumulated in the G2/M phase of the cell cycle with higher efficacy than either single agent alone. The combination also resulted in enhanced γH2AX intranuclear accumulation as a result of DNA damage induction. In vivo, the combination of trabectedin and rucaparib significantly enhanced progression-free survival with an increased percentage of tumor necrosis. CONCLUSION: The combination of PARP inhibitor and trabectedin is beneficial in pre-clinical models of soft-tissue sarcoma and deserves further exploration in the clinical setting.

HIPK1 drives p53 activation to limit colorectal cancer cell growth.

HIPK1 (homeodomain interacting protein kinase 1) is a serine/threonine kinase that belongs to the CMGC superfamily. Emerging data point to the role of HIPK1 in cancer, but it is still not clear whether it acts as a tumor suppressor or promoter. Here we identified HIPK1 as a kinase that is significantly overexpressed in colorectal cancer (CRC) and whose expression is stage-dependent. Being abundantly expressed at the onset of the disease, the HIPK1 level gradually decreased as tumor stage progressed. To further uncover how this factor regulates tumorigenesis and establish whether it constitutes an early factor necessary for neoplastic transformation or for cellular defense, we studied the effect of its overexpression in vitro by investigating various cancer-related signaling cascades. We found that HIPK1 mostly regulates the p53 signaling pathway both in HCT116 and HeLa cells. By phosphorylating p53 on its serine-15, HIPK1 favored its transactivation potential, which led to a rise in p21 protein level and a decline in cell proliferation. Assuming that HIPK1 could impede CRC growth by turning on the p53/p21 pathway, we then checked p21 mRNA levels in patients. Interestingly, p21 transcripts were only increased in a subset of patients expressing high levels of HIPK1. Unlike the rest of the cohort, the majority of these patients hosted a native p53 protein, meaning that such a pro-survival pathway (HIPK1+ > p53 > p21) is active in patients, and that HIPK1 acts rather as a tumor suppressor.

Synthesis and evaluation of apoptosis induction of thienopyrimidine compounds on KRAS and BRAF mutated colorectal cancer cell lines.

Monoclonal antibodies (MoAb) and tyrosine kinase inhibitors (TKI) targeting the EGFR (Epidermal Growth Factor Receptor) pathways are currently used in colorectal cancer treatment. Despite the improvement of median overall survival, resistance is observed notably due to KRAS and BRAF gene mutations. We synthesized four series of thienopyrimidines whose scaffold is structurally close to TKI used in clinical practice. We evaluated apoptosis induced by these compounds using flow cytometry on KRAS and BRAF mutated cell lines. Our results confirm that the mutated cell lines (HCT116 and HT29) are more resistant to apoptosis than the non-mutated cell line (Hela). Interestingly, among the 13 compounds tested, three of them (5b, 6b and 6d) and gefitinib exhibited a noteworthy pro-apoptotic effect, especially on mutated cell lines with an IC(50) value between 70 and 110µM. These three compounds seem particularly attractive for the development of novel treatments for colorectal cancer patients harboring EGFR pathway mutations.

Non-antioxidant properties of α-tocopherol reduce the anticancer activity of several protein kinase inhibitors in vitro.

The antioxidant properties of α-tocopherol have been proposed to play a beneficial chemopreventive role against cancer. However, emerging data also indicate that it may exert contrasting effects on the efficacy of chemotherapeutic treatments when given as dietary supplement, being in that case harmful for patients. This dual role of α-tocopherol and, in particular, its effects on the efficacy of anticancer drugs remains poorly documented. For this purpose, we studied here, using high throughput flow cytometry, the direct impact of α-tocopherol on apoptosis and cell cycle arrest induced by different cytotoxic agents on various models of cancer cell lines in vitro. Our results indicate that physiologically relevant concentrations of α-tocopherol strongly compromise the cytotoxic and cytostatic action of various protein kinase inhibitors (KI), while other classes of chemotherapeutic agents or apoptosis inducers are unaffected by this vitamin. Interestingly, these anti-chemotherapeutic effects of α-tocopherol appear to be unrelated to its antioxidant properties since a variety of other antioxidants were completely neutral toward KI-induced cell cycle arrest and cell death. In conclusion, our data suggest that dietary α-tocopherol could limit KI effects on tumour cells, and, by extent, that this could result in a reduction of the clinical efficacy of anti-cancer treatments based on KI molecules.

Tissue microarray cytometry reveals positive impact of homeodomain interacting protein kinase 2 in colon cancer survival irrespective of p53 function.

The human p53 gene is a tumor suppressor mutated in half of colon cancers. Although p53 function appears important for proliferation arrest and apoptosis induced by cancer therapeutics, the prognostic significance of p53 mutations remains elusive. This suggests that p53 function is modulated at a posttranslational level and that dysfunctions affecting its modulators can have a prognostic impact. Among p53 modulators, homeodomain interacting protein kinase (HIPK) 2 emerges as a candidate "switch" governing p53 transition from a cytostatic to a proapoptotic function. Thus, we investigated the possible prognostic role of HIPK2 on a retrospective series of 80 colon cancer cases by setting up a multiplexed cytometric approach capable of exploring correlative protein expression at the single tumor cell level on TMA. Crossing the data with quantitative PCR and p53 gene sequencing and p53 functional assays, we observed the following: despite a strong impact on p21 transcription, the presence of disabling p53 mutations has no prognostic value, and the increased expression of the HIPK2 protein in tumor cells compared with paired normal tissue cells has a strong impact on survival. Unexpectedly, HIPK2 effect does not appear to be mediated by p53 function because it is also observed in p53-disabling mutated backgrounds. Thus, our results point to a prominent and p53-independent role of HIPK2 in colon cancer survival.

Two isoforms of human RNA polymerase III with specific functions in cell growth and transformation.

Transcription in eukaryotic nuclei is carried out by DNA-dependent RNA polymerases I, II, and III. Human RNA polymerase III (Pol III) transcribes small untranslated RNAs that include tRNAs, 5S RNA, U6 RNA, and some microRNAs. Increased Pol III transcription has been reported to accompany or cause cell transformation. Here we describe a Pol III subunit (RPC32beta) that led to the demonstration of two human Pol III isoforms (Pol IIIalpha and Pol IIIbeta). RPC32beta-containing Pol IIIbeta is ubiquitously expressed and essential for growth of human cells. RPC32alpha-containing Pol IIIalpha is dispensable for cell survival, with expression being restricted to undifferentiated ES cells and to tumor cells. In this regard, and most importantly, suppression of RPC32alpha expression impedes anchorage-independent growth of HeLa cells, whereas ectopic expression of RPC32alpha in IMR90 fibroblasts enhances cell transformation and dramatically changes the expression of several tumor-related mRNAs and that of a subset of Pol III RNAs. These results identify a human Pol III isoform and isoform-specific functions in the regulation of cell growth and transformation.

Recombinant differential anchorage probes that tower over the spatial dimension of intracellular signals for high content screening and analysis.

Recombinant fluorescent probes allow the detection of molecular events inside living cells. Many of them exploit the intracellular space to provide positional signals and, thus, require detection by single cell imaging. We describe here a novel strategy based on probes capable of encoding the spatial dimension of intracellular signals into "all-or-none" fluorescence intensity changes (differential anchorage probes, DAPs). The resulting signals can be acquired in single cells at high throughput by automated flow cytometry, (i) bypassing image acquisition and analysis, (ii) providing a direct quantitative readout, and (iii) allowing the exploration of large experimental series. We illustrate our purpose with DAPs for Bax and the effector caspases 3 and 7, which are keys players in apoptotic cell death, and show applications in basic research, high content multiplexed library screening, compound characterization, and drug profiling.