BackgroundMultiplexed ion beam imaging (MIBI) combines time-of-flight secondary ion mass spectrometry (ToF-SIMS) with metal labeled antibodies to image 40+ proteins in a single scan at subcellular spatial resolution. Here, we show that the recently released MIBIscope provides improved sensitivity for detecting immune checkpoint markers and offers greater throughput at higher resolution than the alpha instrument.MethodsSerial sections from three FFPE NSCLC samples, in addition to a control slide consisting of various unremarkable tissues, were stained with a panel of 25 metal labeled antibodies. The tissue was imaged at subcellular resolution using the MIBIscope and the alpha instrument. Masses of detected species were assigned to target biomolecules given the unique label of each antibody and multi-step processing was used to create images. Cell classification was performed using two complementary methods that differed in the need for cell segmentation to phenotypically characterize the tissue environments and quantify marker expression.ResultsReplicate regions of interest (ROIs) were collected on both instruments with similarly sized ROIs acquired in 17 minutes with the MIBIscope compared to 280 minutes with the alpha instrument. Fourier Ring Correlation (FRC) showed the resolution to be greater on the MIBIscope as compared to the alpha instrument with FRC also demonstrating uniform resolution across an ROI 2.5X greater in size. Even with the 16X greater speed of the MIBIscope, the signal of the 25 markers across replicate ROIs was increased (y=x^1.07) and showed similar expression patterns to those observed on the alpha instrument (figure 1). This resulted in greater sensitivity to markers with low expression, such as checkpoint markers. Eleven cell populations were classified across the ROIs utilizing two methods, with both methods showing a similar frequency of tumor cells and B, T, and myeloid cell subsets between instruments. Segmentation enabled the number of cells within a population to be calculated but defining boundaries is laborious and signal from neighboring cells can result in misclassification. Performing classification at the pixel level, without segmentation, enabled the fraction of the tissue that is tumor or any other cell type to be rapidly determined.Abstract 48 Figure 1Comparison of images acquired between instrumentsThe signal intensity is greater on the MIBIscope and shows a similar staining pattern as achieved by the alpha instrument. Shown are 3 overlays from a single scan from replicate ROIs of an NSCLC sample displayed with the same contrast settings.ConclusionsThe MIBIscope enables the phenotypic characterization of tumor and non-tumor microenvironments. Co-expression of markers can be used to classify tumor and immune populations and to quantify the expression of markers associated with immune suppression. The increased sensitivity and throughput of the MIBIscope, in combination with the 40-parameter capability and subcellular resolution, provides a platform uniquely suited to understanding the complex tumor immune landscape.
STK11 loss of function mutations occur in 12-15% of lung adenocarcinoma and have been shown to drive resistance to immune checkpoint blockade in patients and preclinical models. STK11-deficient syngeneic mouse tumor models reflect this biology and are insensitive to anti-PD1 treatment. Informed by in vivo CRISPR-based screens, histone deacetylase 1 (HDAC1) was identified as a target gene, which when knocked out in tumor cells, reverses anti-PD1 resistance driven by STK11 tumor suppressor gene loss. Here, we describe the discovery and development of TNG260 as a potent and selective inhibitor of the HDAC1-containing complex CoREST. TNG260 inhibits CoREST deacetylase activity with 500-fold selectivity over the other HDAC1-containing complexes, NuRD and Sin3, and the HDAC3-containing complex NCoR. In preclinical studies, selective CoREST inhibition by TNG260 results in transcriptional reprogramming of STK11-mutant tumor cells, altering tumor cell cytokine secretion and markedly reducing recruitment of suppressive Treg cells to STK11-mutant tumors. Moreover, TNG260, in combination with anti-PD1 treatment, drives durable tumor regressions in multiple syngeneic STK11-mutant xenograft models. TNG260 has no anti-tumor efficacy in athymic mice, indicating the responses with TNG260 are mediated by T cells. Unlike previously developed pan-HDAC inhibitors, which are directly cytotoxic to cancer (and immune) cells, IND-enabling toxicology studies in rat and dog showed that TNG260 was well-tolerated at exposures predicted to be efficacious in humans, with bone marrow suppression only detectable at doses where TNG260 is no longer selective for CoREST inhibition. TNG260 clinical development will be among the first to combine the power of genetic patient selection and immunotherapy, evaluating patients with STK11 mutant cancers in a trial combining TNG260 and a checkpoint inhibitor. Citation Format: Leanne G. Ahronian, Minjie Zhang, Chengyin Min, Alice W. Tsai, Jacques Ermolieff, Patrick McCarren, Margaret Wyman, David Guerin, Ye Wang, Alborz Bejnood, Kenjie Amemiya, Brian McMillan, Nikitha Das, Preksha Shahagadkar, Brian Doyon, Andre Mignault, Colin Liang, Vassil Elitzin, Samuel R. Meier, Ashley Choi, Yi Yu, John P. Maxwell, Brian B. Haines, Jannik N. Andersen, Heather DiBenedetto, Aaron Weitzman, Alan Huang, Xinyuan Wu. TNG260: A novel, orally active, CoREST-selective deacetylase inhibitor for the treatment of STK11-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND12.
3542 Background: Achieving major molecular response (MMR) is an important milestone in chronic myeloid leukemia (CML) therapy. MMR has been defined as a 3-log reduction in BCR-ABL transcript levels from a standardized baseline (BL) established in the IRIS trial (Hughes TP, N Engl J Med. 2003). Standardization has been achieved through the development of an IS, which defines MMR as BCR-ABLIS = 0.1%. In contrast, the NCCN defines MMR as a 3-log reduction in BCR-ABL transcript levels but is indefinite on the definition of BL. Here, using reconstructed samples emulating CML patient BCR-ABL levels, the pairwise concordance of MMR determination was examined within and between 3 labs using the IS-standardized GeneXpert® (GX) system and 3 labs using laboratory-developed tests (LDTs). For comparative purposes, this analysis assumes BL is established at the time of diagnosis. Methods: 100 virtual patients (VPs) were emulated based on data from the REVEAL BCR-ABL Methods Comparison Study, in which 8 discrete levels of blinded K562 cell–spiked blood corresponding to BCR-ABLIS ratios ranging from ∼10% to ∼0.01% were analyzed by 3 labs using the IS-standardized GX system and 3 labs using non-IS LDTs. VP emulations were guided by actual patient outcomes in landmark analyses of 7- treatment response (Hughes TP, Blood. 2010). Treatment response profiles over an 18-month time horizon were modeled by assigning one of the 8 BCR-ABL levels ranging from approximately 10%-0.01% IS sampled in the REVEAL study to each of 4 virtual time points (eg, 3, 6, 12, and 18 months). BL levels were selected from quartiles representing pretreatment BCR- ABL ratios between 50–150%; results based on BL levels observed in the IRIS clinical trial will also be presented. 600 VP transcript profiles (VTPs) were then reconstructed using data from each of the 6 laboratories for all 100 VPs. The final 18-month time point in each VTP provided the BCR-ABL level against which the IS or NCCN objective criterion was applied to make MMR determinations. MMR concordance was evaluated by inspecting all possible inter-lab pairwise comparisons among the 100 VPs. Results: Pairwise concordance in MMR as determined by NCCN criterion among all 6 labs is shown in Fig 1A. MMR determinations among the 3 GX labs were concordant in 88% to 93% of VPs. In contrast, MMR determinations among the LDTs were concordant in 43% to 80% of VPs, and MMR determinations were concordant in 53% to 91% of VPs when compared between GX labs and LDTs. When MMR determination based on IS criterion for GX was considered, MMR concordance improved to 93% to 96% among the GX labs in contrast to 51% to 92% concordance observed between the GX and LDT sites (Fig 1B). It is noteworthy that Lab D results more closely approximated the IS than results from the other LDTs examined in the REVEAL study (data not shown). Although Lab D does not report results per the IS, it does report results relative to a median diagnostic BL, similar to the approach used in the IRIS trial. A healthcare system based on LDTs without any attempted IS standardization resulted in MMR concordance of only 43%. Potential sources of discordance among tests will be discussed in detail. Conclusions: These results illustrate that the NCCN criterion for MMR determination is not adequate for inter-lab comparisons of BCR-ABL transcript levels near the clinically important level of MMR. In contrast, standardization to the IS improves inter-lab concordance in MMR determination. Taken together, these results highlight the discrepancies that may result when comparing molecular responses between labs not standardized to the IS. As attainment of MMR is a critical milestone of CML therapy, errors in MMR determination may have an adverse impact on CML disease management. Disclosures: Reddy: Novartis: Research Funding, as Presenting Author, sponsorship to attend ASH. Höfling:Novartis: Employment. Manning:Novartis: Employment. Mignault:Novartis: Employment. Mullaney:Novartis: Employment. Ossa:Novartis: Employment. Stein:Novartis: Employment. Wang:Novartis: Employment. Yang:Novartis: Employment.
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