Quantitative Measurement of HER2 Expression in Non-Small Cell Lung Cancer With a High-Sensitivity Assay.

Recently, low human epidermal growth factor receptor 2 (HER2) protein expression has been proposed as a predictive biomarker for response to the antibody-drug conjugate trastuzumab deruxtecan (T-DXd) in metastatic breast cancer. HER2 expression in non-small cell lung cancer (NSCLC) patients has never been carefully measured, and little is known about the frequency of cases with unamplified but detectable levels of the protein. Although some HER2-targeted therapies have been studied in NSCLC patients, they have been restricted to those with genomic ERBB2 gene alterations, which only represent relatively rare cases of NSCLC. Still, emerging investigations of T-DXd in NSCLC have shown promise in patients with unamplified HER2. Taken together, we hypothesize that there may be many cases of NSCLC with levels of HER2 protein expression comparable with levels seen in breast cancer that benefit from T-DXd. Here, we used a previously validated, analytic, quantitative immunofluorescence (QIF) assay that is more sensitive than legacy clinical HER2 immunohistochemistry assays. We measured HER2 protein levels in NSCLC cases to determine the proportion of cases with detectable HER2 expression. Using cell line calibration microarrays alongside our QIF method enabled us to convert HER2 signal into units of attomoles per mm2. We found that over 63% of the 741 analyzed NSCLC cases exhibited HER2 expression above the limit of detection, with more than 17% of them exceeding the lower limit of quantification. Although the threshold for response to T-DXd in breast cancer is still unknown, many cases of NSCLC have expression in a range comparable to breast cancer cases with immunohistochemistry scores of 1+ or 2+. Our assay could potentially select NSCLC cases with a detectable target (ie, HER2) that might benefit from HER2 antibody-drug conjugates, irrespective of ERBB2 genomic alterations.

Ursids evolved dietary diversity without major alterations in metabolic rates.

The diets of the eight species of ursids range from carnivory (e.g., polar bears, Ursus maritimus) to insectivory (e.g., sloth bears, Melursus ursinus), omnivory (e.g., brown bears, U. arctos), and herbivory (e.g., giant pandas, Ailuropoda melanoleuca). Dietary energy availability ranges from the high-fat, highly digestible, calorically dense diet of polar bears (~ 6.4 kcal digestible energy/g fresh weight) to the high-fiber, poorly digestible, calorically restricted diet (~ 0.7) of giant pandas. Thus, ursids provide the opportunity to examine the extent to which dietary energy drives evolution of energy metabolism in a closely related group of animals. We measured the daily energy expenditure (DEE) of captive brown bears in a relatively large, zoo-type enclosure and compared those values to previously published results on captive brown bears, captive and free-ranging polar bears, and captive and free-ranging giant pandas. We found that all three species have similar mass-specific DEE when travel distances and energy intake are normalized even though their diets differ dramatically and phylogenetic lineages are separated by millions of years. For giant pandas, the ability to engage in low-cost stationary foraging relative to more wide-ranging bears likely provided the necessary energy savings to become bamboo specialists without greatly altering their metabolic rate.

Polar bear energetic and behavioral strategies on land with implications for surviving the ice-free period.

Declining Arctic sea ice is increasing polar bear land use. Polar bears on land are thought to minimize activity to conserve energy. Here, we measure the daily energy expenditure (DEE), diet, behavior, movement, and body composition changes of 20 different polar bears on land over 19-23 days from August to September (2019-2022) in Manitoba, Canada. Polar bears on land exhibited a 5.2-fold range in DEE and 19-fold range in activity, from hibernation-like DEEs to levels approaching active bears on the sea ice, including three individuals that made energetically demanding swims totaling 54-175 km. Bears consumed berries, vegetation, birds, bones, antlers, seal, and beluga. Beyond compensating for elevated DEE, there was little benefit from terrestrial foraging toward prolonging the predicted time to starvation, as 19 of 20 bears lost mass (0.4-1.7 kg•day-1). Although polar bears on land exhibit remarkable behavioral plasticity, our findings reinforce the risk of starvation, particularly in subadults, with forecasted increases in the onshore period.

Circadian gene transcription plays a role in cellular metabolism in hibernating brown bears, Ursus arctos.

Hibernation is a highly seasonal physiological adaptation that allows brown bears (Ursus arctos) to survive extended periods of low food availability. Similarly, daily or circadian rhythms conserve energy by coordinating body processes to optimally match the environmental light/dark cycle. Brown bears express circadian rhythms in vivo and their cells do in vitro throughout the year, suggesting that these rhythms may play important roles during periods of negative energy balance. Here, we use time-series analysis of RNA sequencing data and timed measurements of ATP production in adipose-derived fibroblasts from active and hibernation seasons under two temperature conditions to confirm that rhythmicity was present. Culture temperature matching that of hibernation body temperature (34 °C) resulted in a delay of daily peak ATP production in comparison with active season body temperatures (37 °C). The timing of peaks of mitochondrial gene transcription was altered as were the amplitudes of transcripts coding for enzymes of the electron transport chain. Additionally, we observed changes in mean expression and timing of key metabolic genes such as SIRT1 and AMPK which are linked to the circadian system and energy balance. The amplitudes of several circadian gene transcripts were also reduced. These results reveal a link between energy conservation and a functioning circadian system in hibernation.

Feeding during hibernation shifts gene expression toward active season levels in brown bears (Ursus arctos).

Hibernation in bears involves a suite of metabolical and physiological changes, including the onset of insulin resistance, that are driven in part by sweeping changes in gene expression in multiple tissues. Feeding bears glucose during hibernation partially restores active season physiological phenotypes, including partial resensitization to insulin, but the molecular mechanisms underlying this transition remain poorly understood. Here, we analyze tissue-level gene expression in adipose, liver, and muscle to identify genes that respond to midhibernation glucose feeding and thus potentially drive postfeeding metabolical and physiological shifts. We show that midhibernation feeding stimulates differential expression in all analyzed tissues of hibernating bears and that a subset of these genes responds specifically by shifting expression toward levels typical of the active season. Inferences of upstream regulatory molecules potentially driving these postfeeding responses implicate peroxisome proliferator-activated receptor gamma (PPARG) and other known regulators of insulin sensitivity, providing new insight into high-level regulatory mechanisms involved in shifting metabolic phenotypes between hibernation and active states.

A multi-tissue gene expression dataset for hibernating brown bears.

OBJECTIVES: Complex physiological adaptations often involve the coordination of molecular responses across multiple tissues. Establishing transcriptomic resources for non-traditional model organisms with phenotypes of interest can provide a foundation for understanding the genomic basis of these phenotypes, and the degree to which these resemble, or contrast, those of traditional model organisms. Here, we present a one-of-a-kind gene expression dataset generated from multiple tissues of two hibernating brown bears (Ursus arctos). DATA DESCRIPTION: This dataset is comprised of 26 samples collected from 13 tissues of two hibernating brown bears. These samples were collected opportunistically and are typically not possible to attain, resulting in a highly unique and valuable gene expression dataset. In combination with previously published datasets, this new transcriptomic resource will facilitate detailed investigation of hibernation physiology in bears, and the potential to translate aspects of this biology to treat human disease.

Multi-Institutional Study of Pathologist Reading of the Programmed Cell Death Ligand-1 Combined Positive Score Immunohistochemistry Assay for Gastric or Gastroesophageal Junction Cancer.

The assessment of the expression of programmed cell death ligand-1 (PD-L1) using immunohistochemistry (IHC) has been controversial since its introduction. The methods of assessment and the range of assays and platforms contribute to confusion. Perhaps the most challenging aspect of PD-L1 IHC is the combined positive score (CPS) method of interpretation of IHC results. Although the CPS method is prescribed for more indications than any other PD-L1 scoring system, its reproducibility has never been rigorously assessed. In this study, we collected a series of 108 gastric or gastroesophageal junction cancer cases, stained them using the Food and Drug Administration-approved 22C3 assay, scanned them, and then circulated them to 14 pathologists at 13 institutions for the assessment of interpretative concordance for the CPS system. We found that higher cut points (10 or 20) performed better than a CPS of <1 or >1. We used the Observers Needed to Evaluate Subjective Tests algorithm to assess how the CPS system might perform in the real-world setting and found that the cut points of <1 or >1 showed an overall percent agreement of only 30% among the pathologist raters, with a plateau occurring at 8 raters. The raters performed better at higher cut points. However, the best cut point of <20 versus that of >20 was still disappointing, with a plateau at an overall percent agreement of 70% (at 7 raters). Although there is no ground truth for CPS, we compared the score with quantitative messenger RNA measurement and showed no relationship between the score (at any cut point) and messenger RNA amount. In summary, we showed that CPS shows high subjective variability among pathologist readers and is likely to perform poorly in the real-world setting. This system may be the root cause of the poor specificity and relatively low predictive value of IHC companion diagnostic tests for PD-1 axis therapies that use the CPS system.

Multi-institutional Assessment of Pathologist Scoring HER2 Immunohistochemistry.

The HercepTest was approved 20+ years ago as the companion diagnostic test for trastuzumab in human epidermal growth factor 2 (HER2) or ERBB2 gene-amplified/overexpressing breast cancers. Subsequent HER2 immunohistochemistry (IHC) assays followed, including the now most common Ventana 4B5 assay. Although this IHC assay has become the clinical standard, its reliability, reproducibility, and accuracy have largely been approved and accepted on the basis of concordance among small numbers of pathologists without validation in a real-world setting. In this study, we evaluated the concordance and interrater reliability of scoring HER2 IHC in 170 breast cancer biopsies by 18 breast cancer-specialized pathologists from 15 institutions. We used the Observers Needed to Evaluate Subjective Tests method to determine the plateau of concordance and the minimum number of pathologists needed to estimate interrater agreement values for large numbers of raters, as seen in the real-world setting. We report substantial discordance within the intermediate categories (<1% agreement for 1+ and 3.6% agreement for 2+) in the 4-category HER2 IHC scoring system. The discordance within the IHC 0 cases is also substantial with an overall percent agreement (OPA) of only 25% and poor interrater reliability metrics (0.49 Fleiss' kappa, 0.55 intraclass correlation coefficient). This discordance can be partially reduced by using a 3-category system (28.8% vs 46.5% OPA for 4-category and 3-category scoring systems, respectively). Observers Needed to Evaluate Subjective Tests plots suggest that the OPA for the task of determining a HER2 IHC score 0 from not 0 plateaus statistically around 59.4% at 10 raters. Conversely, at the task of scoring HER2 IHC as 3+ or not 3+ pathologists' concordance was much higher with an OPA that plateaus at 87.1% with 6 raters. This suggests that legacy HER2 IHC remains valuable for finding the patients in whom the ERBB2 gene is amplified but unacceptably discordant in assigning HER2-low or HER2-negative status for the emerging HER2-low therapies.

Serum plays an important role in reprogramming the seasonal transcriptional profile of brown bear adipocytes.

Understanding how metabolic reprogramming happens in cells will aid the progress in the treatment of a variety of metabolic disorders. Brown bears undergo seasonal shifts in insulin sensitivity, including reversible insulin resistance in hibernation. We performed RNA-sequencing on brown bear adipocytes and proteomics on serum to identify changes possibly responsible for reversible insulin resistance. We observed dramatic transcriptional changes, which depended on both the cell and serum season of origin. Despite large changes in adipocyte gene expression, only changes in eight circulating proteins were identified as related to the seasonal shifts in insulin sensitivity, including some that have not previously been associated with glucose homeostasis. The identified serum proteins may be sufficient for shifting hibernation adipocytes to an active-like state.

Ursids evolved early and continuously to be low-protein macronutrient omnivores.

The eight species of bears world-wide consume a wide variety of diets. Some are specialists with extensive anatomical and physiological adaptations necessary to exploit specific foods or environments [e.g., polar bears (Ursus maritimus), giant pandas (Ailuropoda melanoleuca), and sloth bears (Melursus ursinus)], while the rest are generalists. Even though ursids evolved from a high-protein carnivore, we hypothesized that all have become low-protein macronutrient omnivores. While this dietary strategy has already been described for polar bears and brown bears (Ursus arctos), a recent study on giant pandas suggested their macronutrient selection was that of the ancestral high-protein carnivore. Consumption of diets with inappropriate macronutrient profiles has been associated with increased energy expenditure, ill health, failed reproduction, and premature death. Consequently, we conducted feeding and preference trials with giant pandas and sloth bears, a termite and ant-feeding specialist. Both giant pandas and sloth bears branched off from the ursid lineage a million or more years before polar bears and brown bears. We found that giant pandas are low-protein, high-carbohydrate omnivores, whereas sloth bears are low-protein, high-fat omnivores. The preference for low protein diets apparently occurred early in the evolution of ursids and may have been critical to their world-wide spread.

Examining the Effects of Hibernation on Germline Mutation Rates in Grizzly Bears.

A male mutation bias is observed across vertebrates, and, where data are available, this bias is accompanied by increased per-generation mutation rates with parental age. While continuing mitotic cell division in the male germline post puberty has been proposed as the major cellular mechanism underlying both patterns, little direct evidence for this role has been found. Understanding the evolution of the per-generation mutation rate among species requires that we identify the molecular mechanisms that change between species. Here, we study the per-generation mutation rate in an extended pedigree of the brown (grizzly) bear, Ursus arctos horribilis. Brown bears hibernate for one-third of the year, a period during which spermatogenesis slows or stops altogether. The reduction of spermatogenesis is predicted to lessen the male mutation bias and to lower the per-generation mutation rate in this species. However, using whole-genome sequencing, we find that both male bias and per-generation mutation rates are highly similar to that expected for a non-hibernating species. We also carry out a phylogenetic comparison of substitution rates along the lineage leading to brown bear and panda (a non-hibernating species) and find no slowing of the substitution rate in the hibernator. Our results contribute to accumulating evidence that suggests that male germline cell division is not the major determinant of mutation rates and mutation biases. The results also provide a quantitative basis for improved estimates of the timing of carnivore evolution.

A Beary Good Genome: Haplotype-Resolved, Chromosome-Level Assembly of the Brown Bear (Ursus arctos).

The brown bear (Ursus arctos) is the second largest and most widespread extant terrestrial carnivore on Earth and has recently emerged as a medical model for human metabolic diseases. Here, we report a fully phased chromosome-level assembly of a male North American brown bear built by combining Pacific Biosciences (PacBio) HiFi data and publicly available Hi-C data. The final genome size is 2.47 Gigabases (Gb) with a scaffold and contig N50 length of 70.08 and 43.94 Megabases (Mb), respectively. Benchmarking Universal Single-Copy Ortholog (BUSCO) analysis revealed that 94.5% of single copy orthologs from Mammalia were present in the genome (the highest of any ursid genome to date). Repetitive elements accounted for 44.48% of the genome and a total of 20,480 protein coding genes were identified. Based on whole genome alignment to the polar bear, the brown bear is highly syntenic with the polar bear, and our phylogenetic analysis of 7,246 single-copy orthologs supports the currently proposed species tree for Ursidae. This highly contiguous genome assembly will support future research on both the evolutionary history of the bear family and the physiological mechanisms behind hibernation, the latter of which has broad medical implications.

Temporal Analysis of Gene Expression and Isoform Switching in Brown Bears (Ursus arctos).

Hibernation in brown bears is an annual process involving multiple physiologically distinct seasons-hibernation, active, and hyperphagia. While recent studies have characterized broad patterns of differential gene regulation and isoform usage between hibernation and active seasons, patterns of gene and isoform expression during hyperphagia remain relatively poorly understood. The hyperphagia stage occurs between active and hibernation seasons and involves the accumulation of large fat reserves in preparation for hibernation. Here, we use time-series analyses of gene expression and isoform usage to interrogate transcriptomic regulation associated with all three seasons. We identify a large number of genes with significant differential isoform usage (DIU) across seasons and show that these patterns of isoform usage are largely tissue-specific. We also show that DIU and differential gene-level expression responses are generally non-overlapping, with only a small subset of multi-isoform genes showing evidence of both gene-level expression changes and changes in isoform usage across seasons. Additionally, we investigate nuanced regulation of candidate genes involved in the insulin signaling pathway and find evidence of hyperphagia-specific gene expression and isoform regulation that may enhance fat accumulation during hyperphagia. Our findings highlight the value of using temporal analyses of both gene- and isoform-level gene expression when interrogating complex physiological phenotypes and provide new insight into the mechanisms underlying seasonal changes in bear physiology.

Quantitative measurement of HER2 expression to subclassify ERBB2 unamplified breast cancer.

The efficacy of the antibody drug conjugate (ADC) Trastuzumab deruxtecan (T-DXd) in HER2 low breast cancer patients suggests that the historical/conventional assays for HER2 may need revision for optimal patient care. Specifically, the conventional assay is designed to distinguish amplified HER2 from unamplified cases but is not sensitive enough to stratify the lower ranges of HER2 expression. Here we determine the optimal dynamic range for unamplified HER2 detection in breast cancer and then redesign an assay to increase the resolution of the assay to stratify HER2 expression in unamplified cases. We used the AQUA™ method of quantitative immunofluorescence to test a range of antibody concentrations to maximize the sensitivity within the lower range of HER2 expression. Then, using a cell line microarray with HER2 protein measured by mass spectrometry we determined the amount of HER2 protein in units of attomols/mm2. Then by calculation of the limits of detection, quantification, and linearity of this assay we determined that low HER2 range expression in unamplified cell lines is between 2 and 20 attomol/mm2. Finally, application of this assay to a serial collection of 364 breast cancer cases from Yale shows 67% of the population has HER2 expression above the limit of quantification and below the levels seen in HER2 amplified breast cancer. In the future, this assay could be used to determine the levels of HER2 required for response to T-DXd or similar HER2 conjugated ADCs.

Development of an immunohistochemical assay for Siglec-15.

Siglec-15, a member of sialic-acid binding immunoglobulin type lectins, is normally expressed by myeloid cells and upregulated in some human cancers and represents a promising new target for immunotherapy. While PD-L1 blockade is an important strategy for immunotherapy, its effectiveness is limited. The expression of Siglec-15 has been demonstrated to be predominantly mutually exclusive to PD-L1 in certain cancer histologies. Thus, there is significant opportunity for Siglec-15 as an immunotherapeutic target for patients that do not respond to PD-1/PD-L1 inhibition. The aim of this study was to prospectively develop an immunohistochemical (IHC) assay for Siglec-15 to be used as a companion diagnostic for future clinical trials. Here, we create and validate an IHC assay with a novel recombinant antibody to the cytoplasmic domain of Siglec-15. To find an enriched target, this antibody was first used in a quantitative fluorescence (QIF) assay to screen a broad range of tumor histologies to determine tumor types where Siglec-15 demonstrated high expression. Based on this and previous data, we focused on development of a chromogenic IHC assay for lung cancer. Then we developed a scoring system for this assay that has high concordance amongst pathologist readers. We then use this chromogenic IHC assay to test the expression of Siglec-15 in two cohorts of NSCLC. We found that this assay shows a higher level of staining in both tumor and immune cells compared to previous QIF assays utilizing a polyclonal antibody. However, similar to that study, only a small percentage of positive Siglec-15 cases showed high expression for PD-L1. This validated assay for Siglec-15 expression may support development of a companion diagnostic assay to enrich for patients expressing the Siglec-15 target for therapy.

Real-time luminescence enables continuous drug-response analysis in adherent and suspension cell lines.

The drug-induced proliferation (DIP) rate is a metric of in vitro drug response that avoids inherent biases in commonly used metrics such as 72 h viability. However, DIP rate measurements rely on direct cell counting over time, a laborious task that is subject to numerous challenges, including the need to fluorescently label cells and automatically segment nuclei. Moreover, it is incredibly difficult to directly count cells and accurately measure DIP rates for cell populations in suspension. As an alternative, we use real-time luminescence measurements derived from the cellular activity of NAD(P)H oxidoreductase to efficiently estimate drug response in both adherent and suspension cell populations to a panel of known anticancer agents. For the adherent cell lines, we collect both luminescence reads and direct cell counts over time simultaneously to assess their congruency. Our results demonstrate that the proposed approach significantly speeds up data collection, avoids the need for cellular labels and image segmentation, and opens the door to significant advances in high-throughput screening of anticancer drugs.

Long-read isoform sequencing reveals tissue-specific isoform expression between active and hibernating brown bears (Ursus arctos).

Understanding hibernation in brown bears (Ursus arctos) can provide insight into some human diseases. During hibernation, brown bears experience periods of insulin resistance, physical inactivity, extreme bradycardia, obesity, and the absence of urine production. These states closely mimic aspects of human diseases such as type 2 diabetes, muscle atrophy, as well as renal and heart failure. The reversibility of these states from hibernation to active season enables the identification of mediators with possible therapeutic value for humans. Recent studies have identified genes and pathways that are differentially expressed between active and hibernation seasons in bears. However, little is known about the role of differential expression of gene isoforms on hibernation physiology. To identify both distinct and novel mRNA isoforms, full-length RNA-sequencing (Iso-Seq) was performed on adipose, skeletal muscle, and liver from three individual bears sampled during both active and hibernation seasons. The existing reference genome annotation was improved by combining it with the Iso-Seq data. Short-read RNA-sequencing data from six individuals were mapped to the new reference annotation to quantify differential isoform usage (DIU) between tissues and seasons. We identified differentially expressed isoforms in all three tissues, to varying degrees. Adipose had a high level of DIU with isoform switching, regardless of whether the genes were differentially expressed. Our analyses revealed that DIU, even in the absence of differential gene expression, is an important mechanism for modulating genes during hibernation. These findings demonstrate the value of isoform expression studies and will serve as the basis for deeper exploration into hibernation biology.

Changing lanes: seasonal differences in cellular metabolism of adipocytes in grizzly bears (Ursus arctos horribilis).

Obesity is among the most prevalent of health conditions in humans leading to a multitude of metabolic pathologies such as type 2 diabetes and hyperglycemia. However, there are many wild animals that have large seasonal cycles of fat accumulation and loss that do not result in the health consequences observed in obese humans. One example is the grizzly bear (Ursus arctos horribilis) that can have body fat content > 40% that is then used as the energy source for hibernation. Previous in vitro studies found that hibernation season adipocytes exhibit insulin resistance and increased lipolysis. Yet, other aspects of cellular metabolism were not addressed, leaving this in vitro model incomplete. Thus, the current studies were performed to determine if the cellular energetic phenotype-measured via metabolic flux-of hibernating bears was retained in cultured adipocytes and to what extent that was due to serum or intrinsic cellular factors. Extracellular acidification rate and oxygen consumption rate were used to calculate proton efflux rate and total ATP defined as both ATP from glycolysis and from mitochondrial respiration. Hibernation adipocytes treated with hibernation serum produced less ATP and exhibited lower maximal respiration and glycolysis rates than active season adipocytes. These effects were reversed with serum from the opposite season. Insulin had little influence on total ATP production and lipolysis in both hibernation and active serum-treated adipocytes. Together, these results suggest that the metabolic suppression occurring in hibernation adipocytes are downstream of insulin signaling and likely due to a combined reduction in mitochondria number and/or function and glycolytic processes. Future elucidation of the serum components and the cellular mechanisms that enable alterations in mitochondrial function could provide a novel avenue for the development of treatments for human metabolic diseases.

Fatty acid profiles of feeding and fasting bears: estimating calibration coefficients, the timeframe of diet estimates, and selective mobilization during hibernation.

Accurate information on diet composition is central to understanding and conserving carnivore populations. Quantitative fatty acid signature analysis (QFASA) has emerged as a powerful tool for estimating the diets of predators, but ambiguities remain about the timeframe of QFASA estimates and the need to account for species-specific patterns of metabolism. We conducted a series of feeding experiments with four juvenile male brown bears (Ursus arctos) to (1) track the timing of changes in adipose tissue composition and QFASA diet estimates in response to a change in diet and (2) quantify the relationship between consumer and diet FA composition (i.e., determine "calibration coefficients"). Bears were fed three compositionally distinct diets for 90-120 days each. Two marine-based diets were intended to approximate the lipid content and composition of the wild diet of polar bears (U. maritimus). Bear adipose tissue composition changed quickly in the direction of the diet and showed evidence of stabilization after 60 days. During hibernation, FA profiles were initially stable but diet estimates after 10 weeks were sensitive to calibration coefficients. Calibration coefficients derived from the marine-based diets were broadly similar to each other and to published values from marine-fed mink (Mustela vison), which have been used as a model for free-ranging polar bears. For growing bears on a high-fat diet, the temporal window for QFASA estimates was 30-90 days. Although our results reinforce the importance of accurate calibration, the similarities across taxa and diets suggest it may be feasible to develop a generalized QFASA approach for mammalian carnivores.