Histopathology and levels of proteins in plasma associate with survival after colorectal cancer diagnosis

Funding Information: The authors thank the subjects who have donated their time and their samples that were used in this research. Publisher Copyright: © 2023, The Author(s).Background: The TNM system is used to assess prognosis after colorectal cancer (CRC) diagnosis. Other prognostic factors reported include histopathological assessments of the tumour, tumour mutations and proteins in the blood. As some of these factors are strongly correlated, it is important to evaluate the independent effects they may have on survival. Methods: Tumour samples from 2162 CRC patients were visually assessed for amount of tumour stroma, severity of lymphocytic infiltrate at the tumour margins and the presence of lymphoid follicles. Somatic mutations in the tumour were assessed for 2134 individuals. Pre-surgical levels of 4963 plasma proteins were measured in 128 individuals. The associations between these features and prognosis were inspected by a Cox Proportional Hazards Model (CPH). Results: Levels of stroma, lymphocytic infiltration and presence of lymphoid follicles all associate with prognosis, along with high tumour mutation burden, high microsatellite instability and TP53 and BRAF mutations. The somatic mutations are correlated with the histopathology and none of the somatic mutations associate with survival in a multivariate analysis. Amount of stroma and lymphocytic infiltration associate with local invasion of tumours. Elevated levels of two plasma proteins, CA-125 and PPP1R1A, associate with a worse prognosis. Conclusions: Tumour stroma and lymphocytic infiltration variables are strongly associated with prognosis of CRC and capture the prognostic effects of tumour mutation status. CA-125 and PPP1R1A may be useful prognostic biomarkers in CRC.Peer reviewe

Respiratory syncytial virus reverses airway hyperresponsiveness to methacholine in ovalbumin-sensitized mice.

Each year, approximately 20% of asthmatics in the United States experience acute symptom exacerbations, which commonly result from pulmonary viral infections. The majority of asthma exacerbations in very young children follow infection with respiratory syncytial virus (RSV). However, pathogenic mechanisms underlying induction of asthma exacerbations by RSV are not well understood. We therefore investigated the effect of post-sensitization RSV infection on lung function in ovalbumin (OVA)-sensitized BALB/c mice as a model of RSV asthma exacerbations. OVA sensitization of uninfected female BALB/c mice increased bronchoalveolar lavage fluid (BALF) eosinophil levels and induced airway hyperresponsiveness to the muscarinic agonist methacholine, as measured by the forced-oscillation technique. In contrast, intranasal infection with replication-competent RSV strain A2 for 2-8 days reduced BALF eosinophil counts and reversed airway hyperresponsiveness in a pertussis toxin-sensitive manner. BALF levels of the chemokine keratinocyte cytokine (KC; a murine homolog of interleukin-8) were elevated in OVA-sensitized, RSV-infected mice and reversal of methacholine hyperresponsiveness in these animals was rapidly inhibited by KC neutralization. Hyporesponsiveness could be induced in OVA-sensitized, uninfected mice by recombinant KC or the Gαi agonist melittin. These data suggest that respiratory syncytial virus induces KC-mediated activation of Gαi, resulting in cross-inhibition of Gαq-mediated M(3)-muscarinic receptor signaling and reversal of airway hyperresponsiveness. As in unsensitized mice, KC therefore appears to play a significant role in induction of airway dysfunction by respiratory syncytial virus. Hence, interleukin-8 may be a promising therapeutic target to normalize lung function in both asthmatics and non-asthmatics with bronchiolitis. However, the OVA-sensitized, RSV-infected mouse may not be an appropriate model for investigating the pathogenesis of viral asthma exacerbations

Special Issue on Digital Pathology, Tissue Image Analysis, Artificial Intelligence, and Machine Learning: Approximation of the Effect of Novel Technologies on Toxicologic Pathology

For decades, it has been postulated that digital pathology is the future. By now it is safe to say that we are living that future. Digital pathology has expanded into all aspects of pathology, including human diagnostic pathology, veterinary diagnostics, research, drug development, regulatory toxicologic pathology primary reads, and peer review. Digital tissue image analysis has enabled users to extract quantitative and complex data from digitized whole-slide images. The following editorial provides an overview of the content of this special issue of Toxicologic Pathology to highlight the range of key topics that are included in this compilation. In addition, the editors provide a commentary on important current aspects to consider in this space, such as accessibility of publication content to the machine learning-novice pathologist, the importance of adequate test set selection, and allowing for data reproducibility

Keratinocyte cytokine released in response to RSV infection reverses hyperresponsiveness to methacholine in OVA-sensitized mice.

<p>(<b>A</b>) Bronchoalveolar lavage fluid keratinocyte cytokine (KC; ng/ml) levels in unsensitized, uninfected mice (UNSENS/UNINF; <i>n</i> = 5), OVA-sensitized, uninfected mice (OVA/UNINF; <i>n</i> = 11), OVA-sensitized, uninfected mice treated with 50 µg/ml heat-inactivated recombinant murine KC (OVA/UNINF + HI-KC; <i>n</i> = 5), OVA-sensitized, uninfected mice treated with 50 µg/ml recombinant murine KC (OVA/UNINF + KC; <i>n</i> = 7), OVA-sensitized mice infected with RSV (10⁶ pfu/mouse) for 2 days (OVA/DAY 2; <i>n</i> = 6), OVA-sensitized mice “infected” with UV-inactivated RSV for 2 days (OVA/UVx DAY 2; <i>n</i> = 4), and OVA-sensitized mice infected with RSV for 8 days (OVA/DAY 8; <i>n</i> = 6). *<i>P</i><0.05, ***<i>P</i><0.0005, <i>vs</i>. UNSENS/UNINF mice. (<b>B</b>) Bronchoconstrictive responses to increasing doses of nebulized methacholine (MCH) in OVA-sensitized, RSV-infected mice following nebulization of normal rat IgG (50 µg/ml; <i>n</i> = 5), KC-neutralizing monoclonal antibody (ANTI-KC, 50 µg/ml; <i>n</i> = 5), pretreatment with pertussis toxin and IgG (PTX + IgG; <i>n</i> = 6), or pretreatment with pertussis toxin and KC neutralizing antibody (PTX + ANTI-KC; <i>n</i> = 8). ***MCH dose-response curve differs significantly (<i>P</i><0.0005) from UNSENS/UNINF mice (<i>n</i> = 16).</p

RSV infection reverses airway hyperresponsiveness to methacholine in OVA-sensitized mice.

<p>Bronchoconstrictive responses to increasing doses of nebulized methacholine (MCH) in: (<b>A</b>) unsensitized, uninfected mice analyzed on day 0 (UNSENS/UNINF; <i>n</i> = 6), OVA-sensitized, uninfected mice (OVA/UNINF; <i>n</i> = 8), and OVA-sensitized mice infected with RSV (10⁶ pfu/mouse) for 2 days (OVA/DAY 2; <i>n</i> = 16), or 8 days (OVA/DAY 8; <i>n</i> = 8); (<b>B</b>) OVA/UNINF mice and OVA-sensitized mice “infected” with UV-inactivated RSV for 2 days (OVA/UVx DAY 2; <i>n</i> = 5) or 8 days (OVA/UVx DAY 8; <i>n</i> = 9). ***MCH dose-response curve differs significantly (<i>P</i><0.0005) from UNSENS/UNINF mice.</p

Keratinocyte cytokine exposure and Gαi activation are both sufficient to reverse methacholine hyperresponsiveness in OVA-sensitized, uninfected mice.

<p>Bronchoconstrictive responses to increasing doses of nebulized methacholine (MCH) following nebulization of: (<b>A</b>) heat-inactivated recombinant murine keratinocyte cytokine (HI-rmKC, 50 µg/ml; <i>n</i> = 6) or recombinant murine keratinocyte cytokine (rmKC, 50 µg/ml; <i>n</i> = 7); and (<b>B</b>) Melittin (100 µM; <i>n</i> = 7). ***MCH dose-response curve differs significantly (<i>P</i><0.0005) from OVA/UNINF mice (<i>n</i> = 8).</p

Schematic timeline of the OVA sensitization/challenge and RSV infection protocol.

<p>Mice were sensitized by intraperitoneal (i.p.) injection of OVA in alum at days −28 and −14. From −7 to −3 days, mice were challenged daily by intranasal (i.n.) OVA instillation. Animals were infected with RSV 3 days after the last OVA challenge (day 0). Airway responsiveness to methacholine (MCH) was measured at 2–8 days post-infection (d.p.i.).</p

RSV infection reverses hyperresponsiveness to methacholine in OVA-sensitized mice via a pertussis toxin-sensitive pathway.

<p>Bronchoconstrictive response to increasing doses of nebulized methacholine (MCH) following pretreatment with saline (100 µl i.p.; <i>n</i> = 4) or pertussis toxin (PTX, 100 µg/kg in 100 µl saline i.p.; <i>n</i> = 9). ***MCH dose-response curve differs significantly (<i>P</i><0.0005) from OVA/DAY 2 mice (OVA-sensitized mice infected with 10⁶ pfu/mouse RSV A2 for 2 days; <i>n</i> = 16).</p

RSV infection reduces bronchoalveolar lavage fluid cell counts in OVA-sensitized mice.

<p>Effect of OVA sensitization on (<b>A</b>) Alveolar macrophage (AM), eosinophil (EO) and small lymphocyte (SL) counts in uninfected mice (OVA/UNINF; <i>n</i> = 8) and unsensitized, uninfected controls (UNSENS/UNINF; <i>n</i> = 8); (<b>B</b>) Total cell counts in OVA-sensitized mice after mock infection for 2 days (M2; <i>n</i> = 5), 4 days (M4; <i>n</i> = 4), or 8 days (M8; <i>n</i> = 8), and infection with RSV (10⁶ pfu/mouse) for 2 days (<i>n</i> = 16), 4 days (<i>n</i> = 10), or 8 days (<i>n</i> = 6); (<b>C</b>) AM, EO, and SL counts after mock infection for 2, 4, or 8 days and infection with RSV for 2–8 days; and (<b>D</b>) Neutrophil (PMN) counts after infection with RSV for 2–8 days. No PMNs were detected in bronchoalveolar lavage fluid from uninfected or mock-infected mice at any timepoint (not shown). *<i>P</i><0.05, **<i>P</i><0.005, ***<i>P</i><0.001. N.D.: None detected.</p