Background. The Nod‐Like‐Receptor‐Protein‐3 (NLRP3) inflammasome and the Interleukin‐6 (IL‐6) pathways are central mechanisms of the inflammatory response in myocardial reperfusion injury. Expanding our knowledge about the inflammasome signaling axis is important to improve treatment options. In a cross‐sectional study, we aimed to study presence, localization, and genetic expression of inflammasome‐ and IL‐6‐ signaling‐related proteins in coronary thrombi and circulating leukocytes from ST‐elevation myocardial infarction (STEMI) patients, with relation to myocardial injury and time from symptoms to PCI. Methods. Intracoronary thrombi were aspirated from 33 STEMI patients. Blood samples were drawn. mRNA of Toll‐Like‐Receptor‐4 (TLR4), NLRP3, caspase 1, Interleukin‐1β (IL1‐β), Interleukin‐18 (IL‐18), IL‐6, IL‐6‐receptor (IL‐6R), and glycoprotein 130 (gp130) were isolated from thrombi and circulating leukocytes and relatively quantified by RT‐PCR. A part of each thrombus was embedded in paraffin for histology and immunohistochemistry analyses. Results. Genes encoding the 8 markers were present in 76‐100% of thrombi. Expression of TLR4 in thrombi significantly correlated to troponin T (r = 0.455, p = 0.013), as did NLRP3 (r = 0.468, p = 0.024). Troponin T correlated with expression in circulating leukocytes of TLR4 (r = 0.438, p = 0.011), NLRP3 (r = 0.420, p = 0.0149), and IL‐1β (r = 0.394, p = 0.023). IL‐6R expression in thrombi correlated significantly to troponin T (r = 0.434, p = 0.019), whereas gp130 was inversely correlated (r = −0.398, p = 0.050). IL‐6 in circulating leukocytes correlated inversely to troponin T (r = −0.421, p = 0.015). There were no significant correlations between genes expressed in thrombi and time from symptom to PCI. Conclusions. The inflammasome signaling pathway was actively regulated in coronary thrombi and in circulating leukocytes from patients with STEMI, in association with myocardial damage measured by troponin T. This supports the strategy of medically targeting this pathway in treating myocardial infarction and contributes to sort out optimal timing and targets for anti‐inflammatory treatment. The study is registered at clinicaltrials.gov with identification number NCT02746822.

Objective: The purpose of this study was to investigate and classify the molecular subtypes of high-grade ductal carcinoma in situ (DCIS) and identify possible high-risk subtypes. The heterogenicity of DCIS with variable clinical and histopathological presentations has been recognized. Nevertheless, only histopathological grading and diameter are currently implemented in clinical decision-making following the diagnosis of DCIS. The molecular subtypes of DCIS and their IHC surrogate markers have not been defined in conventional treatment guidelines and recommendations. We applied the definitions of molecular subtypes according to the IHC surrogate markers defined for IBC and subclassified high-grade DCIS, accordingly. Methods: Histopathological specimens were collected, revised, and regraded from 494 patients diagnosed with DCIS between 1996 and 2018. Other in situ and papillary lesions observed in breast biopsies were excluded from this study. 357 high-grade DCIS cases were submitted to IHC analysis. The markers investigated were ER, PR, HER2, and Ki67. Results: 45 cases were classified as grade 1, 19 as grade 2, and 430 as grade 3. Sixty patients with high-grade DCIS had an additional invasive component in the surgical specimen. Thirty-three patients were diagnosed with recurrent DCIS or invasive cancer (minimum one year after their primary DCIS diagnosis). The proportions of luminal A and luminal B HER2-negative subtypes varied depending on whether 2011 or 2013 St. Gallen Consensus Conference guidelines were adopted. Luminal A was the most prevalent subtype, according to both classifications. The luminal B HER2-positive subtype was found in 22.1% of cases, HER2-enriched subtype in 21.8%, and TPN subtype in 5.6%. There were strong indications that HER2-enriched subtype was significantly more frequent among DCIS with invasive component (p = 0.0169). Conclusions: High-grade DCIS exhibits all the molecular subtypes previously identified in IBC, but with a somewhat different distribution in our cohort. HER2-enriched subtype is substantially related to the presence of an invasive component in DCIS; consequently, it is regarded as a high-risk entity.

Simple summary: We investigated the role of Ki67, a ubiquitous marker in cancer, within the context of ductal carcinoma in situ (DCIS), a precursor of invasive breast cancer. Through rigorous analysis of histopathological and immunopathological samples from a substantial cohort, this study revealed robust correlations between heightened Ki67 expression, diminished progesterone (PR) levels, and HER2 overexpression, indicative of aggressive DCIS phenotypes. These findings offer novel insights into the surrogate immunomolecular subtyping landscape of DCIS, potentially refining risk stratification and therapeutic approaches. This elucidation underscores the translational significance of Ki67 as a prognostic and predictive biomarker in DCIS, with implications for personalized treatment paradigms and patient outcomes.Background: The Ki67 proliferation index is widely used in various tumors, including invasive breast carcinoma (IBC). However, its prognostic utility is often constrained by technical complexity. Its diagnostic and clinical significance in ductal carcinoma in situ (DCIS) remains uncertain. We studied Ki67 immunohistochemistry interobserver diagnostic agreement at different cutoff values in high-grade DCIS. Additionally, we investigated the associations between Ki67 expression, PR levels, and human epidermal growth factor receptor 2 (HER2) in high-grade DCIS among various subtypes (Luminal (Lum) A, LumB HER2^-, LumB HER2⁺, HER2-enriched, and triple-negative)).Methods: Using histopathological specimens from 484 patients diagnosed with DCIS between 1996 and 2018, we implemented the 2013 St. Gallen recommendations for surrogate immunomolecular subtyping of IBC. Subtypes were classified, and the Ki67 interobserver diagnostic agreement between Counting Pathologist 1 (CP1) and CP2 was calculated using Cohen’s kappa coefficient at various cutoff values.Results: The Cohen’s kappa coefficient for interobserver agreement between CP1 and CP2 was κ = 0.586, indicating moderate agreement. Ki67 levels varied significantly among subtypes (p < 0.0001), with a median Ki67% being higher in cases with invasive components (p = 0.0351). Low PR combined with high Ki67% was significantly associated with HER2 overexpression (p = 0.0107).Conclusions: Interobserver agreement for the Ki67 count was moderate. Ki67 expression showed considerable variability in high-grade DCIS. Low PR levels combined with high Ki67 expression were linked to HER2 overexpression, showing possible clinical implications for identifying high-risk DCIS.

Inflammation contributes to myocardial injury in ST-elevation myocardial infarction (STEMI). Interleukin-6 receptor (IL-6R) inhibition has been shown to mitigate myocardial injury and reduce levels of the prothrombotic and inflammatory mediator, neutrophil extracellular traps (NETs). The enzyme peptidylarginine deiminase 4 (PAD4) is central in NET formation. We hypothesized that PAD4 links IL-6R activation and NET formation. Methods: We conducted thrombus aspiration and peripheral blood sampling in 33 STEMI patients. In thrombi and leukocytes, we quantified the mRNA of IL-6, IL-6R, and PAD4. In peripheral blood, the protein levels of IL-6, IL-6R, PAD4, dsDNA, H3Cit, MPO-DNA, and troponin T were quantified. Results: In thrombi and circulating leukocytes, PAD4 mRNA was associated with IL-6R mRNA (thrombi: β = 0.34, 95% CI [0.16–0.53], p = 0.001, circulating leukocytes: β = 0.92, 95% CI [0.07–1.77], p = 0.036). There were no correlations between PAD4 and IL-6 in thrombi and leukocytes. The protein levels of IL-6R were associated with the NET marker H3Cit (r_s = 0.40, p = 0.02). In thrombi, PAD4 mRNA was associated with high levels of troponin T (β = 1.15 95% CI [0.27–2.04], p = 0.013). Conclusion: We demonstrate an association between PAD4, IL-6R, and troponin release in STEMI patients. Our findings indicate a PAD4-mediated connection between IL-6R and NET formation and highlight PAD4 as a potential treatment target for mitigating inflammation and myocardial injury in STEMI.

Objective: In this study, we investigated pivotal molecular markers in human high-grade breast ductal carcinoma in situ (DCIS). Expression status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth receptor 2 (HER2) was measured among various subtypes (Luminal (Lum) A, LumB HER2^-, LumB HER2⁺, HER2-enriched and triple-negative).Methods: In total, 357 DCIS cases were classified into respective subtypes, according to the 2013 St. Gallen guidelines. Each subtype was categorized into three subcategories: “Pure” (those without an invasive component), “W/invasive” (those with an invasive component), and “All” (the entire group of the given subtype). ER and PR expression were registered as intervals. Equivocal HER2 immunohistochemistry (IHC) cases (2+) were further investigated using dual-color in situ hybridization.Results: The majority of patients (71%) were over the age of 50. We discovered no significant differences in the proportion of age between the “Pure” and “W/invasive” groups. There was no significant difference in ER/PR expression between “Pure” luminal subtypes of DCIS and “W/invasive” cases. We compared the HER2 IHC scores of “0”, “1+”, and “2+” among LumA and LumB HER2 subtypes and identified no statistically significant differences between “Pure” and “W/invasive” (p = 0.603). ER and PR expression ≥ 50% cutoff value was present in > 90% of all LumA cases. The incidences of cases with ER expression at cutoff values of < 10% and ≥ 50% in LumA were significantly different compared to other luminal subtypes (p < 0.0001). The proportion of cases with PR expression < 20% showed significant differences in the various luminal subtypes. In luminal B subtypes, low PR expression (< 20%) was significantly associated with both strong HER2 expression (3+) and the presence of an invasive component (p = 0.0001 and p = 0.0365, respectively).Conclusions: ER and PR expression at ≥ 50% cutoff values were found in more than 90% of LumA cases. Samples with ER < 10% and ≥ 50% in LumA were significantly different compared to other luminal subtypes (p < 0.0001). Low PR expression in high-grade DCIS was strongly associated with HER2 overexpression (3+) and an invasive component (p = 0.0001 and p = 0.0365, respectively).