Probes for INS

Black men are two to three times more likely to die from prostate cancer (PCa) than White men. This disparity is due in part to discrepancies in socioeconomic status and access to quality care. Studies also suggest that differences in the prevalence of innate immune cells and heightened function in the tumor microenvironment of Black men may promote PCa aggressiveness.We evaluated the spatial localization of and quantified CD66ce+ neutrophils by immunohistochemistry and CD68+ (pan), CD80+ (M1), and CD163+ (M2) macrophages by RNA in situ hybridization on formalin-fixed paraffin-embedded tissues from organ donor "normal" prostate (n = 9) and radical prostatectomy (n = 38) tissues from Black and White men. Neutrophils were quantified in PCa and matched benign tissues in tissue microarray (TMA) sets comprised of 560 White and 371 Black men. Likewise, macrophages were quantified in TMA sets comprised of tissues from 60 White and 120 Black men. The phosphatase and tensin homolog (PTEN) and ETS transcription factor ERG (ERG) expression status of each TMA PCa case was assessed via immunohistochemistry. Finally, neutrophils and macrophage subsets were assessed in a TMA set comprised of distant metastatic PCa tissues collected at autopsy (n = 6) sampled across multiple sites.CD66ce+ neutrophils were minimal in normal prostates, but were increased in PCa compared to benign tissues, in low grade compared to higher grade PCa, in PCa tissues from White compared to Black men, and in PCa with PTEN loss or ERG positivity. CD163+ macrophages were the predominant macrophage subset in normal organ donor prostate tissues from both Black and White men and were significantly more abundant in organ donor compared to prostatectomy PCa tissues. CD68,+  CD80,+ and CD163+ macrophages were significantly increased in cancer compared to benign tissues and in cancers with ERG positivity. CD68+ and CD163+ macrophages were increased in higher grade cancers compared to low grade cancer and CD80 expression was significantly higher in benign prostatectomy tissues from Black compared to White men.Innate immune cell infiltration is increased in the prostate tumor microenvironment of both Black and White men, however the composition of innate immune cell infiltration may vary between races.

Abstract: Aims: To investigate the frequency and transcriptional activity of HPV and its correlation to p16 and p21 expression in basaloid squamous cell carcinoma (BSCC) of the larynx. Methods: We evaluated tissues from 29 patients with BSCC of the larynx for the expressions of p16 and p21 proteins by immunohistochemistry (IHC) and for HPV E6 and E7 mRNA by RNA in situ hybridization (ISH). The presence of genotype-specific HPV DNA was evaluated using PCR-RDB in formalin-fixed paraffin-embedded tissues. P16 and p21 expression and HPV DNA status were correlated with clinicopathological features. Results: HPV DNA was detected in 8 of 29 (27.59%) patients, with HPV-16 being the predominant genotype. P16 and p21-positivity were observed in 7/29 (24.14%) and 8/29 (27.59%) patients, respectively. HPV was not correlated with p16 expression (P > 0.05). However, p21 expression was significantly higher in HPV-positive tumors than in HPV-negative tumors (P < 0.05). No cases exhibited transcriptionally active HPV in our series. Conclusion: Our findings suggest that a small fraction of BSCC of the larynx is HPV DNA-positive in this Chinese population, p21 expression was significantly higher in HPV-positive tumors, and no cases were HPV transcriptionally active in this small cohort. Further research of HPV and its role in BSCC of the larynx are warranted.

High CD103+ intratumoral immune cell (ITIC) abundance is associated with better prognosis in unselected patients with human papilloma virus associated oropharyngeal squamous cell carcinoma(HPV-associated OPSCC) treated with cisplatin and radiotherapy(CIS/RT). Substituting cetuximab(CETUX) for CIS with RT in HPV-associated OPSCC resulted in inferior efficacy. Our aim was to determine if quantification of ITIC CD103 could be used to identify a population of HPV-associated OPSCC with superior prognosis.We pooled data from the TROG 12.01 and De-ESCALaTE randomised trials that compared CETUX/70GyRT with CIS/70GyRT in low risk HPV-associated OPSCC: AJCC 7th Stage III (excluding T1-2N1) or stage IV (excluding N2b-c if smoking history >10 pack years and/or distant metastases), including all patients with available tumor samples. The primary endpoint was failure-free survival (FFS) in patients receiving CETUX/ RT comparing CD103+ ITIC high (>30%) versus low (<30%). High/low CD103 were compared using Cox regression adjusting for age, stage and trial.Tumor samples were available in 159/182 patients on TROG 12.01 and 145/334 on De-ESCALaTE. CD103+ ITIC abundance was high in 27% of patients. The median follow-up was 3.2 years. The 3-year FFS in patients treated with CETUX/RT were 93% (95% CI: 79-98%) in high CD103 and 74% (95% CI: 63-81%) in low CD103, adjusted HR 0.22 (95% CI: 0.12-0.41); p<0.001. The 3-year overall survival in patients treated with CETUX/RT was 100% in high CD103 and 86% (95% CI: 76-92%) in low CD103, p<0.001. In patients treated with CIS/RT there was no significant difference in FFS.CD103+ ITIC expression separates CETUX/RT treated low risk HPV-associated OPSCC into excellent and poor prognosis subgroups. The high CD103 population is a rational target for de-intensification trials.

Succinate is released by skeletal muscle during exercise and activates SUCNR1/GPR91. Signaling of SUCNR1 is involved in metabolite-sensing paracrine communication in skeletal muscle during exercise. However, the specific cell types responding to succinate and the directionality of communication are unclear. We aim to characterize the expression of SUCNR1 in human skeletal muscle. De novo analysis of transcriptomic datasets demonstrated that SUCNR1 mRNA is expressed in immune, adipose, and liver tissues, but scarce in skeletal muscle. In human tissues, SUCNR1 mRNA was associated with macrophage markers. Single-cell RNA sequencing and fluorescent RNAscope demonstrated that in human skeletal muscle, SUCNR1 mRNA is not expressed in muscle fibers but coincided with macrophage populations. Human M2-polarized macrophages exhibit high levels of SUCNR1 mRNA and stimulation with selective agonists of SUCNR1 triggered Gq- and Gi-coupled signaling. Primary human skeletal muscle cells were unresponsive to SUCNR1 agonists. In conclusion, SUCNR1 is not expressed in muscle cells and its role in the adaptive response of skeletal muscle to exercise is most likely mediated via paracrine mechanisms involving M2-like macrophages within the muscle.

RNA plays a vital role in the physiological and pathological processes of cells and tissues. However, RNA in situ hybridization applications in clinical diagnostics are still limited to a few examples. In this study, we developed a novel in situ hybridization assay for human papillomavirus (HPV) E6/E7 mRNA by taking advantage of specific padlock probing and rolling circle amplification, combined with chromogenic readout. We designed padlock probes for 14 types of high-risk HPV and demonstrated that E6/E7 mRNA could be visualized in situ as discrete dot-like signals using bright-field microscopy. Overall, the results are consistent with the clinical diagnostics lab's hematoxylin and eosin (H&E) staining and p16 immunohistochemistry test results. Our work thus shows the potential applications of RNA in situ hybridization for clinical diagnostics using chromogenic single-molecule detection, offering an alternative technical option to the current commercially available kit based on branched DNA technology. IMPORTANCE In situ detection of viral mRNA expression in tissue samples is of great value for pathological diagnosis to access viral infection status. Unfortunately, conventional RNA in situ hybridization assays lack sensitivity and specificity for clinical diagnostic purposes. Currently, the commercially available branched DNA technology-based single-molecule RNA in situ detection method offers satisfactory results. Here, we present our padlock probe- and rolling circle amplification-based RNA in situ hybridization assay for detecting HPV E6/E7 mRNA expression in formalin-fixed paraffin-embedded tissue sections, providing an alternative yet robust method for viral RNA in situ visualization that is also applicable to different types of diseases.

The aim of the study was to investigate prevalence of high-risk human papillomavirus (HR-HPV) infection in sinonasal carcinomas by immunohistochemistry, in situ hybridization, and polymerase chain reaction, detecting p16INK4a protein (p16) expression and presence of both HPV DNA and HPV E6/E7 messenger RNA (mRNA). The study comprised 47 males and 26 females, aged 23-83 years (median 62 years), mostly (67 %) with a squamous cell carcinoma (SCC). Of the tumors, 53 % arose in the nasal cavity, 42 % in the maxillary sinus, and 5 % in the ethmoid complex. The follow-up period ranged 1-241 months (median 19 months). HPV16, HPV18, or HPV35 were detected in 18/73 (25 %) tumors, 17 SCCs, and 1 small cell neuroendocrine carcinoma. There was a strong correlation between results of HPV detection methods and p16 expression (p < 0.005). HPV-positive SCCs occurred more frequently in smokers (p = 0.04) and were more frequently p16-positive (p < 0.0001) and nonkeratinizing (p = 0.02), the latter occurring more commonly in nasal cavity (p = 0.025). Median survival for HPV-positive SCC patients was 30 months, while for HPV-negative SCC patients was 14 months (p = 0.23). In summary, we confirm that HR-HPV is actively involved in the etiopathogenesis of a significant subset of sinonasal SCCs. p16 may be used as a reliable surrogate marker for determination of HPV status also in sinonasal SCCs. Although we observed a trend toward better overall survival in HPV-positive SCCs, the prognostic impact of HPV status in sinonasal carcinomas needs to be elucidated by further studies.

High-risk human papillomavirus (hrHPV) is an etiologic agent in squamous cell carcinoma (SqCC) arising in the oropharynx and cervix, and a proven prognostic factor in oropharyngeal SqCC. Many studies have found HPV in non-small cell lung carcinoma (NSCLC). Recent studies advocate the detection of mRNA transcripts of E6/E7 as more reliable evidence of transcriptively active HPV in tumor cells. The clinical significance of finding HPV remains unclear in NSCLC. This study sought to determine the prevalence of biologically active HPV infection in NSCLC comparing different methodologies. Surgical pathology material from resected primary lung adenocarcinoma (ADC; n = 100) and SqCC (n = 96) were retrieved to construct tissue microarrays. In-situ hybridization (ISH) for hrHPV DNA (DNA-ISH), hrHPV E6/E7 RNA (RNA-ISH), and p16 immunohistochemistry (IHC) were performed. Cases of oropharyngeal SqCC with known HPV infection were used as positive controls. Expression of p16 was scored as positive if at least 70% of tumor cells showed diffuse and strong nuclear and cytoplasmic staining. Punctate nuclear hybridization signals by DNA-ISH in the malignant cells defined an HPV-positive carcinoma. Of the 196 patients (range 33-87 years; 108 men), p16 was positive in 19 ADC and 9 SqCC, but HPV DNA-ISH and RNA-ISH were negative in all cases. Our study did not detect HPV infection by DNA-ISH or RNA-ISH in any cases of primary NSCLC despite positive p16 expression in a portion of ADC and SqCC. p16 should therefore not be used as a surrogate marker for HPV infection in NSCLC.

Although a strong etiologic relationship between human papillomavirus (HPV) and a majority of oropharyngeal squamous cell carcinomas has been established, the role of HPV in non-oropharyngeal head and neck carcinomas is much less clear. Here, we investigated the prevalence and clinicopathologic significance of HPV and its reported biomarkers, CDKN2A(p16) and CDKN1A(p21), in laryngeal squamous cell carcinomas in patients treated either with primary surgery and postoperative radiation or with definitive radiation-based therapy. Nearly all of 76 tumors were keratinizing and none displayed the nonkeratinizing morphology that is typically associated with HPV infection in the oropharynx. However, CDKN2A(p16) immunohistochemistry was positive in 21 cases (28%) and CDKN1A(p21) in 34 (45%). CDKN2A(p16) and CDKN1A(p21) status strongly correlated with each other (P=0.0038). Yet, only four cases were HPV positive by DNA in situ hybridization or by reverse transcriptase PCR E6/E7 mRNA (all four were CDKN2A(p16) and CDKN1A(p21) positive). Unexpectedly, 9 additional tumors out of 20 CDKN2A(p16) positive cases harbored high-risk HPV DNA by PCR. For further investigation of this unexpected result, in situ hybridization for E6/E7 mRNA was performed on these nine cases and all were negative, confirming the absence of transcriptionally active virus. Patients with CDKN1A(p21)-positive tumors did have better overall survival (69% at 3 years) than those with CDKN1A(p21)-negative tumors (51% at 3 years) (P=0.045). There was also a strong trend towards better overall survival in the CDKN2A(p16)-positive group (P=0.058). Thus, it appears that the role of HPV is more complex in the larynx than in the oropharynx, and that CDKN2A(p16) and CDKN1A(p21) expression may not reflect HPV-driven tumors in most cases. Because of this, CDKN2A(p16) should not be used as a definitive surrogate marker of HPV-driven tumors in the larynx.

Abstract BACKGROUND: Folate receptor alpha (FOLR1/FRA) is expressed in a number of epithelial cancers and in particular epithelial ovarian cancer (EOC), especially of the serous histotype. Recent studies have shown that EOC originates from the fallopian tube fimbriae rather than from epithelial cells lining the ovary. We have previously shown by immunohistochemistry a strong correlation between FRA expression in EOC and normal and fallopian adenocarcinoma. Folate receptor beta (FOLR2/FRB) has been described to be expressed by macrophages both in inflammatory disorders and certain epithelial cancers. Given the high sequence identity of these two folate receptor family members we sought to investigate the architectural and cell-specific expression of these two receptors in gynecologic tissues. METHODS: RNA scope, a novel chromogenic in situ hybridization assay tool, was used to examine expression of the alpha (FOLR1) and beta (FOLR2) isoforms of folate receptor relative to each other as well as to the macrophage markers CD11b and CD68, in samples of normal fallopian tube and fallopian adenocarcinoma as well as normal ovary and EOC. RESULTS: We demonstrated expression of both FOLR1 and FOLR2 in EOC, normal fallopian tube and fallopian adenocarcinoma tissue while very little expression of either marker was observed in normal ovary. Furthermore, FOLR2 was shown to be expressed almost exclusively in macrophages, of both the M1 and M2 lineages, as determined by co-expression of CD11b and/or CD68, with little or no expression in epithelial cells. CONCLUSIONS: These findings further substantiate the hypothesis that the cell of origin of EOC is tubal epithelium and that the beta isoform of folate receptor is primarily restricted to macrophages. Further, macrophages expressing FOLR2 may represent tumor associated or infiltrating macrophages (TAMs) in epithelial cancers.

The tumor microenvironment evolves during malignant progression, with major changes in nonmalignant cells, cytokine networks, and the extracellular matrix (ECM). In this study, we aimed to understand how the ECM changes during neoplastic transformation of serous tubal intraepithelial carcinoma lesions (STIC) into high-grade serous ovarian cancers (HGSOC). Analysis of the mechanical properties of human fallopian tubes (FT) and ovaries revealed that normal FT and fimbria had a lower tissue modulus, a measure of stiffness, than normal or diseased ovaries. Proteomic analysis of the matrisome fraction between FT, fimbria, and ovaries showed significant differences in the ECM protein TGF beta induced (TGFBI, also known as βig-h3). STIC lesions in the fimbria expressed high levels of TGFBI, which was predominantly produced by CD163-positive macrophages proximal to STIC epithelial cells. In vitro stimulation of macrophages with TGFβ and IL4 induced secretion of TGFBI, whereas IFNγ/LPS downregulated macrophage TGFBI expression. Immortalized FT secretory epithelial cells carrying clinically relevant TP53 mutations stimulated macrophages to secrete TGFBI and upregulated integrin αvβ3, a putative TGFBI receptor. Transcriptomic HGSOC datasets showed a significant correlation between TGFBI expression and alternatively activated macrophage signatures. Fibroblasts in HGSOC metastases expressed TGFBI and stimulated macrophage TGFBI production in vitro. Treatment of orthotopic mouse HGSOC tumors with an anti-TGFBI antibody reduced peritoneal tumor size, increased tumor monocytes, and activated β3-expressing unconventional T cells. In conclusion, TGFBI may favor an immunosuppressive microenvironment in STICs that persists in advanced HGSOC. Furthermore, TGFBI may be an effector of the tumor-promoting actions of TGFβ and a potential therapeutic target. SIGNIFICANCE: Analysis of ECM changes during neoplastic transformation reveals a role for TGFBI secreted by macrophages in immunosuppression in early ovarian cancer.

Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation.UB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice.In vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy.These findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing.