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Davide Giordano,1 Maria Gabriella Raso,2 Carmine Pernice,1 Vanni Agnoletti,3 Verter Barbieri1 1Otorhinolaryngology Unit, Department of Surgery, Arcispedale Santa Maria Nuova – IRCCS, Reggio Emilia, 2Anesthesiology, Intensive Care, and Pain Medicine Unit, Department of Surgical Sciences, University Hospital of Parma, Parma, 3Anesthesiology and Intensive Care Unit, Department of Cardiology, Thoracic and Vascular Surgery, and Critical Care Medicine, Arcispedale Santa Maria Nuova – IRCCS, Reggio Emilia, Italy Abstract: In recent years, the popularity of aesthetic and cosmetic procedures, often performed in outpatient settings, has strongly renewed interest in topical anesthetics. A number of different options are widely used, alone or in combination, in order to minimize the pain related to surgery. Moreover, interest in local anesthetics in the treatment of some painful degenerative conditions such as myofascial trigger point pain, shoulder impingement syndrome, or patellar tendinopathy is increasing. Numerous clinical trials have shown that lidocaine–tetracaine combination, recently approved for adults aged 18 or older, is effective and safe in managing pain. The present paper gives an overview of the recent literature regarding the efficacy and safety of lidocaine–tetracaine combination use. Keywords: lidocaine, tetracaine, local anesthetics, efficacy, safety

Background/objectives: Cranioplasty (CP) is the main surgical procedure aiming to repair a morphological defect in the skull. It has been shown that early CP is useful for patients with traumatic brain injury (TBI) to achieve functional recovery, whereas few studies have investigated the clinical effects of ultra-late CP on TBI outcomes., Methods: Here, we describe the clinical course over 2 years of a TBI patient who underwent CP 19 months after fronto-parietal decompressive craniectomy (DC) of a limited size., Results: We found that after ultra-late CP, a meaningful functional recovery (cognitive and motor), with emergence from a minimally conscious state and recovery of functional communication, was revealed., Conclusions: Our preliminary findings contribute to the actual debate on the timing of CP for this neurosurgical procedure's therapeutic success, as early CP has already been shown.

Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3ζ creates a platform that recruits critical kinases, such as lymphocyte-specific protein tyrosine kinase (LCK) and zeta-chain-associated protein kinase 70 (ZAP70), initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy. Significance: We demonstrated that incorporation of the T-cell-centric costimulatory molecule CD28, which is normally absent in mature natural killer (NK) cells, into the chimeric antigen receptor (CAR) construct recruits key kinases including lymphocyte-specific protein tyrosine kinase and zeta-chain-associated protein kinase 70 and results in enhanced CAR-NK cell persistence and sustained antitumor cytotoxicity., (©2024 American Association for Cancer Research.)

Background: Adenoid cystic carcinoma is a rare subtype of triple-negative breast carcinoma. These low-grade tumours, which are treated by simple mastectomy and have an excellent prognosis compared to other triple-negative breast carcinomas. Solid-variant adenoid cystic carcinomas have basaloid features and are difficult to distinguish morphologically from other triple-negative breast cancers. Breast adenoid cystic carcinoma exhibits MYB protein overexpression, which can be detected by immunohistochemistry (IHC)., Aim: We compared the IHC expression of MYB in solid-variant adenoid cystic carcinoma with that in other triple-negative breast cancers., Methods: We conducted IHC staining of 210 samples of triple-negative breast cancers, including solid-variant adenoid cystic carcinoma (n = 17), metaplastic breast carcinoma (n = 44), basaloid triple-negative breast cancer (n = 21), and other triple-negative invasive ductal carcinoma (n = 128). We classified nuclear staining of MYB as diffuse/strong (3+), focal moderate (2+), focal weak (1+), or none (0)., Results: All 17 solid/basaloid adenoid cystic carcinoma cases exhibited 3+ MYB expression. Of the 21 solid/basaloid triple-negative breast cancers, one (5%) had 2+ expression, seven (33%) 1+ expression, and 13 (62%) 0 expression. Of the 44 metaplastic carcinoma cases, 39 cases (89%) had no (0) staining, and the other five cases had focal weak (1+) or moderate (2+) staining. Among the 128 triple-negative invasive ductal carcinoma cases, 92 cases (72%) had no (0) staining, 36 cases (28%) exhibited focal weak (1+) or moderate (2+) staining., Conclusions: Our study revealed diffuse/strong MYB staining (3+) only in solid/basaloid adenoid cystic carcinomas. Thus, we recommend routine MYB IHC staining in triple-negative breast carcinoma with solid/basaloid morphology to improve diagnostic accuracy., (© 2024 John Wiley & Sons Ltd.)

Purpose: Radiation therapy is an underinvestigated tool for priming the immune system in intact human breast cancers. We sought here to investigate if a preoperative radiation therapy boost delivered was associated with a significant change in tumor-infiltrating lymphocytes (TILs) in the tumor in estrogen receptor positive, HER2Neu nonamplified breast cancers., Methods and Materials: A total of 20 patients were enrolled in a phase 2 clinical trial and received either 7.5 Gy × 1 fraction or 2 Gy × 5 fractions, completed 6 to 8 days before surgery. Percent stromal TILs were evaluated on hematoxylin and eosin-stained samples. Short-term safety was assessed based on time to surgery, toxicities, and cosmesis up to 6 months after boost., Results: Stromal TIL increased 6 to 8 days after completion of boost radiation therapy (median 3.0 [IQR, 1.0-6.5]) before radiation therapy versus median 5.0 (IQR, 1.5-8.0) after radiation therapy, P = .0037. Zero grade ≥3 toxicities up to 6 months after boost were experienced. In all, 94% (16/17) patients with 6-month follow-up cosmetic assessment after breast conservation had good-excellent cosmesis by physician assessment., Conclusion: In this phase 2 trial, preoperative radiation therapy boost resulted in a short-term increase in stromal TIL with minimal toxicities. Preoperative breast radiation therapy appears to be safe and may be a feasible means for priming the tumor microenvironment., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Patient-derived xenografts (PDX) model human intra- and intertumoral heterogeneity in the context of the intact tissue of immunocompromised mice. Histologic imaging via hematoxylin and eosin (H&E) staining is routinely performed on PDX samples, which could be harnessed for computational analysis. Prior studies of large clinical H&E image repositories have shown that deep learning analysis can identify intercellular and morphologic signals correlated with disease phenotype and therapeutic response. In this study, we developed an extensive, pan-cancer repository of >1,000 PDX and paired parental tumor H&E images. These images, curated from the PDX Development and Trial Centers Research Network Consortium, had a range of associated genomic and transcriptomic data, clinical metadata, pathologic assessments of cell composition, and, in several cases, detailed pathologic annotations of neoplastic, stromal, and necrotic regions. The amenability of these images to deep learning was highlighted through three applications: (i) development of a classifier for neoplastic, stromal, and necrotic regions; (ii) development of a predictor of xenograft-transplant lymphoproliferative disorder; and (iii) application of a published predictor of microsatellite instability. Together, this PDX Development and Trial Centers Research Network image repository provides a valuable resource for controlled digital pathology analysis, both for the evaluation of technical issues and for the development of computational image-based methods that make clinical predictions based on PDX treatment studies. Significance: A pan-cancer repository of >1,000 patient-derived xenograft hematoxylin and eosin-stained images will facilitate cancer biology investigations through histopathologic analysis and contributes important model system data that expand existing human histology repositories., (©2024 The Authors; Published by the American Association for Cancer Research.)

Background: Risk of recurrence and progression of ductal carcinoma in situ (DCIS) to invasive cancer remains uncertain, emphasizing the need for developing predictive biomarkers of aggressive DCIS., Methods: Human cell lines and mouse models of disease progression were analyzed for candidate risk predictive biomarkers identified and validated in two independent DCIS cohorts., Results: RNA profiling of normal mammary and DCIS tissues (n = 48) revealed that elevated SOX11 expression correlates with MKI67, EZH2, and DCIS recurrence score. The 21T human cell line model of DCIS progression to invasive cancer and two mouse models developing mammary intraepithelial neoplasia confirmed the findings. AKT activation correlated with chromatin accessibility and EZH2 enrichment upregulating SOX11 expression. AKT and HER2 inhibitors decreased SOX11 expression along with diminished mammosphere formation. SOX11 was upregulated in HER2+ and basal-like subtypes (P < 0.001). Longitudinal DCIS cohort (n = 194) revealed shorter recurrence-free survival in SOX11+ than SOX11- patients (P = 0.0056 in all DCIS; P < 0.0001 in HER2+ subtype) associated with increased risk of ipsilateral breast event/IBE (HR = 1.9, 95%CI = 1.2-2.9; P = 0.003)., Discussion: Epigenetic activation of SOX11 drives recurrence of DCIS and progression to invasive cancer, suggesting SOX11 as a predictive biomarker of IBE., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The Wnt receptor ROR1 has generated increased interest as a cancer therapeutic target. Research on several therapeutic approaches involving this receptor is ongoing; however, ROR1 tissue expression remains understudied. We performed an immunohistochemistry analysis of ROR1 protein expression in a large cohort of multiple tumor and histologic types. We analyzed 12 anonymized multi-tumor tissue microarrays (TMAs), including mesothelioma, esophageal and upper gastrointestinal carcinomas, and uterine endometrioid carcinoma, among other tumor types. Additionally, we studied 5 different sarcoma types of TMAs and 6 patient-derived xenografts (PDX) TMAs developed from 19 different anatomic sites and tumor histologic types. A total of 1142 patient cases from different histologic types and 140 PDXs placed in TMAs were evaluated. Pathologists assessed the percentage of tumor cells in each case that were positive for ROR1 and the intensity of staining. For determining the prevalence of staining for each tumor type, a case was considered positive if >1% of its tumor cells showed ROR1 staining. Our immunohistochemistry assays revealed a heterogeneous ROR1 expression profile. A high prevalence of ROR1 expression was found in mesothelioma (84.6%), liposarcoma (36.1%), gastrointestinal stromal tumors (33.3%), and uterine endometrioid carcinoma (28.9%). Other histologic types such as breast, lung, renal cell, hepatocellular, urothelial carcinoma, and colon carcinomas; glioblastoma; cholangiocarcinoma; and leiomyosarcoma showed less ROR1 overall expression, ranging between 0.9 and 13%. No ROR1 expression was seen in mesenchymal chondrosarcoma, rhabdomyosarcoma, or gastric adenocarcinoma cases. Overall, ROR1 expression was relatively infrequent and low in most tumor types investigated; however, ROR1 expression was infrequent but high in selected tumor types, such as gastroesophageal GIST, suggesting that ROR1 prescreening may be preferable for those indications. Further, mesothelioma exhibited frequent and high levels of ROR1 expression, which represents a previously unrecognized therapeutic opportunity. These findings can contribute to the development of ROR1-targeted therapies.

Zanidatamab is a bispecific human epidermal growth factor receptor 2 (HER2)-targeted antibody that has demonstrated antitumor activity in a broad range of HER2-amplified/expressing solid tumors. We determined the antitumor activity of zanidatamab in patient-derived xenograft (PDX) models developed from pretreatment or postprogression biopsies on the first-in-human zanidatamab phase I study (NCT02892123). Of 36 tumors implanted, 19 PDX models were established (52.7% take rate) from 17 patients. Established PDXs represented a broad range of HER2-expressing cancers, and in vivo testing demonstrated an association between antitumor activity in PDXs and matched patients in 7 of 8 co-clinical models tested. We also identified amplification of MET as a potential mechanism of acquired resistance to zanidatamab and demonstrated that MET inhibitors have single-agent activity and can enhance zanidatamab activity in vitro and in vivo. These findings provide evidence that PDXs can be developed from pretreatment biopsies in clinical trials and may provide insight into mechanisms of resistance., Significance: We demonstrate that PDXs can be developed from pretreatment and postprogression biopsies in clinical trials and may represent a powerful preclinical tool. We identified amplification of MET as a potential mechanism of acquired resistance to the HER2 inhibitor zanidatamab and MET inhibitors alone and in combination as a therapeutic strategy. This article is featured in Selected Articles from This Issue, p. 695., (©2024 The Authors; Published by the American Association for Cancer Research.)

Hematopoietic stem and progenitor cells (HSPCs) maintain blood-forming and immune activity, yet intrinsic regulators of HSPCs remain elusive. STAT3 function in HSPCs has been difficult to dissect as Stat3-deficiency in the hematopoietic compartment induces systemic inflammation, which can impact HSPC activity. Here, we developed mixed bone marrow (BM) chimeric mice with inducible Stat3 deletion in 20% of the hematopoietic compartment to avoid systemic inflammation. Stat3-deficient HSPCs were significantly impaired in reconstitution ability following primary or secondary bone marrow transplantation, indicating hematopoietic stem cell (HSC) defects. Single-cell RNA sequencing of Lin - ckit + Sca1 + BM cells (LSKs) revealed aberrant activation of cell cycle, p53, and interferon (IFN) pathways in Stat3-deficient HSPCs. Stat3-deficient LSKs accumulated γH2AX and showed increased expression of DNA sensors and type-I IFN (IFN-I), while treatment with A151-ODN inhibited expression of IFN-I and IFN-responsive genes. Further, the blockade of IFN-I receptor signaling suppressed aberrant cell cycling, STAT1 activation, and nuclear p53 accumulation. Collectively, our results show that STAT3 inhibits a deleterious autocrine IFN response in HSCs to maintain long-term HSC function. These data signify the importance of ensuring therapeutic STAT3 inhibitors are targeted specifically to diseased cells to avoid off-target loss of healthy HSPCs., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Breast cancer is the most common cancer among women. Metaplastic breast carcinoma (MpBC) is a rare, heterogeneous group of invasive breast carcinomas, which are classified as predominantly triple-negative breast carcinomas (TNBCs; HR-negative/HER2-negative). Histologically, MpBC is classified into six subtypes. Two of these are considered low-grade and the others are high-grade. MpBCs seem to be more aggressive, less responsive to neoadjuvant chemotherapy, and have higher rates of chemoresistance than other TNBCs. MpBCs have a lower survival rate than expected for TNBCs. MpBC treatment represents a challenge, leading to a thorough exploration of the tumor immune microenvironment, which has recently opened the possibility of new therapeutic strategies. The epithelial-mesenchymal transition in MpBC is characterized by the loss of intercellular adhesion, downregulation of epithelial markers, underexpression of genes with biological epithelial functions, upregulation of mesenchymal markers, overexpression of genes with biological mesenchymal functions, acquisition of fibroblast-like (spindle) morphology, cytoskeleton reorganization, increased motility, invasiveness, and metastatic capabilities. This article reviews and summarizes the current knowledge and translational aspects of MpBC.

Despite the successes of immunotherapy in cancer treatment over recent decades, less than <10%-20% cancer cases have demonstrated durable responses from immune checkpoint blockade. To enhance the efficacy of immunotherapies, combination therapies suppressing multiple immune evasion mechanisms are increasingly contemplated. To better understand immune cell surveillance and diverse immune evasion responses in tumor tissues, we comprehensively characterized the immune landscape of more than 1,000 tumors across ten different cancers using CPTAC pan-cancer proteogenomic data. We identified seven distinct immune subtypes based on integrative learning of cell type compositions and pathway activities. We then thoroughly categorized unique genomic, epigenetic, transcriptomic, and proteomic changes associated with each subtype. Further leveraging the deep phosphoproteomic data, we studied kinase activities in different immune subtypes, which revealed potential subtype-specific therapeutic targets. Insights from this work will facilitate the development of future immunotherapy strategies and enhance precision targeting with existing agents., Competing Interests: Declaration of interests R. Sebra is currently a paid consultant and equity holder at GeneDx. L.C.C. is a founder and member of the board of directors of Agios Pharmaceuticals; is a founder and receives research support from Petra Pharmaceuticals; has equity in and consults for Cell Signaling Technologies, Volastra, Larkspur, and 1 Base Pharmaceuticals; and consults for Loxo-Lilly. J.L.J. has received consulting fees from Scorpion Therapeutics and Volastra Therapeutics. T.M.Y. is a co-founder and stockholder of DeStroke., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Small cell lung cancer (SCLC) is an aggressive malignancy composed of distinct transcriptional subtypes, but implementing subtyping in the clinic has remained challenging, particularly due to limited tissue availability. Given the known epigenetic regulation of critical SCLC transcriptional programs, we hypothesized that subtype-specific patterns of DNA methylation could be detected in tumor or blood from SCLC patients. Using genomic-wide reduced-representation bisulfite sequencing (RRBS) in two cohorts totaling 179 SCLC patients and using machine learning approaches, we report a highly accurate DNA methylation-based classifier (SCLC-DMC) that can distinguish SCLC subtypes. We further adjust the classifier for circulating-free DNA (cfDNA) to subtype SCLC from plasma. Using the cfDNA classifier (cfDMC), we demonstrate that SCLC phenotypes can evolve during disease progression, highlighting the need for longitudinal tracking of SCLC during clinical treatment. These data establish that tumor and cfDNA methylation can be used to identify SCLC subtypes and might guide precision SCLC therapy., Competing Interests: Declaration of interests S.H., C.M.G., L.A.B., and J.V.H. own intellectual property on the classification of SCLC from DNA methylation and gene expression. D.F., A.W., A.S., and C.A.S. are full time employees of Nucleix and own stocks and stock options of Nucleix. Furthermore, S.H. reports consulting fees from Guardant Health, AstraZeneca, Boehringer Ingelheim, and Qiagen. C.M.G. is a member of the advisory board at Jazz Pharmaceuticals, AstraZeneca, and Bristol Myers Squibb and served as speaker for AstraZeneca and BeiGene. P.R. received travel support from AstraZeneca, BMS, and MSD. E.A. reports consulting fees from Eli Lilly, AstraZeneca, BMS, Boehringer Ingelheim, Takeda, Roche, and MSD, speaker’s fees from AstraZeneca, BMS, Boehringer Ingelheim, Roche, and MSD, research funding from Roche and AstraZeneca and travel support from AstraZeneca and Takeda. P.H. reports research grants from Thermo Fisher Scientific and Biocartis, and speakers’ fees from AstraZeneca, Roche, Novartis, Bristol-Myers Squibb, Pfizer, Bayer, Illumina, Biocartis, Thermo Fisher Scientific, AbbVie, Amgen, Janssen, Eli Lilly, Daiichi Sankyo, Pierre Fabre, and Guardant. V.H. reports speakers’ fees from BMS. C.M.L. reports personal fees from Amgen, Arrivent, AstraZeneca, Blueprints Medicine, Cepheid, D2G Oncology, Daiichi Sankyo, Eli Lilly, EMD Serono, Foundation Medicine, Genentech, Janssen, Medscape, Novartis, Pfizer, Puma, Syros, and Takeda. N.V. receives consulting fees from Sanofi, Regeneron, Oncocyte, and Eli Lilly, and research funding from Mirati. M.B.N. receives royalties and licensing fees from Spectrum Pharmaceuticals. I.H. received personal as well as institutional funding from Nucleix. J.Z. served on advisory board for AstraZeneca and Geneplus and received speaker’s fees from BMS, Geneplus, OrigMed, Innovent and grants from Merck, Johnson and Johnson. L.A.B received consulting fees and research funding from AstraZeneca, GenMab, Sierra Oncology, research funding from ToleroPharmaceuticals and served as advisor or consultant for PharmaMar, AbbVie, Bristol-Myers Squibb, Alethia, Merck, Pfizer, Jazz Pharmaceuticals, Genentech, and Debiopharm Group. J.V.H. served as advisor for AstraZeneca, EMD Serono, Boehringer-Ingelheim, Catalyst, Genentech, GlaxoSmithKline, Guardant Health, Foundation medicine, Hengrui Therapeutics, Eli Lilly, Novartis, Spectrum, Sanofi, Takeda, Mirati Therapeutics, BMS, BrightPath Biotherapeutics, Janssen Global Services, Nexus Health Systems, Pneuma Respiratory, Kairos Venture Investments, Roche, Leads Biolabs, RefleXion, Chugai Pharmaceuticals, received research support from AstraZeneca, GlaxoSmithKline, Spectrum as well as royalties and licensing fees from Spectrum., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Inactivating TP53 mutations leads to a loss of function of p53, but can also often result in oncogenic gain-of-function (GOF) of mutant p53 (mutp53) proteins which promotes tumor development and progression. The GOF activities of TP53 mutations are well documented, but the mechanisms involved remain poorly understood. Here, we study the mutp53 interactome and find that by targeting minichromosome maintenance complex components (MCMs), GOF mutp53 predisposes cells to replication stress and chromosomal instability (CIN), leading to a tumor cell-autonomous and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent cytosolic DNA response that activates downstream non-canonical nuclear factor kappa light chain enhancer of activated B cell (NC-NF-κB) signaling. Consequently, GOF mutp53-MCMs-CIN-cytosolic DNA-cGAS-STING-NC-NF-κB signaling promotes tumor cell metastasis and an immunosuppressive tumor microenvironment through antagonizing interferon signaling and regulating genes associated with pro-tumorigenic inflammation. Our findings have important implications for understanding not only the GOF activities of TP53 mutations but also the genome-guardian role of p53 and its inactivation during tumor development and progression., (© 2024. The Author(s).)

Introduction: The Wessex Head Injury Matrix (WHIM) was developed to assess patients with disorders of consciousness (DOC) and was tested in terms of inter-rater reliability (IRR) and test-retest reliability (TRR) in the year 2000. The American Congress of Rehabilitation and Medicine reported that IRR and TRR were unproven. We aim to assess the reliability of the WHIM in prolonged DOC patients (PDOC). Methods: A total of 51 PDOC patients (32 unresponsive wakefulness syndrome (UWS/VS) and 19 minimally conscious state (MCS)) who were hosted in a dedicated unit for long-term brain injury care were enrolled. The time from injury ranged from 182 to 3325 days. Two raters administered the Coma Recovery Scale-Revised (CRS-R) and the WHIM to test the IRR and TRR. The TRR was administered two weeks after the first assessment. Results: For the CRS-R, the agreement in IRR and TRR was perfect between the two raters. The agreement for the WHIM ranged from substantial to almost perfect for IRR and from fair to substantial for the TRR. Conclusions: The WHIM showed a strong IRR when administered by expert raters and strongly correlated with the CRS-R. This study provides further evidence of the psychometric qualities of the WHIM and the importance of its use in PDOC patients., Competing Interests: The authors declare no conflicts of interest.

Epithelial-mesenchymal transition (EMT) empowers epithelial cells with mesenchymal and stem-like attributes, facilitating metastasis, a leading cause of cancer-related mortality. Hybrid epithelial-mesenchymal (E/M) cells, retaining both epithelial and mesenchymal traits, exhibit heightened metastatic potential and stemness. The mesenchymal intermediate filament, vimentin, is upregulated during EMT, enhancing the resilience and invasiveness of carcinoma cells. The phosphorylation of vimentin is critical to its structure and function. Here, we identify that stabilizing vimentin phosphorylation at serine 56 induces multinucleation, specifically in hybrid E/M cells with stemness properties but not epithelial or mesenchymal cells. Cancer stem-like cells are especially susceptible to vimentin-induced multinucleation relative to differentiated cells, leading to a reduction in self-renewal and stemness. As a result, vimentin-induced multinucleation leads to sustained inhibition of stemness properties, tumor initiation, and metastasis. These observations indicate that a single, targetable phosphorylation event in vimentin is critical for stemness and metastasis in carcinomas with hybrid E/M properties., Competing Interests: Declaration of interests The authors declare no potential conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

As the second most common subtype of breast carcinoma, Invasive Lobular Carcinoma (ILC) microenvironment features have not been thoroughly explored. ILC has different histological subtypes and elucidating differences in their microenvironments could lead to a comprehensive development of cancer therapies. We designed a custom-made cancer associated fibroblast (CAFs) panel and used multiplex immunofluorescence to identify the differences in tumor microenvironment between Classic ILC and Pleomorphic ILC., Materials and Methods: Multiplex immunofluorescence were performed on formalin fixed paraffin embedded tissues using Opal-7 color kit. The antibodies used for phenotyping CAFs were Pan CK (AE1/AE3), CD45, A-SMA, FAP, S100, Thy-1 with optimized dilutions. The images were acquired and analyzed using Vectra 3.0 imaging system and InForm software respectively., Results: We studied 19 different CAFs colocalized phenotypes in the tumor, stroma and overall tissue compartments between classic and pleomorphic ILC. Total A-SMA+, A-SMA+FAP+S100+ and A-SMA+S100+ CAFs demonstrated higher densities in classic ILC cases while FAP+S100+ and S-100+ CAFs were increased in the pleomorphic subtype samples., Conclusion: Our study explores multiple CAFs phenotypes between classical and pleomorphic ILC. We showed that CAFs subset differ between Classic ILC and Pleomorphic ILC. A-SMA CAFs are more prevalent in the TME of classic ILCs whereas Pleomorphic ILCs are dominated by CAFs without A-SMA expression. This also iterates the importance of exploring this particular type of breast carcinoma in more detail, paving the way for meaningful translational research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Batra, Ding, Pandurengan, Ibarguen, Rabassedas, Sahin, Wistuba, Parra and Raso.)

The second most common breast carcinoma, invasive lobular carcinoma, accounts for approximately 15% of tumors of breast origin. Its incidence has increased in recent times due in part to hormone replacement therapy and improvement in diagnostic modalities. Although believed to arise from the same cell type as their ductal counterpart, invasive lobular carcinomas (ILCs) are a distinct entity with different regulating genetic pathways, characteristic histologies, and different biology. The features most unique to lobular carcinomas include loss of E-Cadherin leading to discohesion and formation of a characteristic single file pattern on histology. Because most of these tumors exhibit estrogen receptor positivity and Her2 neu negativity, endocrine therapy has predominated to treat these tumors. However novel treatments like CDK4/6 inhibitors have shown importance and antibody drug conjugates may be instrumental considering newer categories of Her 2 Low breast tumors. In this narrative review, we explore multiple pathological aspects and translational features of this unique entity. In addition, due to advancement in technologies like spatial transcriptomics and other hi-plex technologies, we have tried to enlist upon the characteristics of the tumor microenvironment and the latest associated findings to better understand the new prospective therapeutic options in the current era of personalized treatment.

Purpose: Cyclin E (CCNE1) has been proposed as a biomarker of sensitivity to adavosertib, a Wee1 kinase inhibitor, and a mechanism of resistance to HER2-targeted therapy., Experimental Design: Copy number and genomic sequencing data from The Cancer Genome Atlas and MD Anderson Cancer Center databases were analyzed to assess ERBB2 and CCNE1 expression. Molecular characteristics of tumors and patient-derived xenografts (PDX) were assessed by next-generation sequencing, whole-exome sequencing, fluorescent in situ hybridization, and IHC. In vitro, CCNE1 was overexpressed or knocked down in HER2+ cell lines to evaluate drug combination efficacy. In vivo, NSG mice bearing PDXs were subjected to combinatorial therapy with various treatment regimens, followed by tumor growth assessment. Pharmacodynamic markers in PDXs were characterized by IHC and reverse-phase protein array., Results: Among several ERBB2-amplified cancers, CCNE1 co-amplification was identified (gastric 37%, endometroid 43%, and ovarian serous adenocarcinoma 41%). We hypothesized that adavosertib may enhance activity of HER2 antibody-drug conjugate trastuzumab deruxtecan (T-DXd). In vitro, sensitivity to T-DXd was decreased by cyclin E overexpression and increased by knockdown, and adavosertib was synergistic with topoisomerase I inhibitor DXd. In vivo, the T-DXd + adavosertib combination significantly increased γH2AX and antitumor activity in HER2 low, cyclin E amplified gastroesophageal cancer PDX models and prolonged event-free survival (EFS) in a HER2-overexpressing gastroesophageal cancer model. T-DXd + adavosertib treatment also increased EFS in other HER2-expressing tumor types, including a T-DXd-treated colon cancer model., Conclusions: We provide rationale for combining T-DXd with adavosertib in HER2-expressing cancers, especially with co-occuring CCNE1 amplifications. See related commentary by Rolfo et al., p. 4317., (©2023 The Authors; Published by the American Association for Cancer Research.)

Background: Lupus nephritis (LN) constitutes the most severe organ manifestations of systemic lupus erythematosus (SLE), where pathogenic T cells have been identified to play an essential role in 'helping' B cells to make autoantibodies and produce inflammatory cytokines that drive kidney injury in SLE. Regulatory T cells (Tregs), responsible for decreasing inflammation, are defective and decreased in SLE and have been associated with disease progression. We hypothesize that treatment with allogeneic, healthy Tregs derived from umbilical cord blood (UCB) may arrest such an inflammatory process and protect against kidney damage., Methods: UCB-Tregs function was examined by their ability to suppress CellTrace Violet-labeled SLE peripheral blood mononuclear cells (PBMCs) or healthy donor (HD) conventional T cells (Tcons); and by inhibiting secretion of inflammatory cytokines by SLE PBMCs. Humanized SLE model was established where female Rag2 -/- γc -/- mice were transplanted with 3 × 10 6 human SLE-PBMCs by intravenous injection on day 0, followed by single or multiple injection of UCB-Tregs to understand their impact on disease development. Mice PB was assessed weekly by flow cytometry. Phenotypic analysis of isolated cells from mouse PB, lung, spleen, liver and kidney was performed by flow cytometry. Kidney damage was assessed by quantifying urinary albumin and creatinine secretion. Systemic disease was evaluated by anti-dsDNA IgG Ab analysis as well as immunohistochemistry analysis of organs. Systemic inflammation was determined by measuring cytokine levels., Results: In vitro , UCB-Tregs are able to suppress HD Tcons and pathogenic SLE-PBMCs to a similar extent. UCB-Tregs decrease secretion of several inflammatory cytokines including IFN-γ, IP-10, TNF-α, IL-6, IL-17A, and sCD40L by SLE PBMCs in a time-dependent manner, with a corresponding increase in secretion of suppressor cytokine, IL-10. In vivo , single or multiple doses of UCB-Tregs led to a decrease in CD8 + T effector cells in different organs and a decrease in circulating inflammatory cytokines. Improvement in skin inflammation and loss of hair; and resolution of CD3 + , CD8 + , CD20 + and Ki67 + SLE-PBMC infiltration was observed in UCB-Treg recipients with a corresponding decrease in plasma anti-double stranded DNA IgG antibody levels and improved albuminuria., Conclusions: UCB-Tregs can decrease inflammatory burden in SLE, reduce auto-antibody production and resolve end organ damage especially, improve kidney function. Adoptive therapy with UCB-Tregs should be explored for treatment of lupus nephritis in the clinical setting., Competing Interests: JDK received research funding from Stemline Therapeutics, Angle, and Kiromic BioPharm. SP has an equity interest in, holds patents for, receives royalties and research funding from, and is a member of the board of directors/advisory committee for Cellenkos Inc. CRF received research funding from 4D, AbbVie, Acerta, Adaptimmune, Allogene, Amgen, Bayer, Celgene, Cellectis, EMD, Gilead, Genentech/Roche, Guardant, Iovance, Janssen Pharmaceutical, Kite, Morphosys, Nektar, Novartis, Pfizer, Pharmacyclics, Sanofi, Takeda, TG Therapeutics, Xencor, Ziopharm, Burroughs Wellcome Fund, and Eastern Cooperative Oncology Group, National Cancer Institute, and Cancer Prevention and Research Institute of Texas: CPRIT Scholar in Cancer Research and and consulting fees from AbbVie, Bayer, BeiGene, Celgene, Denovo, Biopharma, Epizyme, Genentech/Roche, Genmab, Gilead, Karyopharm, Pharmacyclics/Janssen, SeaGen, and Spectrum. TS was employed by the company Cellenkos Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lyu, Tang, Khoury, Raso, Huang, Zeng, Nishimoto, Ma, Sadeghi, Flowers and Parmar.)

Background: The principal conditions differentiating disorders of consciousness (DOC) patients are the unresponsive wakefulness syndrome/vegetative state (UWS/VS) and the minimally conscious state (MCS). Many individuals who suffer from sudden-onset severe brain injury move through stages of UWS/VS and MCS before regaining full awareness. In some patients, the DOC condition is protracted for years (PDOC). In this study, we observed PDOC patients for 6 months to assess possible changes in their level of consciousness., Methods: We enrolled 40 PDOC patients, 23 UWS/VS and 17 MCS hosted in a dedicated unit for long-term brain injury care. The time from injury was 472 ± 533 days for UWS/VS and 1090 ± 1079 days for MCS. The Wessex Head Injury Matrix (WHIM), Coma Recovery Scale-R (CRS-R), and Nociception Coma Scale were administered monthly for 6 months., Results: During the period of assessment, the percentage of UWS/VS shifted from 58 to 45%, while for the MCS, from 42 to 55%. A positive correlation was found for the UWS/VS patients between the months of observation with the CRS-R total score and WHIM total numbers of behaviors (TNB). In the UWS/VS group, the CRS-R auditive and visual subscales correlated positively with the observation time. During the whole period of observation, 8 patients had constant CRS-R total scores while the WHIM TNB changed in 7 of them., Conclusion: Our findings demonstrated that the monthly assessment of PDOC by means of the CRS-R and WHIM was able to detect also subtle changes in consciousness level., (© 2023. The Author(s).)

TROP2 antibody drug conjugates (ADCs) are under active development. We seek to determine whether we can enhance activity of TROP2 ADCs by increasing TROP2 expression. In metaplastic breast cancers (MpBC), there is limited expression of TROP2, and downregulating transcription factor ZEB1 upregulates E-cad and TROP2, thus sensitizing cancers to TROP2 ADC sacituzumab govitecan (SG). Demethylating agent decitabine decreases DNA methyltransferase expression and TROP2 promoter methylation and subsequently increases TROP2 expression. Decitabine treatment as well as overexpression of TROP2 significantly enhance SG antitumor activity. Decitabine also increases SLFN11, a biomarker of topoisomerase 1 inhibitor (TOP1) sensitivity and is synergistic with SG which has a TOP1 payload, in TROP2-expressing SLFN11-low BC cells. In conclusion, TROP2 and SLFN11 expression can be epigenetically modulated and the combination of demethylating agent decitabine with TROP2 ADCs may represent a novel therapeutic approach for tumors with low TROP2 or SLFN11 expression., (© 2023. Springer Nature Limited.)

Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)-mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4-mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs., (© 2022 Zhou et al.)

Triple negative breast cancer (TNBC) is a disease of poor prognosis, with the majority classified as the basal-like subtype associated with epithelial-mesenchymal transition and metastasis. Because basal breast cancers originate from proliferative luminal progenitor-like cells upon dysregulation of proper luminal differentiation, genes regulating luminal-basal transition are critical to elucidate novel therapeutic targets to improve TNBC outcomes. Herein we demonstrate that the tumor suppressor DEAR1/TRIM62 is a critical regulator of luminal cell fate. DEAR1 loss in human mammary epithelial cells results in significantly enhanced mammosphere formation that is accelerated in the presence of TGF-β/SMAD3 signaling. Mammospheres formed following DEAR1 loss are enriched for ALDH1A1 and CK5 expression, EpCAM - /CD49f + and CD44 high /24 low basal-like epithelial cells, indicating that DEAR1 regulates stem/progenitor cell properties and luminal-basal progenitor transition. We show that DEAR1 maintains luminal differentiation as a novel ubiquitin ligase for SNAI2/SLUG, a master regulator driving stemness and generation of basal-like progenitor populations. We also identify a significant inverse correlation between DEAR1 and SNAI2 expression in a 103 TNBC case cohort and show that low DEAR1 expression significantly correlates with young age of onset and shorter time to metastasis, suggesting DEAR1 could serve as a biomarker to stratify early onset TNBCs for targeted stem cell therapies., (© 2022. The Author(s).)

Lung cancer is a highly aggressive and metastatic disease responsible for approximately 25% of all cancer-related deaths in the United States. Using high-throughput in vitro and in vivo screens, we have previously established Impad1 as a driver of lung cancer invasion and metastasis. Here we elucidate that Impad1 is a direct target of the epithelial microRNAs (miRNAs) miR-200 and miR∼96 and is de-repressed during epithelial-to-mesenchymal transition (EMT); thus, we establish a mode of regulation of the protein. Impad1 modulates Golgi apparatus morphology and vesicular trafficking through its interaction with a trafficking protein, Syt11. These changes in Golgi apparatus dynamics alter the extracellular matrix and the tumor microenvironment (TME) to promote invasion and metastasis. Inhibiting Impad1 or Syt11 disrupts the cancer cell secretome, regulates the TME, and reverses the invasive or metastatic phenotype. This work identifies Impad1 as a regulator of EMT and secretome-mediated changes during lung cancer progression., Competing Interests: Declaration of interests D.L.G. declares advisory board work for AstraZeneca, Eli Lilly, Menarini Richerche, 4D Pharma, and Sanofi. D.L.G. has received research grant funding from AstraZeneca, Janssen, Astellas, Ribon Therapeutics, NGM Biotherapeutics, and Takeda., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutant KRAS (KM), the most common oncogene in lung cancer (LC), regulates fatty acid (FA) metabolism. However, the role of FA in LC tumorigenesis is still not sufficiently characterized. Here, we show that KMLC has a specific lipid profile, with high triacylglycerides and phosphatidylcholines (PC). We demonstrate that FASN, the rate-limiting enzyme in FA synthesis, while being dispensable in EGFR-mutant or wild-type KRAS LC, is required for the viability of KMLC cells. Integrating lipidomic, transcriptomic and functional analyses, we demonstrate that FASN provides saturated and monounsaturated FA to the Lands cycle, the process remodeling oxidized phospholipids, such as PC. Accordingly, blocking either FASN or the Lands cycle in KMLC, promotes ferroptosis, a reactive oxygen species (ROS)- and iron-dependent cell death, characterized by the intracellular accumulation of oxidation-prone PC. Our work indicates that KM dictates a dependency on newly synthesized FA to escape ferroptosis, establishing a targetable vulnerability in KMLC., (© 2022. The Author(s).)

Effects of waterpipe smoking on lung pathobiology and carcinogenesis remain sparse despite the worldwide emergence of this tobacco vector. To address this gap, we investigated the effects of chronic waterpipe smoke (WPS) exposure on lung pathobiology, host immunity, and tumorigenesis using an experimental animal model that is prone to tobacco carcinogens and an exploratory observational analysis of human waterpipe smokers and nonsmokers. Mice exhibited elevated incidence of lung tumors following heavy WPS exposure (5 days/week for 20 weeks) compared to littermates with light WPS (once/week for 20 weeks) or control air. Lungs of mice exposed to heavy WPS showed augmented CD8+ and CD4+ T cell counts along with elevated protumor immune phenotypes including increased IL17A in T/B cells, PD-L1 on tumor and immune cells, and the proinflammatory cytokine IL1β in myeloid cells. RNA-sequencing (RNA-seq) analysis showed reduced antitumor immune gene signatures in animals exposed to heavy WPS relative to control air. We also performed RNA-seq analysis of airway epithelia from bronchial brushings of cancer-free waterpipe smokers and nonsmokers undergoing diagnostic bronchoscopy. Transcriptomes of normal airway cells in waterpipe smokers, relative to waterpipe nonsmokers, harbored gene programs that were associated with poor clinical outcomes in patients with lung adenocarcinoma, alluding to a WPS-associated molecular injury, like that established in response to cigarette smoking. Our findings support the notion that WPS exhibits carcinogenic effects and constitutes a possible risk factor for lung cancer as well as warrant future studies that can guide evidence-based policies for mitigating waterpipe smoking., Prevention Relevance: Potential carcinogenic effects of waterpipe smoking are very poorly understood despite its emergence as a socially acceptable form of smoking. Our work highlights carcinogenic effects of waterpipe smoking in the lung and, thus, accentuate the need for inclusion of individuals with exclusive waterpipe smoking in prevention and smoking cessation studies., (©2022 American Association for Cancer Research.)

Significant anti-spike protein receptor-binding domain (S-RBD) antibody responses have been demonstrated in patients with chronic disorder of consciousness (DOC) completing a COVID-19 vaccine regime with BNT162b2 (Pfizer-BioNTech). We now provide further prospective data on the immunogenicity of these patients followed by heterologous booster injection with mRNA-1273 (Moderna). These patients were compared with two different demographically comparable healthcare workers (HCW) groups who underwent homologous booster injection with BNT162b2 vaccine or heterologous booster injection with mRNA-1273. Antibody responses were evaluated at 21 days after the administration of the booster dose of vaccination. Results: No severe adverse reactions were reported after each type of vaccination. Heterologous boosting with mRNA-1273 elicited a higher increase of S-RBD IgG levels than homologous boosting with BNT162b2 both in DOC patients and HCW who had previously received two doses of BNT162b2. No significant difference was detected between DOC and HCW patients who received heterologous boosting. Conclusions: Despite the small sample size, our preliminary results suggest that heterologous boosting with mRNA-1273, following initial vaccination with BNT162b2, is safe and tends to be more immunogenic than homologous boosting, either in fragile people or in healthy controls.

Triple-negative breast cancer (TNBC) with high tumour-infiltrating lymphocytes (TILs) has been associated with a promising prognosis. To better understand the prognostic value of immune cell subtypes in TNBC, we characterised TILs and the interaction between tumour cells and immune cell subtypes. A total of 145 breast cancer tissues were stained by multiplex immunofluorescence (mIF), including panel 1 (PD-L1, PD-1, CD3, CD8, CD68 and CK) and panel 2 (Foxp3, Granzyme B, CD45RO, CD3, CD8 and CK). Phenotypes were analysed and quantified by pathologists using InForm software. We found that in the ER-negative (ER <1% and HER2-negative) group and the ER/PR-low positive (ER 1-9% and HER2-negative) group, 11.2% and 7.1% of patients were PD-L1 + by the tumour cell score, 29.0% and 28.6% were PD-L1 + by the modified immune cell score and 30.8% and 32.1% were PD-L1 + by the combined positive score. We combined ER-negative and ER/PR-low positive cases for the survival analysis since a 10% cut-off is often used in clinical practice for therapeutic purposes. The densities of PD-L1 + tumour cells (HR: 0.366, 95% CI: 0.138-0.970; p = 0.043) within the tumour compartment and CD3 + immune cells in the total area (tumour and stromal compartments combined) (HR: 0.213, 95% CI: 0.070-0.642; p = 0.006) were favourable prognostic biomarkers for overall survival (OS) in TNBC. The density of effector/memory cytotoxic T cells (CD3 + CD8 + CD45RO + ) in the tumour compartment was an independent prognostic biomarker for OS (HR: 0.232, 95% CI: 0.086-0.628; p = 0.004) and DFS (HR: 0.183, 95% CI: 0.1301-0.744; p = 0.009) in TNBC. Interestingly, spatial data suggested that patients with a higher density of PD-L1 + tumour cells had shorter cell-cell distances from tumour cells to cytotoxic T cells (p < 0.01). In conclusion, we found that phenotyping tumour immune cells by mIF is highly informative in understanding the immune microenvironment in TNBC. PD-L1 + tumour cells, total T cells and effector/memory cytotoxic T cells are promising prognostic biomarkers in TNBC., (© 2021. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Objective: In the last year, a large amount of research has investigated the anti-spike protein receptor-binding domain (S-RBD) antibody responses in patients at high risk of developing severe acute respiratory syndrome because of COVID-19 infection. However, no data are available on the chronic disorder of consciousness (DOC)., Methods: Here, we evaluated anti-S-RBD IgG levels after vaccination in chronic DOC patients compared with demographically matched healthy controls (HC) by indirect chemiluminescence immunoassay. All individuals completed a two-dose-cycle vaccination with Pfizer mRNA vaccine (BNT162b2), and antibody responses were evaluated at 30 and 180 days after the administration of the second dose of vaccination., Results: We compared 32 DOC patients with 34 demographically matched healthy controls. Both DOC and HC groups showed a similar antibody response at 30 days, whereas at follow-up (180 days) DOC patients were characterized by lower S-RBD IgG levels with respect to controls. Additional multiple regression analyses including demographical and clinical comorbidities as predictors revealed that age was the only factor associated with the decrease in S-RBD IgG levels at follow-up (180 days). Elderly individuals of both groups were characterized by a reduction in the antibody responses with respect to younger individuals., Conclusions: Our results show an efficacy seroconversion in DOC patients in the first period after vaccination, which significantly declines over time with respect to healthy controls.

Prostate cancer (PCa) that progresses after androgen deprivation therapy (ADT) remains incurable. The underlying mechanisms that account for the ultimate emergence of resistance to ADT, progressing to castrate-resistant prostate cancer (CRPC), include those that reactivate androgen receptor (AR), or those that are entirely independent or cooperate with androgen signaling to underlie PCa progression. The intricacy of metabolic pathways associated with PCa progression spurred us to develop a metabolism-centric analysis to assess the metabolic shift occurring in PCa that progresses with low AR expression. We used PCa patient-derived xenografts (PDXs) to assess the metabolic changes after castration of tumor-bearing mice and subsequently confirmed main findings in human donor tumor that progressed after ADT. We found that relapsed tumors had a significant increase in fatty acids and ketone body (KB) content compared with baseline. We confirmed that critical ketolytic enzymes (ACAT1, OXCT1, BDH1) were dysregulated after castrate-resistant progression. Further, these enzymes are increased in the human donor tissue after progressing to ADT. In an in silico approach, increased ACAT1, OXCT1, BDH1 expression was also observed for a subset of PCa patients that relapsed with low AR and ERG (ETS-related gene) expression. Further, expression of these factors was also associated with decreased time to biochemical relapse and decreased progression-free survival. Our studies reveal the key metabolites fueling castration resistant progression in the context of a partial or complete loss of AR dependence., (© 2021. The Author(s).)

Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and 14 multiregion normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8 + T cells, antigen-presenting macrophages, and inflammatory dendritic cells. We further find that the LUAD ligand-receptor interactome harbors increased expression of epithelial CD24 , which mediates protumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment. SIGNIFICANCE: The geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multiregion single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecologic evolution of LUAD from the lung that comprise high-potential targets for early interception. This article is highlighted in the In This Issue feature, p. 2355 ., (©2021 American Association for Cancer Research.)

Lack of sustained response to therapeutic agents in patients with KRAS-mutant lung cancer poses a major challenge and arises partly due to intratumor heterogeneity that defines phenotypically distinct tumor subpopulations. To attain better therapeutic outcomes, it is important to understand the differential therapeutic sensitivities of tumor cell subsets. Epithelial-mesenchymal transition is a biological phenomenon that can alter the state of cells along a phenotypic spectrum and cause transcriptional rewiring to produce distinct tumor cell subpopulations. We utilized functional shRNA screens, in in vitro and in vivo models, to identify and validate an increased dependence of mesenchymal tumor cells on cyclin-dependent kinase 4 (CDK4) for survival, as well as a mechanism of resistance to MEK inhibitors. High zinc finger E-box binding homeobox 1 levels in mesenchymal tumor cells repressed p21, leading to perturbed CDK4 pathway activity. Increased dependence on CDK4 rendered mesenchymal cancer cells particularly vulnerable to selective CDK4 inhibitors. Coadministration of CDK4 and MEK inhibitors in heterogeneous tumors effectively targeted different tumor subpopulations, subverting the resistance to either single-agent treatment.

Lung squamous cell carcinoma (LSCC) remains a leading cause of cancer death with few therapeutic options. We characterized the proteogenomic landscape of LSCC, providing a deeper exposition of LSCC biology with potential therapeutic implications. We identify NSD3 as an alternative driver in FGFR1-amplified tumors and low-p63 tumors overexpressing the therapeutic target survivin. SOX2 is considered undruggable, but our analyses provide rationale for exploring chromatin modifiers such as LSD1 and EZH2 to target SOX2-overexpressing tumors. Our data support complex regulation of metabolic pathways by crosstalk between post-translational modifications including ubiquitylation. Numerous immune-related proteogenomic observations suggest directions for further investigation. Proteogenomic dissection of CDKN2A mutations argue for more nuanced assessment of RB1 protein expression and phosphorylation before declaring CDK4/6 inhibition unsuccessful. Finally, triangulation between LSCC, LUAD, and HNSCC identified both unique and common therapeutic vulnerabilities. These observations and proteogenomics data resources may guide research into the biology and treatment of LSCC., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis., Competing Interests: Declaration of interests D.L.G. serves on scientific advisory committees for AstraZeneca, GlaxoSmithKline, Sanofi, and Janssen; provides consultation to Ribon Therapeutics; and receives research support from Janssen, Takeda, and AstraZeneca. I.I.W. serves on advisory boards for Genentech/Roche, Bristol-Myers Squibb, Medscape, Astra Zeneca/Medimmune, HTG Molecular, Merck, GlaxoSmithKline, and MSD and receives research support from Genentech, Oncoplex, HTG Molecular, DepArray, Merck, Bristol-Myers Squibb, Medimmune, Adaptive, Adaptimmune, EMD Serono, Pfizer, Takeda, Amgen, Karus, Johnson & Johnson, Bayer, 4D, Novartis, and Perkin-Elmer (Akoya). G.D.L. has received financial support from Pfizer-CTI. J.M.K. has received consulting fees from Halozyme. P.B. has received consulting fees from ExpertConnect., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Telehealth systems have shown success in the remote management of several neurological disorders, but there is a paucity of evidence in disorders of consciousness (DOC). In this study, we explore the effectiveness of a new telemonitoring system, for monitoring Vegetative State (VS) and Minimally Conscious State (MCS) patients. This was a prospective, mono-center randomized controlled study. We included only traumatic brain injury (TBI) patients who required long-term motor/cognitive assistance having a stable clinical condition. We examined their clinical evolution over ~4 years of the follow-up period. Twenty-two TBI patients were enrolled and equally divided into two groups: one telemonitored at home with our service and the second admitted to a standard long-stay hospitalization (LSH) program. Patients enrolled in the telehealth service (age: 49.9 ± 20.4; 45% female; diagnosis: 36% VS/64% MCS) were demographically and clinically-matched with those admitted to the LSH program (age: 55.1 ± 15; 18% female; diagnosis: 54% VS/46% MCS). Thirty-six percent of patients in the LSH program died before completing follow up evaluation with respect to 18% of death in the group of TBI patients telemonitored at home. At follow-up, patients in LSH and telemonitoring groups showed similar clinical progression, as measured by CRS-r, NCS, WHIM, and LCF scales, as well as by the number of medical complications (i.e., bedsores, infections). Finally, we estimated the total daily cost per patient. Severe TBI patients enrolled in the conventional LSH program cost 262€ every single day, whereas the cost per patient in the telehealth service resulted to be less expensive (93€). Here, we highlight that our telehealth monitoring service is as efficacious as in-person usual care to manage a severe neurological disorder such as TBI in a cost-effective way., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Raso, Arcuri, Liperoti, Mercurio, Mauro, Cusato, Romania, Serra, Pignolo, Tonin and Cerasa.)

Currently there is no highly specific and sensitive marker to identify breast cancer-the most common malignancy in women. Breast cancer can be categorized as estrogen receptor (ER)/progesterone receptor (PR)-positive luminal, human epidermal growth factor receptor 2 (HER2)-positive, or triple-negative breast cancer (TNBC) types based on the expression of ER, PR, and HER2. Although GATA3 is the most widely used tumor marker at present to determine the breast origin, which has been shown to be an excellent marker for ER-positive and low-grade breast cancer, but it does not work well for TNBC with sensitivity as low as <20% in metaplastic breast carcinoma. In the current study, through TCGA data mining we identified trichorhinophalangeal syndrome type 1 (TRPS1) as a specific gene for breast carcinoma across 31 solid tumor types. Moreover, high mRNA level of TRPS1 was found in all four subtypes of breast carcinoma including ER/PR-positive luminal A and B types, HER2-positive type, and basal-type/TNBC. We then analyzed TRPS1 expression in 479 cases of various types of breast cancer using immunochemistry staining, and found that TRPS1 and GATA3 had comparable positive expression in ER-positive (98% vs. 95%) and HER2-positive (87% vs. 88%) breast carcinomas. However, TRPS1 which was highly expressed in TNBC, was significantly higher than GATA3 expression in metaplastic (86% vs. 21%) and nonmetaplastic (86% vs. 51%) TNBC. In addition, TRPS1 expression was evaluated in 1234 cases of solid tumor from different organs. In contrast to the high expression of GATA3 in urothelial carcinoma, TRPS1 showed no or little expression in urothelial carcinomas or in other tumor types including lung adenocarcinoma, pancreatic adenocarcinoma, colon and gastric adenocarcinoma, renal cell carcinoma, melanoma, and ovarian carcinoma. These findings suggest that TRPS1 is a highly sensitive and specific marker for breast carcinoma and can be used as a great diagnostic tool, especially for TNBC.

Lung cancer is consistently the leading cause of cancer-related death worldwide, and it ranks as the second most frequent type of new cancer cases diagnosed in the United States, both in males and females. One subtype of lung cancer, small cell lung carcinoma (SCLC), is an aggressive, poorly differentiated, and high-grade neuroendocrine carcinoma that accounts for 13% of all lung carcinomas. SCLC is the most frequent neuroendocrine lung tumor, and it is commonly presented as an advanced stage disease in heavy smokers. Due to its clinical presentation, it is typically diagnosed in small biopsies or cytology specimens, with routine immunostaining only. However, immunohistochemistry markers are extremely valuable in demonstrating neuroendocrine features of SCLC and supporting its differential diagnosis. The 2015 WHO classification grouped all pulmonary neuroendocrine carcinomas in one category and maintained the SCLC combined variant that was previously recognized. In this review, we explore multiple aspects of the pathologic features of this entity, as well as clinically relevant immunohistochemistry markers expression and its molecular characteristics. In addition, we will focus on characteristics of the tumor microenvironment, and the latest pathogenesis findings to better understand the new therapeutic options in the current era of personalized therapy.

Rationale: Early pathogenesis of lung adenocarcinoma (LUAD) remains largely unknown. We found that, relative to wild-type littermates, the innate immunomodulator Lcn2 (lipocalin-2) was increased in normal airways from mice with knockout of the airway lineage gene Gprc5a ( Gprc5a -/- ) and that are prone to developing inflammation and LUAD. Yet, the role of LCN2 in lung inflammation and LUAD is poorly understood. Objectives: Delineate the role of Lcn2 induction in LUAD pathogenesis. Methods: Normal airway brushings, uninvolved lung tissues, and tumors from Gprc5a -/- mice before and after tobacco carcinogen exposure were analyzed by RNA sequencing. LCN2 mRNA was analyzed in public and in-house data sets of LUAD, lung squamous cancer (LUSC), chronic obstructive pulmonary disease (COPD), and LUAD/LUSC with COPD. LCN2 protein was immunohistochemically analyzed in a tissue microarray of 510 tumors. Temporal lung tumor development, gene expression programs, and host immune responses were compared between Gprc5a -/- and Gprc5a -/- / Lcn2 -/- littermates. Measurements and Main Results: Lcn2 was progressively elevated during LUAD development and positively correlated with proinflammatory cytokines and inflammation gene sets. LCN2 was distinctively elevated in human LUADs, but not in LUSCs, relative to normal lungs and was associated with COPD among smokers and patients with LUAD. Relative to Gprc5a -/- mice, Gprc5a -/- / Lcn2 -/- littermates exhibited significantly increased lung tumor development concomitant with reduced T-cell abundance (CD4 + ) and richness, attenuated antitumor immune gene programs, and increased immune cell expression of protumor inflammatory cytokines. Conclusions: Augmented LCN2 expression is a molecular feature of COPD-associated LUAD and counteracts LUAD development in vivo by maintaining antitumor immunity.

Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIβ (PI4KIIIβ). Molecular, biochemical, and cell biological studies show that PI4KIIIβ-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIβ-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIβ antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIβ-dependent secretion for cancer cell survival and tumor progression., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Post-traumatic cricothyroid joint dislocation is reported in the literature. Trauma of the laryngeal framework is the most common cause. As a result, spontaneous non-traumatic cricothyroid joint dislocation is very rarely described. We report the case of a patient with spontaneous cricothyroid joint dislocation with the aim of reporting the diagnostic and therapeutic findings related to this rare entity. History and physical examination may suggest diagnosis. Computed tomography scan can confirm diagnosis and detect coexistence of anatomic anomalies of the neck. Reduction of dislocation may be easily performed in outpatient setting, producing immediate relief from dysphonia and neck pain. As CTJ dislocation is a speech-related disorder, appropriate speech therapy is advisable in order to avoid relapse., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: The association between malnutrition and worse outcomes as pressure ulcers and mortality is well established in a variety of setting. Currently none investigation was conducted in patients with long-term consequences of the acquired brain injury in which recovery from brain injury could be influenced by secondary complications. The aim of this study was to investigate the association between various nutritional status parameters (in particular albumin) and pressure ulcers formation and short-term mortality in minimal conscious state patients., Methods: In this prospective, observational study of 5-months duration, a 30 patients sample admitted to a Neurological Institute was considered. All patients underwent a complete medical examination. Anthropometric parameters like mid-arm circumference and mid-arm muscle circumference and nutritional parameters as serum albumin and blood hemoglobin concentration were assessed., Results: At univariate and logistic regression analysis, mid-arm circumference (p = 0.04; beta = -0.89), mid-arm muscle circumference (p = 0.050; beta = -1.29), hemoglobin (p = 0.04, beta -1.1) and albumin (p = 0.04, beta -7.91) were inversely associated with pressure ulcers. The area under the ROC curve for albumin to predict sores was 0.76 (p = 0.02) and mortality was 0.83 (p = 0.03). Patient with lower albumin had significantly higher short-term mortality than those with higher serum albumin (p = 0.03; χ(2) test = 6.47)., Conclusion: Albumin, haemoglobin and mid-arm circumference are inversely associated with pressure ulcers. Albumin is a prognostic index in MCS patients. Since albumin and haemoglobin could be affected by a variety of factors, this association suggests to optimize nutrition and investigate on other mechanism leading to mortality and pressure ulcers.

Background and Aim Laboratory blood tests for hospitalized patients are often overused. Excessive costs and no proof of benefit suggest re-evaluating the current approach to laboratory test ordering. The aim of the study is to improve the decision-making process of test ordering and to investigate what effect a rational, evidence-based use of laboratory test ordering in surgical wards would have on costs and healthcare resources. Methods Three-phase experimental prospective study carried out at the tertiary referral teaching hospital of Parma. Phase 1 (baseline status). The baseline status of laboratory test ordering was evaluated by recording the number of biochemical tests requested for patients undergoing elective surgery. Laboratory tests were grouped in "recommended" (RT) and "non recommended" (nRT) tests on the basis of pertinent literature. Phase 2 (improvement action): new guidelines were introduced into clinical practice. Phase 3 (feedback): Prospective data collection for first and second feedback was performed with no advance notice. Results A highly significant reduction in test ordering was found on occasion of the phases 2 and 3 of the study. The overall number of tests decreased, largely due to a decrease in the use of nRT. Conclusions Analysis was justified by the fact that most test requests proved not to be supported by clinical evidence. Inappropriate ordering of laboratory tests results in an unnecessarily high number of requests, which do not in turn improve patient management. Moreover, more appropriate, evidence-based laboratory test ordering for patients undergoing elective surgery may produce a significant reduction in costs, particularly in high-cost settings. (www.actabiomedica.it).

Background: Sphenoid sinus lesions are rare entities, occurring in 2 - 3% out of all paranasal sinus lesions. Isolated oculomotor nerve palsy due to acute sphenoid sinusitis is very rare, with only few cases reported in literature., Methods: Retrospective report about a case of isolated acute sphenoid sinusitis in a child with a left-sided third cranial nerve paralysis as the only sign at presentation., Results: Isolated oculomotor nerve palsy can be the initial sign of an isolated acute sphenoid sinusitis in children and it requires a high index of suspicion in order to avoid a delay in diagnosis., Conclusions: Magnetic resonance imaging should be promptly performed. Functional endoscopic sinus surgery represents the treatment of choice in order to restore sinus drainage and avoid further intracranial or ocular complications. The need of serial postoperative debridement under general anesthesia should be adequately scheduled and previously discussed with parents.

Lidocaine is commonly used for topical anesthesia of the upper airway in patient with anticipated difficult tracheal intubation undergoing awake fiberoptic intubation. Lidocaine toxicity is dose related and proportional to its plasma level. Although neurologic toxicity has been frequently observed with intravenous use, it has also been reported for topical use. We report on a case of a patient with base tongue abscess who developed sudden seizures and coma during application of topical anesthesia with lidocaine for awake fiberoptic intubation. The presence of a deep neck infection that causes hyperemia and edema of the pharyngolaryngeal mucosa may enhance transmucosal systemic absorption of local anesthetic. Moreover, conditions such as hypercarbia, dysphagia, or hepatic diseases are known to facilitate onset of lidocaine neurologic toxicity with serum concentration lower than normal. These findings should be kept in mind before administering topical anesthesia of the upper airway. In the presence of any of these conditions above, either the total dose of local anesthetic or its concentration should be reduced as much as possible., (© 2013 Elsevier Inc. All rights reserved.)

Purpose: New prognostic markers to guide treatment decisions in early stage non-small cell lung cancer are necessary to improve patient outcomes. In this report, we assess the utility of a predefined mRNA expression signature of cell-cycle progression genes (CCP score) to define 5-year risk of lung cancer-related death in patients with early stage lung adenocarcinoma., Experimental Design: A CCP score was calculated from the mRNA expression levels of 31 proliferation genes in stage I and stage II tumor samples from two public microarray datasets [Director's Consortium (DC) and GSE31210]. The same gene set was tested by quantitative PCR in 381 formalin-fixed paraffin-embedded (FFPE) primary tumors. Association of the CCP score with outcome was assessed by Cox proportional hazards analysis., Results: In univariate analysis, the CCP score was a strong predictor of cancer-specific survival in both the Director's Consortium cohort (P = 0.00014; HR = 2.08; 95% CI, 1.43-3.02) and GSE31210 (P = 0.0010; HR = 2.25; 95% CI, 1.42-3.56). In multivariate analysis, the CCP score remained the dominant prognostic marker in the presence of clinical variables (P = 0.0022; HR = 2.02; 95% CI, 1.29-3.17 in Director's Consortium, P = 0.0026; HR = 2.16; 95% CI, 1.32-3.53 in GSE31210). On a quantitative PCR platform, the CCP score maintained highly significant prognostic value in FFPE-derived mRNA from clinical samples in both univariate (P = 0.00033; HR = 2.10; 95% CI, 1.39-3.17) and multivariate analyses (P = 0.0071; HR = 1.92; 95% CI, 1.18-3.10)., Conclusions: The CCP score is a significant predictor of lung cancer death in early stage lung adenocarcinoma treated with surgery and may be a valuable tool in selecting patients for adjuvant treatment., (©2013 AACR.)

Introduction: RNA-dependent protein kinase (PKR) is an independent prognostic variable in patients with non-small-cell lung cancer (NSCLC). In the current study, we investigated the correlation between PKR and 25 other biomarkers for NSCLC, identified the markers that could further improve the prognostic significance of PKR and elucidated the mechanisms of interaction between these markers and PKR., Methods: Tissue microarray samples obtained from 218 patients with lung cancer were stained with an anti-PKR antibody and antibodies against 25 biomarkers. Immunohistochemical expression was scored and used for Kaplan-Meier survival analysis. The interaction between PKR and EphA2 in NSCLC cell lines was examined., Results: We found that PKR was associated with EphA2 and that the prognostic information regarding NSCLC provided by the combination of PKR and EphA2 (P/E) was significantly more accurate than that provided by either marker alone. The 5-year overall survival rate in patients with PKR/EphA2 (20%) was significantly lower than that of patients with PKR/EphA2 (74%), patients with PKR/EphA2 (55%), and patients with PKR/EphA2 (55%) (p < 0.0001). We also found that the PKR:EphA2 (P/E) ratio was significantly associated with prognosis (p < 0.0001). Univariate and multivariate Cox analyses revealed that this P/E combination or ratio was an independent predictor of overall survival. In addition, induction of PKR expression reduced EphA2 protein expression levels in NSCLC cell lines., Conclusions: PKR/EphA2 is a significant predictor of prognosis for NSCLC. PKR/EphA2 may be a promising approach to improving screening efficiency and predicting prognosis in patients with NSCLC.