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Purpose: RAS genes (HRAS, KRAS, and NRAS) are commonly found to be mutated in cancers, and activating RAS variants are also found in disorders of somatic mosaicism (DoSM). A survey of the mutational spectrum of RAS variants in DoSM has not been performed., Methods: A total of 938 individuals with suspected DoSM underwent high-sensitivity clinical next-generation sequencing-based testing. We investigated the mutational spectrum and genotype-phenotype associations of mosaic RAS variants., Results: In this article, we present a series of individuals with DoSM with RAS variants. Classic hotspots, including Gly12, Gly13, and Gln61 constituted the majority of RAS variants observed in DoSM. Furthermore, we present 12 individuals with HRAS and KRAS in-frame duplication/insertion (dup/ins) variants in the switch II domain. Among the 18.3% individuals with RAS in-frame dup/ins variants, clinical findings were mainly associated with vascular malformations. Hotspots were associated with a broad phenotypic spectrum, including vascular tumors, vascular malformations, nevoid proliferations, segmental overgrowth, digital anomalies, and combinations of these. The median age at testing was higher and the variant allelic fraction was lower in individuals with in-frame dup/ins variants than those in individuals with mosaic RAS hotspots., Conclusion: Our work provides insight into the allelic and clinical heterogeneity of mosaic RAS variants in nonmalignant conditions., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2022 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Context.—: Acute invasive fungal sinusitis (AIFS) can be challenging to diagnose because of its varied clinical presentation., Objectives.—: To evaluate the discrepancies between clinical and autopsy diagnoses in patients with histologic evidence of AIFS at a tertiary care center., Design.—: Autopsy cases with a pathologic autopsy diagnosis of AIFS from the past 20 years at a tertiary hospital were evaluated for clinicopathologic features. Modified Goldman classification was used to classify discrepant cases. Clinical history was also reviewed., Results.—: Of 7071 institutional autopsy cases during 20 years, 9 met inclusion criteria and demonstrated AIFS at autopsy. Angioinvasive fungal elements were histologically identified in all cases, with multiorgan involvement in most cases (6 of 9; 67%). Major clinicopathologic discrepancies were identified in 6 cases (67%)., Conclusions.—: AIFS is an uncommon diagnosis rendered at autopsy. There is a subset of cases that demonstrate discrepancy between the clinical impression and pathologic autopsy diagnosis. Antemortem diagnosis of AIFS can be challenging, especially in cases without prototypical clinical symptoms and positive fungal cultures. At autopsy, there was multiorgan involvement, supporting extensive tissue sampling to evaluate for AIFS, even in the setting of negative fungal cultures or lack of classic symptoms. These findings highlight the importance of the pathologist's role at autopsy in the definitive diagnosis of AIFS, especially in clinically equivocal cases.

Purpose: Nonoperative management with short-course radiation therapy (SCRT) as a component of definitive therapy for oligometastatic rectal cancer has not been previously reported. This single-institution retrospective analysis evaluates treatment with SCRT in combination with chemotherapy (SCRT-CTX) with nonoperative intent for patients with a locoregional clinical complete response (cCR)., Methods and Materials: Thirty-six patients with newly diagnosed oligometastatic rectal cancer were treated with SCRT-CTX between January 1, 2018, and May 31, 2020. Digital rectal examination, endoscopy, and imaging (computed tomography or magnetic resonance imaging) were used to determine cCR. Medically operable patients without cCR underwent surgical resection of the primary rectal tumor. Patients with cCR who experienced a local failure received salvage surgery. Rates of hospitalization related to primary tumor disease and pelvic symptoms were reviewed. Overall survival (OS) and progression free survival were evaluated., Results: Seventeen percent (6/36) of patients achieved cCR after SCRT-CTX. Eleven percent (4) of patients experienced a local failure. OS for all patients was 83% (71%-96%) at 12 months and 57% (41%-80%) at 24 months. Progression free survival for all patients was 56% (41%-74%) at 12 months and 10% (3.1%-35%) at 24 months. On multivariate analysis, having received more than 4 months of chemotherapy (hazard ratio = 0.21; 95% confidence interval, 0.06-0.71; P = .01) and definitive treatment of metastatic site (hazard ratio = 0.17; 95% confidence interval, 0.05-0.66; P = .01) predicted for improved OS. The number of patients requiring hospitalization due to obstruction (8/36, 22%), rectal bleeding (5/36, 14%), or need for permanent ostomy placement (5/36, 14%) was low, and there was a decrease in endorsement of obstructive symptoms and rectal bleeding after completion of SCRT-CTX., Conclusions: SCRT-CTX with nonoperative intent for patients with a locoregional cCR may be a reasonable treatment option for patients with newly diagnosed oligometastatic rectal adenocarcinoma and demonstrates excellent control of pelvic disease and symptoms. Increased duration of chemotherapy within the treatment paradigm may improve oncologic outcomes., (Copyright © 2022 American Society for Radiation Oncology. Published by Elsevier Inc. All rights reserved.)

Introduction: Current recommendations regarding the size of wide local excision (WLE) margins for Merkel cell carcinoma (MCC) are not well established., Methods: WLE and pathologic margins were respectively reviewed from 79 patients with stage I or II MCC, who underwent WLE at Washington University in St Louis from 2005 to 2019. Outcomes included local recurrence-free survival (LRFS), regional recurrence-free survival (RRFS), distant recurrence-free survival (DRFS), disease-free survival (DFS), and disease-specific survival (DSS)., Results: Thirty-two percent of patients received adjuvant radiotherapy (aRT). At 1 year, DFS was 51.3%, 71.4%, and 87.8% for patients with WLE margins < 1 cm, 1-1.9 cm, and ≥ 2 cm, respectively (p = 0.02). At 3 years, the DSS was 57.7%, 82.6%, and 100% for patients with WLE margins < 1 cm, 1-1.9 cm, and ≥ 2 cm, respectively (p = 0.02). Multivariable Cox analysis demonstrated that every 1-cm increase in WLE margins was associated with improved RRFS [hazard ratio (HR) = 0.28, 95% confidence interval (CI): 0.11-0.75], DRFS (HR 0.30, CI 0.08-0.99), DFS (HR 0.42, CI 0.21-0.86), and DSS (HR 0.16, CI 0.04-0.61). WLE and pathologic margin size were moderately-to-strongly correlated (r = 0.66). Close or positive pathologic margins (< 3 mm) were associated with reduced DRFS (HR 6.83, CI 1.80-25.9), DFS (HR 2.98, CI 1.31-6.75), and DSS (HR 3.52, CI 1.14-10.9)., Conclusion: Reduced WLE and pathologic margins were associated with higher risk of relapse and death from MCC. Larger WLE margins are important in populations with lower rates of adjuvant radiation.

Importance: Current recommendations regarding the size of local excision (LE) margins for Merkel cell carcinoma (MCC) have not been well established., Objective: To assess whether larger clinical LE margins and receipt of adjuvant radiotherapy are associated with improvements in overall survival (OS) among patients with localized MCC., Design, Setting, and Participants: This large multicenter retrospective cohort study used records from the National Cancer Database to identify adult patients with localized stage I or stage II MCC who underwent LE between January 1, 2004, and December 31, 2015. Data were analyzed from August 1, 2020, to January 25, 2021., Exposures: Local excision margin size and adjuvant radiotherapy., Main Outcomes and Measures: Overall and net survival were assessed using Cox multivariable regression analysis., Results: A total of 6156 patients with localized MCC (median age at diagnosis, 77 years [range, 27-90 years]; 2500 women [40.6%]). In the multivariable regression analysis, LE clinical margins larger than 1.0 cm were associated with improvements in OS (HR, 0.88; 95% CI, 0.81-0.95; P < .001) compared with margins of 1.0 cm or smaller, regardless of tumor subsite. At 5 years after surgery, LE margins of 1.0 cm or smaller were associated with a net survival of 76.7%, while LE margins larger than 1.0 cm were associated with a net survival of 89.8% (P < .001). Stratification of LE margins into 3 subgroups indicated that LE margins of 1.1 to 2.0 cm (HR, 0.87; 95% CI, 0.76-0.99; P = .047) and larger than 2.0 cm (HR, 0.84; 95% CI, 0.72-0.98; P = .03) were associated with improvements in OS compared with margins of 1.0 cm or smaller. In patients with less aggressive disease (ie, those who were immunocompetent and had tumors ≤1.0 cm, no lymphovascular invasion, and negative pathologic margins), LE margins larger than 1.0 cm were also associated with improvements in OS (HR, 0.87; 95% CI, 0.78-0.97; P = .01). Among patients who received adjuvant radiotherapy, larger LE margins were associated with improvements in OS (HR, 0.87; 95% CI, 0.76-0.98; P = .03). Receipt of adjuvant radiotherapy was also associated with improvements in OS within the 3 LE margin subgroups. Patients who received adjuvant radiotherapy and had LE margins of 1.0 cm or smaller (HR, 0.81; 95% CI, 0.74-0.89; P < .001) experienced OS that was comparable to that in patients who did not receive adjuvant radiotherapy and had LE margins larger than 1.0 cm (HR, 0.80; 95% CI, 0.71-0.89; P = .87)., Conclusions and Relevance: In this study, LE clinical margins larger than 1.0 cm were associated with improvements in OS, and these improvements were independent of tumor subsite, receipt of adjuvant radiotherapy, positive pathologic margins, or adverse pathologic features for stage I to stage II MCC. Patients with LE margins of 1.0 cm or smaller who received adjuvant radiotherapy experienced OS that was similar to that of patients with larger LE margins who did not receive radiotherapy. The combination of LE clinical margins larger than 1.0 cm and adjuvant radiotherapy was associated with the highest OS.

Background: One-third of patients with Merkel cell carcinoma (MCC) present with locally advanced disease involving the regional lymph nodes, but indications for regional lymph node radiation therapy (rLN-RT) are not well established., Materials and Methods: 72 patients with locally advanced MCC were retrospectively reviewed. Regional lymph nodes were addressed with observation, lymph node dissection (LND) alone, definitive nodal radiotherapy (DnRT), or LND plus adjuvant nodal radiotherapy (AnRT). Cox regression was used to compare treatment modalities in terms of regional recurrence-free survival (RRFS), distant recurrence-free survival (DRFS), disease-free survival (DFS) and disease-specific survival (DSS)., Results: rLN-RT, including both DnRT and AnRT, improved RRFS (Hazard ratio (HR): 0.07, 95% confidence interval (CI): 0.01-0.40, p = 0.003), DRFS (HR: 0.28, CI: 0.11-0.76, p = 0.01), DFS (HR: 0.23, CI: 0.09-0.58, p = 0.002), and DSS (HR: 0.23, CI: 0.06-0.90, p = 0.03). AnRT improved DFS and DSS in high-risk subgroups (e.g., extranodal extension (ENE), ≥ 2 positive lymph nodes, or bulkier lymph nodes). The benefit of AnRT increased with higher disease burden. After controlling for these adverse factors, AnRT significantly improved RRFS (HR: 0.04, CI: 0.01-0.37, p = 0.004), DRFS (HR: 0.14, CI: 0.04-0.50, p = 0.003), DFS (HR: 0.09, CI: 0.02-0.33, p < 0.001), and DSS (HR: 0.21, CI: 0.05-0.89, p = 0.03)., Conclusion: rLN-RT, including both DnRT and AnRT, reduces relapse and death from MCC in patients with node-positive disease. AnRT is particularly beneficial for patients with ENE, multiple involved lymph nodes, or larger nodal foci of disease. These results argue for more liberal use of nodal RT for MCC patients who present with node-positive disease., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Infection is a common cause of morbidity and mortality in chronic lymphocytic leukemia and should be considered when examining bone marrow specimens to identify a potentially treatable pathogen., Competing Interests: None declared., (© 2021 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd.)

The oncofetal mRNA-binding protein, IMP1 or insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1), promotes the overexpression of several oncogenic proteins by binding to and stabilizing their mRNAs. IMP1 is frequently overexpressed in melanoma and is associated with a poor prognosis, but the full spectrum of IMP1 target transcripts remains unknown. Here, we report the identification of protein kinase C-α (PKCα), as a novel molecular target of IMP1. Overexpression of IMP1 resulted in increased levels of PKCα, while RNAi knockdown of IMP1 resulted in decreased PKCα mRNA stability, PKCα protein levels, and MAPK/ERK activation. In addition to IMP1 acting as a positive regulator of PKCα mRNA, we also report the identification of miR-340 as a negative regulator of PKCα mRNA. In melanoma cancer cells, inhibition of miR-340 led to increased PKCα protein levels. PKCα plays important roles in numerous signaling pathways including the MAPK/ERK signaling pathway. PKCα activates RAF1, which in turn activates MEK1, and activates downstream transcriptional targets of MAPK through activation of JNK signaling. Together, these pathways provide a way to activate MAPK signaling downstream of BRAF and MEK1 inhibitors, which are commonly used to treat melanoma. Analysis of 117 melanoma tumors samples showed that overexpression of PKCα is associated with poorer overall survival. In patients harboring BRAF or NRAS mutations, PKCα overexpression is associated with an 11-fold increased risk of death. Thus, PKCα mRNA is a novel target of IMP1, which is commonly overexpressed in melanoma and is linked to poorer overall survival.

Anti-programmed death 1 (PD-1) immune checkpoint inhibitors enhance the antitumour activity of the immune system and have produced durable tumour responses in several solid tumours including non-small cell lung cancer (NSCLC). However, PD-1 inhibitors can lead to immune-related adverse events , including pneumonitis, which is typically mild, but can be severe and potentially fatal. Pneumonitis often resolves with steroids, but some cases are steroid refractory, leading to a relapsing and remitting course in milder cases or the need for salvage therapies in more severe cases. Here, we present two patients with NSCLC who developed severe pneumonitis following therapy with nivolumab and pembrolizumab. While one patient improved with steroids and infliximab, the other patient failed to respond to steroids and subsequently died. These cases demonstrate the highly variable presentation and therapeutic responses seen in patients with pneumonitis following anti-PD-1 therapy and illustrate that severe cases can often present refractory to steroid therapy., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2018. No commercial re-use. See rights and permissions. Published by BMJ.)

The oncofetal mRNA-binding protein, IMP1 or insulin-like growth factor-2 mRNA-binding protein 2 (IGF2BP1), binds to and stabilizes c-Myc, β-TrCP1, and other oncogenic mRNAs, leading to increased expression of the proteins encoded by its target mRNAs. IMP1 is frequently overexpressed in cancer and is strongly correlated with a poor prognosis and reduced survival in melanoma, ovarian, breast, colon, and lung cancer. While IMP1 is an attractive anticancer drug target, there are no small molecule inhibitors of IMP1. A fluorescence anisotropy-based assay was used to screen 160,000 small molecules for their ability to inhibit IMP1 binding to fluorescein-labeled c-Myc mRNA. The small molecule, BTYNB, was identified as a potent and selective inhibitor of IMP1 binding to c-Myc mRNA. In cells, BTYNB downregulates several mRNA transcripts regulated by IMP1. BTYNB destabilizes c-Myc mRNA, resulting in downregulation of c-Myc mRNA and protein. BTYNB downregulates β-TrCP1 mRNA and reduces activation of nuclear transcriptional factors-kappa B (NF-κB). The oncogenic translation regulator, eEF2, emerged as a new IMP1 target mRNA, enabling BTYNB to inhibit tumor cell protein synthesis. BTYNB potently inhibited proliferation of IMP1-containing ovarian cancer and melanoma cells with no effect in IMP1-negative cells. Overexpression of IMP1 reversed BTYNB inhibition of cell proliferation. BTYNB completely blocked anchorage-independent growth of melanoma and ovarian cancer cells in colony formation assays. With its ability to target c-Myc and to inhibit proliferation of difficult-to-target melanomas and ovarian cancer cells, and with its unique mode of action, BTYNB is a promising small molecule for further therapeutic evaluation and mechanistic studies., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Recurrent estrogen receptor α (ERα)-positive breast and ovarian cancers are often therapy resistant. Using screening and functional validation, we identified BHPI, a potent noncompetitive small molecule ERα biomodulator that selectively blocks proliferation of drug-resistant ERα-positive breast and ovarian cancer cells. In a mouse xenograft model of breast cancer, BHPI induced rapid and substantial tumor regression. Whereas BHPI potently inhibits nuclear estrogen-ERα-regulated gene expression, BHPI is effective because it elicits sustained ERα-dependent activation of the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR), and persistent inhibition of protein synthesis. BHPI distorts a newly described action of estrogen-ERα: mild and transient UPR activation. In contrast, BHPI elicits massive and sustained UPR activation, converting the UPR from protective to toxic. In ERα(+) cancer cells, BHPI rapidly hyperactivates plasma membrane PLCγ, generating inositol 1,4,5-triphosphate (IP3), which opens EnR IP3R calcium channels, rapidly depleting EnR Ca(2+) stores. This leads to activation of all three arms of the UPR. Activation of the PERK arm stimulates phosphorylation of eukaryotic initiation factor 2α (eIF2α), resulting in rapid inhibition of protein synthesis. The cell attempts to restore EnR Ca(2+) levels, but the open EnR IP3R calcium channel leads to an ATP-depleting futile cycle, resulting in activation of the energy sensor AMP-activated protein kinase and phosphorylation of eukaryotic elongation factor 2 (eEF2). eEF2 phosphorylation inhibits protein synthesis at a second site. BHPI's novel mode of action, high potency, and effectiveness in therapy-resistant tumor cells make it an exceptional candidate for further mechanistic and therapeutic exploration.

Cancer cell proliferation is regulated by oncogenes, such as c-Myc. An alternative approach to directly targeting individual oncogenes is to target IMP-1, an oncofetal protein that binds to and stabilizes messenger RNAs (mRNAs), leading to elevated expression of c-Myc and other oncogenes. Expression of IMP-1 is tightly correlated with a poor prognosis and reduced survival in ovarian, lung, and colon cancer. Small-molecule inhibitors of IMP-1 have not been reported. We established a fluorescence anisotropy/polarization microplate assay (FAMA) for analyzing binding of IMP-1 to a fluorescein-labeled 93 nucleotide c-Myc mRNA target (flMyc), developed the assay as a highly robust (Z' factor = 0.60) FAMA-based high-throughput screen for inhibitors of binding of IMP-1 to flMyc, and carried out a successful pilot screen of 17,600 small molecules. Our studies support rapidly filtering out toxic nonspecific inhibitors using an early cell-based assay in control cells lacking the target protein. The physiologic importance of verified hits from the in vitro high-throughput screen was demonstrated by identification of the first small-molecule IMP-1 inhibitor, a lead compound that selectively inhibits proliferation of IMP-1-positive cancer cells with very little or no effect on proliferation of IMP-1-negative cells.

Human monocytes from patients with patent filarial infections are studded with filarial antigen and express markers associated with alternative activation of macrophages (MΦ). To explore the role of filaria-derived parasite antigen in differentiation of human monocytes, cells were exposed to microfilariae (mf) of Brugia malayi, and their phenotypic and functional characteristics were compared with those of monocytes exposed to factors known to generate either alternatively (interleukin-4 [IL-4]) or classically (macrophage colony-stimulating factor [MCSF]) activated MΦ. IL-4 upregulated mRNA expression of CCL13, CCL15, CCL17, CCL18, CCL22, CLEC10A, MRC1, CADH1, CD274, and CD273 associated with alternative activation of MΦ but not arginase 1. IL-4-cultured monocytes had a diminished ability to promote proliferation of both CD4(+) and CD8(+) T cells compared to that of unexposed monocytes. Similar to results with IL-4, exposure of monocytes to live mf induced upregulation of CCL15, CCL17, CCL18, CCL22, CD274, and CD273 and downregulation of Toll-like receptor 3 (TLR3), TLR5, and TLR7. In contrast to results with MCSF-cultured monocytes, exposure of monocytes to mf resulted in significant inhibition of the phagocytic ability of these cells to the same degree as that seen with IL-4. Our data suggest that short exposure of human monocytes to IL-4 induces a phenotypic characteristic of alternative activation and that secreted filarial products skew monocytes similarly.

APC dysfunction has been postulated to mediate some of the parasite-specific T cell unresponsiveness seen in patent filarial infection. We have shown that live microfilariae of Brugia malayi induce caspase-dependent apoptosis in human monocyte-derived dendritic cells (DCs) in vitro. This study addresses whether apoptosis observed in vitro extends to patent filarial infections in humans and is reflected in the number of circulating myeloid DCs (mDCs; CD11c(-)CD123(lo)) in peripheral blood of infected microfilaremic individuals. Utilizing flow cytometry to identify DC subpopulations (mDCs and plasmacytoid DCs [pDCs]) based on expression of CD11c and CD123, we found a significant increase in numbers of circulating mDCs (CD11c(+)CD123(lo)) in filaria-infected individuals compared with uninfected controls from the same filaria-endemic region of Mali. Total numbers of pDCs, monocytes, and lymphocytes did not differ between the two groups. To investigate potential causes of differences in mDC numbers between the two groups, we assessed chemokine receptor expression on mDCs. Our data indicate that filaria-infected individuals had a lower percentage of circulating CCR1(+) mDCs and a higher percentage of circulating CCR5(+) mDCs and pDCs. Finally, live microfilariae of B. malayi were able to downregulate cell-surface expression of CCR1 on monocyte-derived DCs and diminish their calcium flux in response to stimulation by a CCR1 ligand. These findings suggest that microfilaria are capable of altering mDC migration through downregulation of expression of some chemokine receptors and their signaling functions. These observations have major implications for regulation of immune responses to these long-lived parasites.

Dysregulation of professional APC has been postulated as a major mechanism underlying Ag-specific T cell hyporesponsiveness in patients with patent filarial infection. To address the nature of this dysregulation, dendritic cells (DC) and macrophages generated from elutriated monocytes were exposed to live microfilariae (mf), the parasite stage that circulates in blood and is responsible for most immune dysregulation in filarial infections. DC exposed to mf for 24-96 h showed a marked increase in cell death and caspase-positive cells compared with unexposed DC, whereas mf exposure did not induce apoptosis in macrophages. Interestingly, 48-h exposure of DC to mf induced mRNA expression of the proapoptotic gene TRAIL and both mRNA and protein expression of TNF-alpha. mAb to TRAIL-R2, TNF-R1, or TNF-alpha partially reversed mf-induced cell death in DC, as did knocking down the receptor for TRAIL-R2 using small interfering RNA. The mf also induced gene expression of BH3-interacting domain death agonist and protein expression of cytochrome c in DC; mf-induced cleavage of BH3-interacting domain death agonist could be shown to induce release of cytochrome c, leading to activation of caspase 9. Our data suggest that mf induce DC apoptosis in a TRAIL- and TNF-alpha-dependent fashion.