Preexisting tumor-resident T cells with cytotoxic potential associate with response to neoadjuvant anti-PD-1 in head and neck cancer.

About 50% of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) experience recurrences after definitive therapy. The presurgical administration of anti-programmed cell death protein 1 (PD-1) immunotherapy results in substantial pathologic tumor responses (pTR) within the tumor microenvironment (TME). However, the mechanisms underlying the dynamics of antitumor T cells upon neoadjuvant PD-1 blockade remain unresolved, and approaches to increase pathologic responses are lacking. In a phase 2 trial (NCT02296684), we observed that 45% of patients treated with two doses of neoadjuvant pembrolizumab experienced marked pTRs (≥50%). Single-cell analysis of 17,158 CD8+ T cells from 14 tumor biopsies, including 6 matched pre-post neoadjuvant treatment, revealed that responding tumors had clonally expanded putative tumor-specific exhausted CD8+ tumor-infiltrating lymphocytes (TILs) with a tissue-resident memory program, characterized by high cytotoxic potential (CTX+) and ZNF683 expression, within the baseline TME. Pathologic responses after 5 weeks of PD-1 blockade were consistent with activation of preexisting CTX+ZNF683+CD8+ TILs, paralleling loss of viable tumor and associated tumor antigens. Response was associated with high numbers of CD103+PD-1+CD8+ T cells infiltrating pretreatment lesions, whereas revival of nonexhausted persisting clones and clonal replacement were modest. By contrast, nonresponder baseline TME exhibited a relative absence of ZNF683+CTX+ TILs and subsequent accumulation of highly exhausted clones. In HNSCC, revival of preexisting ZNF683+CTX+ TILs is a major mechanism of response in the immediate postneoadjuvant setting.

Mucoepidermoid carcinoma may be devoid of squamoid cells by immunohistochemistry: expanding the histologic and immunohistochemical spectrum of MAML2- rearranged salivary gland tumours.

Mucoepidermoid carcinoma (MEC) is historically defined by a mix of squamoid, intermediate, and mucous cells, but we have recently encountered several cases lacking immunoreactivity for squamous markers p40, p63, and CK5/6 despite MAML2 fusions. This study will characterise these unique tumours. Ten MEC were collected arising from the parotid gland (n = 4), submandibular gland (n = 2), nasopharynx (n = 1), base of tongue (n = 1), bronchus (n = 1), and trachea (n = 1). Six tumours were low-grade, two intermediate-grade, one high-grade, and one demonstrated low-grade areas with high-grade transformation. Four cases were oncocytic, four had clear-cell features, two had spindle cell features, and one high-grade MEC had prominent solid, cord-like, and micropapillary features. The tumours were negative for p40 (10/10), p63 (10/10), and CK5/6 (9/9). Targeted RNA sequencing demonstrated CRTC1::MAML2 in five cases, CRTC3::MAML2 in two, and a novel MAML2::CEP126 in the unusual high-grade case. In two cases with insufficient RNA, MAML2 fluorescence in situ hybridisation (FISH) showed rearrangement. Genetically-confirmed MEC may lack overt squamous differentiation by histology and immunohistochemistry. While most cases harboured canonical fusions and fit within the spectra of MEC variants with oncocytic, clear cell, and/or spindle cell features, one had a novel MAML2::CEP126 fusion and unusual morphology. In MEC without squamoid cells, the use of immunohistochemistry may hinder, rather than aid, the correct diagnosis. In such cases, MAML2 analysis is most useful. The historical definition of MEC as a carcinoma with squamoid, intermediate and mucous cells should be revisited.

Discrepancies Between Clinical and Anatomic Causes of Death in Acute Invasive Fungal Sinusitis: An Institutional Experience at a Tertiary Care Center.

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.

Regional lymph node irradiation in locally advanced Merkel cell carcinoma reduces regional and distant relapse and improves disease-specific survival.

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.

The Decline of Salivary Adenocarcinoma Not Otherwise Specified as a Tumor Entity: Reclassification Using Contemporary Immunohistochemical Profiling and Diagnostic Criteria.

The classification of salivary gland carcinomas has become increasingly specific over the last decade with the definition of new tumor types, documentation of novel molecular and immunohistochemical findings, and development of more refined diagnostic criteria. In this setting, it is unclear how many salivary tumors still cannot be easily categorized-and whether such tumors represent undifferentiated malignancies or include additional definable entities. Relying largely on current classification schemes and contemporary immunohistochemical panels, we reassessed salivary tumors previously diagnosed as adenocarcinoma, not otherwise specified (ACA NOS) from 2 large academic medical centers. Fifty-seven ACA NOS (72%) could be reclassified as more specific entities including 31 salivary duct carcinomas (39%), 7 polymorphous adenocarcinomas (9%), 5 epithelial-myoepithelial carcinomas (6%), 4 myoepithelial carcinomas (5%), 4 secretory carcinomas (5%), 1 acinic cell carcinoma (1%), 1 basal cell adenocarcinoma (1%), 1 intraductal carcinoma (1%), and 1 clear cell carcinoma (1%) as well as 2 metastatic squamous cell carcinomas (3%). Of reclassified cases, 21 (37%) represented variant histologies within these categories. ACA NOS comprised 11% of salivary malignancies before reclassification, but only 4% after reclassification. The remaining 22 ACA NOS demonstrated heterogeneous features, with an association between histologic grade and clinical outcome. In effect, ACA NOS is becoming a bygone entity as modern classification schemes and ancillary techniques now permit more specific typing of a majority of these tumors, potentially facilitating more specific prognostication and treatment. Additional distinctive entities such as mucinous adenocarcinoma may still be definable within the ACA NOS category.

Hemagglutinin Receptor Binding of a Human Isolate of Influenza A(H10N8) Virus.

Three cases of influenza A(H10N8) virus infection in humans have been reported; 2 of these infected persons died. Characterization of the receptor binding pattern of H10 hemagglutinin from avian and human isolates showed that both interact weakly with human-like receptors and maintain strong affinity for avian-like receptors.

Enhancement of the proapoptotic properties of newcastle disease virus promotes tumor remission in syngeneic murine cancer models.

Newcastle disease virus (NDV) is considered a promising agent for cancer therapy due to its oncolytic properties. These include preferential replication in transformed cells, induction of innate and adaptive immune responses within tumors, and cytopathic effects in infected tumor cells due to the activation of apoptosis. To enhance the latter and thus possibly enhance the overall oncolytic activity of NDV, we generated a recombinant NDV encoding the human TNF receptor Fas (rNDV-B1/Fas). rNDV-B1/Fas replicates to similar titers as its wild-type (rNDV-B1) counterpart; however, overexpression of Fas in infected cells leads to higher levels of cytotoxicity correlated with faster and increased apoptosis responses, in which both the intrinsic and extrinsic pathways are activated earlier. Furthermore, in vivo studies in syngeneic murine melanoma models show an enhancement of the oncolytic properties of rNDV-B1/Fas, with major improvements in survival and tumor remission. Altogether, our data suggest that upregulation of the proapoptotic function of NDV is a viable approach to enhance its antitumor properties and adds to the currently known, rationally based strategies to design optimized therapeutic viral vectors for the treatment of cancer.

Localized oncolytic virotherapy overcomes systemic tumor resistance to immune checkpoint blockade immunotherapy.

Preexisting lymphocytic infiltration of tumors is associated with superior prognostic outcomes in a variety of cancers. Recent studies also suggest that lymphocytic responses may identify patients more likely to benefit from therapies targeting immune checkpoints, suggesting that therapeutic efficacy of immune checkpoint blockade can be enhanced through strategies that induce tumor inflammation. To achieve this effect, we explored the immunotherapeutic potential of oncolytic Newcastle disease virus (NDV). We find that localized intratumoral therapy of B16 melanoma with NDV induces inflammatory responses, leading to lymphocytic infiltrates and antitumor effect in distant (nonvirally injected) tumors without distant virus spread. The inflammatory effect coincided with distant tumor infiltration with tumor-specific CD4(+) and CD8(+) T cells, which was dependent on the identity of the virus-injected tumor. Combination therapy with localized NDV and systemic CTLA-4 blockade led to rejection of preestablished distant tumors and protection from tumor rechallenge in poorly immunogenic tumor models, irrespective of tumor cell line sensitivity to NDV-mediated lysis. Therapeutic effect was associated with marked distant tumor infiltration with activated CD8(+) and CD4(+) effector but not regulatory T cells, and was dependent on CD8(+) cells, natural killer cells, and type I interferon. Our findings demonstrate that localized therapy with oncolytic NDV induces inflammatory immune infiltrates in distant tumors, making them susceptible to systemic therapy with immunomodulatory antibodies, which provides a strong rationale for investigation of such combination therapies in the clinic.

Oncolytic specificity of Newcastle disease virus is mediated by selectivity for apoptosis-resistant cells.

Newcastle disease virus (NDV) is a negative-sense RNA virus that has been shown to possess oncolytic activity. NDV's selective replication in tumor cells has been previously suggested to be due to the lack of a proper antiviral response in these cells. Here we demonstrate that NDV possesses oncolytic activity in tumor cells capable of a robust type I interferon (IFN) response, suggesting that another mechanism underlies NDV's tumor specificity. We show that the oncolytic selectivity of NDV for tumor cells is dependent upon tumor cell resistance to apoptosis. Utilizing the human non-small-cell lung cancer cell line A549 overexpressing the antiapoptotic protein Bcl-xL, we show significant enhancement of oncolytic activity and NDV replication. Interestingly, while the Bcl-xL-overexpressing cells were resistant to apoptotic stimuli induced by chemotherapeutic agents and early viral replication, during the subsequent viral cycles, we observed a paradoxical increase in apoptosis in response to NDV. The increased oncolytic activity seen was secondary to enhanced viral replication and syncytium formation. The induction of a type I IFN response was enhanced in Bcl-xL cells. Overall, these findings propose a new mechanism for cancer cell specificity for NDV, making it an attractive anticancer agent for chemoresistant tumors with enhanced antiapoptotic activity.

Enhancement of oncolytic properties of recombinant newcastle disease virus through antagonism of cellular innate immune responses.

Newcastle disease virus (NDV) has been previously shown to possess oncolytic activity, causing specific lysis of cancerous but not normal cells. Here we show that despite these findings, the oncolytic efficiency of naturally occurring NDV strains can still be relatively low, as many tumors exhibit strong innate immune responses that suppress viral replication and spread. To overcome this problem, we generated a recombinant fusogenic NDV expressing influenza NS1 protein, a protein exhibiting interferon (IFN)-antagonist and antiapoptotic functions in human and mouse cells. Interestingly, the resultant virus was dramatically enhanced in its ability to form syncytia, lyse a variety of human and mouse tumor cell lines, and suppressed the induction of the cellular IFN responses. Using the aggressive syngeneic murine melanoma model, we show that the NDV-NS1 virus is more effective than virus not expressing NS1 in clearing the established footpad tumors and results in higher overall long-term animal survival. In addition, mice treated with NDV-NS1 exhibited no signs of toxicity to the virus and developed tumor-specific cytotoxic T lymphocyte (CTL) responses. These findings demonstrate that modulation of innate immune responses by NDV results in enhancement of its oncolytic properties and warrant further investigation of this strategy in design of oncolytic NDV vectors against human tumors.

Twist is transcriptionally induced by activation of STAT3 and mediates STAT3 oncogenic function.

To explore the basis of metastasis, we compared the human breast cancer lines MCF-7 and MDA-MB453, which have low invasive ability, with their sublines MCF7-I4 and MDA-MB453-I4 with high invasive ability for gene expression and signaling pathways. We previously showed that the I4 lines had dramatically elevated levels of Twist compared with their parental lines. In this study, we observed significantly increased STAT3 Tyr(705) phosphorylation, but not the STAT3 protein levels, in the I4 lines. Activation of STAT3 by interleukin-6 or expression of activated Src induced Twist expression at protein and mRNA levels. Inhibiting STAT3 by a small molecule inhibitor, JSI-124, STAT3 small hairpin RNAs, or dominant negative STAT3 resulted in significant reduction of Twist protein and mRNA expression. STAT3 directly bound to the second proximal STAT3-binding site on the human Twist promoter and activated its transcriptional activity. Inhibition of STAT3 reduced migration, invasion, and colony formation of the I4 cells. Ectopic expression of Twist significantly rescued those phenotypes. Ten normal and 46 tumor specimens of breast tissues were examined for activation of STAT3 and expression of Twist. There was a strong correlation between Tyr(705) p-STAT3 and Twist level in the late stage tumor tissues. Our results indicate that activated STAT3 transcriptionally induces Twist, which plays an important role in promoting migration, invasion, and anchorage-independent growth. Together with our previous observation that Twist transcriptionally induces AKT2 to mediate Twist-promoted oncogenic functions, we conclude that STAT3, Twist, and AKT2 form a functional signaling axis to regulate pivotal oncogenic properties of cancer cells.