Molecular characteristics and biological behaviours of the oncocytic and pancreatobiliary subtypes of intraductal papillary mucinous neoplasms.

Intraductal papillary mucinous neoplasm (IPMN) consists of four epithelial subtypes. Of those, pancreatobiliary and oncocytic types are recently recognized and relatively uncommon, and usually exhibit high-grade dysplasia. The biological properties and molecular characteristics of these two types have not been well documented. The few molecular studies of the oncocytic type showed absence of KRAS mutations commonly seen in the other subtypes, raising the possibility that the oncocytic type is distinct from the other subtypes. Thus, we examined clinicopathological features and molecular alterations of the two subtypes. The study cohort consisted of 12 pancreatobiliary and 18 oncocytic IPMN cases. KRAS, BRAF, and PIK3CA mutations and TP53, SMAD4, and ß-catenin expression were analysed, and the results of molecular and clinicopathological profiles were compared between the two subtypes. KRAS mutations were identified in the oncocytic type, but less frequently than the pancreatobiliary type (17% versus 58%, p = 0.048). BRAF mutation was found in a single oncocytic tumour, and no PIK3CA mutations were seen in any of the study cohort. TP53 overexpression was less frequent in the oncocytic type than in the pancreatobiliary type (11% versus 58%, p = 0.013). Invasive components were present in 50% of the oncocytic and 92% of the pancreatobiliary types, with lymph node metastasis more frequently seen in the latter, corresponding to better outcomes in the former (5-year survival rates: 93% versus 32%, p = 0.014). Our demonstration of KRAS and BRAF mutations in the oncocytic-type IPMN supports a role for the activation of the RAS-MAPK pathway in this tumour type. However, the less frequent TP53 overexpression associated with the significantly lower rates of invasion and nodal disease in the oncocytic type correlates with better outcomes compared to the pancreatobiliary type.

Uncoupled cardiac nitric oxide synthase mediates diastolic dysfunction.

BACKGROUND: Heart failure with preserved ejection fraction is 1 consequence of hypertension and is caused by impaired cardiac diastolic relaxation. Nitric oxide (NO) is a known modulator of cardiac relaxation. Hypertension can lead to a reduction in vascular NO, in part because NO synthase (NOS) becomes uncoupled when oxidative depletion of its cofactor tetrahydrobiopterin (BH(4)) occurs. Similar events may occur in the heart that lead to uncoupled NOS and diastolic dysfunction. METHODS AND RESULTS: In a hypertensive mouse model, diastolic dysfunction was accompanied by cardiac oxidation, a reduction in cardiac BH(4), and uncoupled NOS. Compared with sham-operated animals, male mice with unilateral nephrectomy, with subcutaneous implantation of a controlled-release deoxycorticosterone acetate pellet, and given 1% saline to drink were mildly hypertensive and had diastolic dysfunction in the absence of systolic dysfunction or cardiac hypertrophy. The hypertensive mouse hearts showed increased oxidized biopterins, NOS-dependent superoxide production, reduced NO production, and dephosphorylated phospholamban. Feeding hypertensive mice BH(4) (5 mg/d), but not treating with hydralazine or tetrahydroneopterin, improved cardiac BH(4) stores, phosphorylated phospholamban levels, and diastolic dysfunction. Isolated cardiomyocyte experiments revealed impaired relaxation that was normalized with short-term BH(4) treatment. Targeted cardiac overexpression of angiotensin-converting enzyme also resulted in cardiac oxidation, NOS uncoupling, and diastolic dysfunction in the absence of hypertension. CONCLUSIONS: Cardiac oxidation, independently of vascular changes, can lead to uncoupled cardiac NOS and diastolic dysfunction. BH(4) may represent a possible treatment for diastolic dysfunction.

Angiotensin-converting enzyme N-terminal inactivation alleviates bleomycin-induced lung injury.

Bleomycin has potent anti-oncogenic properties for several neoplasms, but drug administration is limited by bleomycin-induced lung fibrosis. Inhibition of the renin-angiotensin system has been suggested to decrease bleomycin toxicity, but the efficacy of such strategies remains uncertain and somewhat contradictory. Our hypothesis is that, besides angiotensin II, other substrates of angiotensin-converting enzyme (ACE), such as the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), play a significant role in controlling fibrosis. We studied bleomycin-induced lung injury in normotensive mice, termed N-KO and C-KO, which have point mutations inactivating either the N- or C-terminal catalytic sites of ACE, respectively. N-KO, but not C-KO mice, have a marked resistance to bleomycin lung injury as assessed by lung histology and hydroxyproline content. To determine the importance of the ACE N-terminal peptide substrate AcSDKP in the resistance to bleomycin injury, N-KO mice were treated with S-17092, a prolyl-oligopeptidase inhibitor that inhibits the formation of AcSDKP. In response to bleomycin injection, S-17092-treated N-KO mice developed lung fibrosis similar to wild-type mice. In contrast, the administration of AcSDKP to wild-type mice reduced lung fibrosis due to bleomycin administration. This study shows that the inactivation of the N-terminal catalytic site of ACE significantly reduced bleomycin-induced lung fibrosis and implicates AcSDKP in the mechanism of protection. These data suggest a possible means to increase tolerance to bleomycin and to treat fibrosing lung diseases.

Transoral robotic surgery and neck dissection for HPV-positive oropharyngeal carcinoma: Importance of nodal count in survival.

BACKGROUND: In this study we determine the survival in patients with HPV-positive oropharyngeal carcinoma treated with transoral robotic surgery (TORS), neck dissection and risk-adapted adjuvant therapy. METHODS: We retrospectively identified 122 patients with HPV-positive oropharyngeal carcinoma treated with TORS and neck dissection between 2011 and 2018. Survival probability was calculated. We determined the effect of the type of neck dissection performed (modified radical neck dissection-MRND vs. selective neck dissection - SND), extranodal extension (ENE), margin status, and presence of ≥ 5 metastatic nodes on survival. RESULTS: Our patient population had a five-year overall survival of 91.0% (95% C.I. 85-97%). The five-year probability of recurrence or cancer-associated death was 0.0977 (95% C.I. 0.0927-0.1027). The five-year probability of cancer-associated death was 0.0528 (95% C.I. 0.048-0.0570). All patients who died of their disease had distant metastasis. Our PEG dependence rate was 0%. Patients with ENE and positive margins who underwent adjuvant chemoradiation did not have worse survival. Presence of ≥ 5 metastatic nodes portended worse survival after controlling for age, positive ENE and margins. Low yield (<18 nodes) on neck dissection worsened DFS on multivariable analysis. Furthermore, patients who underwent SND did not have worse OS than those who underwent MRND. CONCLUSION: Our study demonstrates that surgery could be simplified by performing TORS with SND rather than MRND. The one true poor prognostic factor in HPV-positive oropharyngeal carcinoma patients who undergo surgery is high nodal burden. Patients with high nodal burden are much more likely to die from their disease.

New insights into the role of angiotensin-converting enzyme obtained from the analysis of genetically modified mice.

Angiotensin-converting enzyme (ACE) has been well-recognized for its role in blood pressure regulation. ACE is made by many tissues, though it is most abundantly expressed on the luminal surface of vascular endothelium. ACE knockout mice show a profound phenotype with low blood pressure, but also with hemopoietic and developmental defects, which complicates understanding the biological functions of ACE in individual tissue types. Using a promoter-swapping strategy, several mouse lines with unique ACE tissue expression patterns were studied. These include mice with ACE expression in the liver (ACE 3/3), the heart (ACE 8/8), and macrophages (ACE 10/10). We also investigated mice with a selective inactivation of either the N- or C-terminal ACE catalytic domain. Our studies indicate that ACE plays a role in many other physiologic processes beyond simple blood pressure control.

Genetic models provide unique insight into angiotensin and bradykinin peptides in the extravascular compartment of the heart in vivo.

1. There is continuing uncertainty about the tissue compartments where angiotensin and bradykinin peptide formation occurs. Mice with angiotensin-converting enzyme (ACE) expression targeted to the cardiomyocyte membrane provide a unique experimental model to detect ACE substrates in the extravascular compartment of the heart in vivo. 2. Angiotensin (Ang) I and II, bradykinin-(1-7) and bradykinin-(1-9) were measured in blood and cardiac ventricles of wild-type (WT) mice, mice with a non-functional somatic ACE gene promoter (KO), mice homozygous (8/8) and heterozygous (1/8) for cardiomyocyte-targeted ACE expression and a non-functional somatic ACE gene promoter, and mice heterozygous for cardiomyocyte-targeted ACE expression and heterozygous for the WT ACE allele (WT/8). 3. Cardiac AngII levels of 8/8, 1/8, WT/8 and WT mice were higher than KO levels. Cardiac AngII levels in 8/8 and 1/8 mice were also higher than WT levels, but the levels in WT/8 mice were similar to WT levels. Cardiac bradykinin-(1-9) levels of WT, but not 8/8 mice, were lower than in KO mice, whereas bradykinin-(1-7) levels in 8/8 mice were lower than in KO mice. 4. We conclude that AngI and bradykinin-(1-7) are present in the cardiac extravascular compartment of mice lacking vascular ACE and that extravascular ACE produces AngII and metabolises bradykinin-(1-7) in this compartment. The data suggest that the vascular compartment is the main site of AngI and bradykinin-(1-9) formation and metabolism and that vascular ACE may limit AngI entry to the extravascular compartment of WT mice.

Tissue specific expression of angiotensin converting enzyme: a new way to study an old friend.

Angiotensin-converting enzyme (ACE) plays a central role in blood pressure regulation by producing the vasoconstrictor angiotensin II. When ACE knockout mice were studied, they presented with a complicated phenotype, including cardiovascular, reproductive, hematologic and developmental defects. The complexity of an ACE knockout mouse emphasizes the advantages and disadvantages of the classic knockout strategy. An animal lacking all ACE is very different from a wild type animal, and can be modeled as representing an extreme phenotype. To understand the role of ACE in a tissue and organ specific fashion, our group used targeted homologous recombination to create mouse models in which a promoter swapping strategy results in very restricted tissue patterns of ACE expression. Mice with ACE expression only in the heart, termed ACE 8/8 mice, present with atria enlargement and electrical conduction defects, but normal ventricular function. Mice with ACE expression only in monocytes and macrophages, termed ACE 10/10 mice, have a marked resistance to the growth of melanoma due to an enhanced immune response characterized by increased tumor specific CD8+ T cells and increased proinflammatory cytokines. These mice may define a new means of augmenting the immune response, potentially useful in human clinical situations. The promoter swapping strategy permits scientific investigation of questions unapproachable by other experimental approaches.

P16-positive cystic squamous cell carcinoma in midline neck: metastasis from oropharynx or primary carcinoma arising from thyroglossal duct cyst?

Cystic squamous cell carcinoma (SCC) of the lateral neck is considered metastatic human papilloma-virus (HPV)-related oropharyngeal SCC (HPV-OPSCC) until proven otherwise. P16 immunohistochemistry is diffusely positive in those carcinomas and is used as a surrogate marker of active human papillomavirus (HPV) infection. Thyroglossal duct cysts (TDC) are one of the differential diagnoses for cystic neck lesions. SCC arising from TDC is extremely rare. In this study, we report a p16-positive cystic SCC located in the midline neck. Radiologic features and the presence of thyroid tissue in the cyst wall indicated that it was a TDC. The morphologic features of the lesion raised the question: is the carcinoma metastatic HPV-OPSCC? The HPV confirmative test, high-risk HPV RNA in situ hybridization, was negative. We then studied p16 immunohistochemistry in the squamous epithelium of benign TDC and found that rare benign TDC can show diffuse and strong p16 positivity.

Correction: STING expression and response to treatment with STING ligands in premalignant and malignant disease.

[This corrects the article DOI: 10.1371/journal.pone.0187532.].

ACE and ACE2 activity in diabetic mice.

ACE-related carboxypeptidase (ACE2) may counterbalance the angiotensin (ANG) II-promoting effects of ACE in tissues where both enzymes are found. Alterations in renal ACE and ACE2 expression have been described in experimental models of diabetes, but ACE2 activity was not assessed in previous studies. We developed a microplate-based fluorometric method for the concurrent determination of ACE and ACE2 activity in tissue samples. Enzymatic activity (relative fluorescence unit [RFU] . microg protein(-1) . h(-1)) was examined in ACE and ACE2 knockout mice and in two rodent models of diabetes, the db/db and streptozotocin (STZ)-induced diabetic mice. In kidney cortex, preparations consisting mainly of proximal tubules and cortical collecting tubules, ACE2 activity had a strong positive correlation with ACE2 protein expression (90-kDa band) in both knockout models and their respective wild-type littermates (r = 0.94, P < 0.01). ACE activity, likewise, had a strong positive correlation with renal cortex ACE protein expression (170-kDa band) (r = 0.838, P < 0.005). In renal cortex, ACE2 activity was increased in both models of diabetes (46.7 +/- 4.4 vs. 22.0 +/- 4.7 in db/db and db/m, respectively, P < 0.01, and 22.1 +/- 2.8 vs. 13.1 +/- 1.5 in STZ-induced diabetic versus untreated mice, respectively, P < 0.05). ACE2 mRNA levels in renal cortex from db/db and STZ-induced diabetic mice, by contrast, were not significantly different from their respective controls. In cardiac tissue, ACE2 activity was lower than in renal cortex, and there were no significant differences between diabetic and control mice (db/db 2.03 +/- 0.23 vs. db/m 1.85 +/- 0.10; STZ-induced diabetic 0.42 +/- 0.04 vs. untreated 0.52 +/- 0.07 mice). ACE2 activity in renal cortex correlated positively with ACE2 protein in db/db and db/m mice (r = 0.666, P < 0.005) as well as in STZ-induced diabetic and control mice (r = 0.621, P < 0.05) but not with ACE2 mRNA (r = -0.468 and r = -0.522, respectively). We conclude that in renal cortex from diabetic mice, ACE2 expression is increased at the posttranscriptional level. The availability of an assay for concurrent measurement of ACE and ACE2 activity should be helpful in the evaluation of kidney-specific alterations in the balance of these two carboxypeptidases, which are involved in the control of local ANG II formation and degradation.

Margin Analysis: Squamous Cell Carcinoma of the Oropharynx.

Because of the common shared risk factors of smoking and heavy alcohol consumption, literature involving oropharyngeal squamous cell carcinoma (OPSCC) is often combined with oral squamous cell carcinoma. Human papilloma virus is now confirmed to be a major risk factor of OPSCC with its distinct epidemiology and favorable treatment outcome. The impact of adjuvant chemoradiation in the setting of positive surgical margins remains unclear but is likely influenced by tumor biology. This article reviews the tumor biology of OPSCC and summarizes recent findings on outcomes following surgical treatment of OPSCC.

STING expression and response to treatment with STING ligands in premalignant and malignant disease.

Human papilloma virus positive (HPV+) tumors represent a large proportion of anal, vulvar, vaginal, cervical and head and neck squamous carcinomas (HNSCC) and late stage invasive disease is thought to originate from a premalignant state. Cyclic dinucleotides that activate STimulator of INterferon Genes (STING) have been shown to cause rapid regression of a range of advanced tumors. We aimed to investigate STING ligands as a novel treatment for papilloma. We tested therapies in a spontaneous mouse model of papilloma of the face and anogenital region that histologically resembles human HPV-associated papilloma. We demonstrate that STING ligands cause rapid regression of papilloma, associated with T cell infiltration, and are significantly more effective than Imiquimod, a current immunotherapy for papilloma. In humans, we show that STING is expressed in the basal layer of normal skin and lost during keratinocyte differentiation. We found STING was expressed in all HPV-associated cervical and anal dysplasia and was strongly expressed in the cancer cells of HPV+ HNSCC but not in HPV-unrelated HNSCC. We found no strong association between STING expression and progressive disease in non-HPV oral dysplasia and oral pre-malignancies that are not HPV-related. These data demonstrate that STING is expressed in basal cells of the skin and is retained in HPV+ pre-malignancies and advanced cancers, but not in HPV-unrelated HNSCC. However, using a murine HNSCC model that does not express STING, we demonstrate that STING ligands are an effective therapy regardless of expression of STING by the cancer cells.

Circulating versus local angiotensin II in blood pressure control: lessons from tissue-specific expression of angiotensin-converting enzyme (ACE).

The renin angiotensin system (RAS) is a central player in blood pressure control. Its effector peptide, angiotensin II, regulates blood pressure through coordinated actions in multiple tissues. The RAS is generally considered to be an endocrine system, and angiotensin II to be a circulating hormone. In recent years, however, a role for locally produced angiotensin II has been proposed. The major site for angiotensin II production is endothelium, where angiotensin-converting enzyme (ACE) is abundantly expressed. To elucidate the relative importance of circulating angiotensin II versus locally produced angiotensin II, one approach is to create a mouse model in which ACE is expressed in a tissue-specific manner. In this review, we discuss strategies to create such a model. In a mouse model we generated using a novel promoter-swapping technique, the endothelial ACE is eliminated and replaced by ectopic production of ACE in the liver. This model specifically addresses the question of whether local production of angiotensin II is essential for RAS function.

Circulating and intratumoral macrophages in patients with hepatocellular carcinoma: correlation with therapeutic approach.

BACKGROUND: Hepatocellular carcinoma arises in an environment of chronic injury, and wound-healing responses may vary by treatment. METHODS: Peripheral blood myeloid populations were quantified in 39 patients with hepatocellular carcinoma treated with surgical or endoluminal therapy. Macrophages were quantified in tissue when available. RESULTS: There was a similar expansion of myeloid populations after operative procedures compared with endoluminal treatments. Immunostaining for CD68 revealed no significant differences in the number of macrophages within benign versus malignant tumors and when tumors were compared with nontumor liver. Cytotoxic CD8+ T cells were rare within tumors compared with the surrounding liver (P < .0001). Progression-free survival was reduced in patients with preoperative peripheral blood monocyte expansion (P < .05). CONCLUSIONS: These data provide preliminary evidence of poor prognostic significance of elevated peripheral blood monocyte counts. We propose that the inflammatory environment of hepatocellular carcinoma may represent a consistent feature to both predict and alter the course of disease.

Inhibition of renin-angiotensin system (RAS) reduces ventricular tachycardia risk by altering connexin43.

Renin-angiotensin system (RAS) activation is associated with arrhythmias. We investigated the effects of RAS inhibition in cardiac-specific angiotensin-converting enzyme (ACE) overexpression (ACE 8/8) mice, which exhibit proclivity to ventricular tachycardia (VT) and sudden death because of reduced connexin43 (Cx43). ACE 8/8 mice were treated with an ACE inhibitor (captopril) or an angiotensin receptor type-1 blocker (losartan). Subsequently, electrophysiological studies were performed, and the hearts were extracted for Cx43 quantification using immunoblotting, immunohistochemistry, fluorescent dye spread method, and sodium current quantification using whole cell patch clamping. VT was induced in 12.5% of captopril-treated ACE 8/8 and in 28.6% of losartan-treated mice compared to 87.5% of untreated mice (P < 0.01). Losartan and captopril treatment increased total Cx43 2.4-fold (P = 0.01) and the Cx43 phosphorylation ratio 2.3-fold (P = 0.005). Treatment was associated with a recovery of gap junctional conductance. Survival in treated mice improved to 0.78 at 10 weeks (95% confidence interval 0.64 to 0.92), compared to the expected survival of less than 0.50. In a model of RAS activation, arrhythmic risk was correlated with reduced Cx43 amount and phosphorylation. RAS inhibition resulted in increased total and phosphorylated Cx43, decreased VT inducibility, and improved survival.

Mice expressing ACE only in the heart show that increased cardiac angiotensin II is not associated with cardiac hypertrophy.

In the heart, angiotensin II has been suggested to regulate cardiac remodeling and promote cardiac hypertrophy. To examine this, we studied compound heterozygous mice, called angiotensin-converting enzyme (ACE) 1/8, in which one ACE allele is null, whereas the other ACE allele (the 8 allele) targets expression to the heart. In this model, cardiac ACE levels are about 15 times those of wild-type mice, and ACE expression is reduced or eliminated in other tissues. ACE 1/8 mice have 58% the cardiac ACE of a previous model, called ACE 8/8, but both ACE 1/8 and ACE 8/8 mice have ventricular angiotensin II levels about twofold those of wild-type controls. Despite equivalent levels of cardiac angiotensin II, ACE 1/8 mice do not develop the marked atrial enlargement or the conduction defects previously reported in the ACE 8/8 mice. Six-month-old ACE 1/8 mice have normal cardiac function, as determined by echocardiography and left ventricular catheterization, despite the elevated levels of angiotensin II. ACE 1/8 mice also have normal levels of connexin 43. Both wild-type and ACE 1/8 mice develop similar degrees of cardiac hypertrophy after aortic banding. These data suggest that a moderate increase of local angiotensin II production in the heart does not produce cardiac dysfunction, at least under basal conditions, and that, in response to aortic banding, cardiac hypertrophy is not augmented by a twofold increase of cardiac angiotensin II.

Angiotensin-converting enzyme C-terminal catalytic domain is the main site of angiotensin I cleavage in vivo.

Angiotensin-converting enzyme (ACE) plays a central role in the production of the vasoconstrictor angiotensin II. ACE is a single polypeptide, but it contains 2 homologous and independent catalytic domains, each of which binds zinc. To understand the in vivo role of these 2 domains, we used gene targeting to create mice with point mutations in the ACE C-domain zinc-binding motif. Such mice, termed ACE13/13, produce a full-length ACE protein with tissue expression identical to wild-type mice. Analysis of ACE13/13 mice showed that they produce ACE having only N-domain catalytic activity, as determined by the hydrolysis of domain specific substrates and by chloride sensitivity. ACE13/13 mice have blood pressure and blood angiotensin II levels similar to wild-type mice. However, plasma renin concentration is increased 2.6-fold and blood angiotensin I levels are increased 7.5-fold. Bradykinin peptide levels are not different from wild-type levels. ACE13/13 mice have a reduced increase of blood pressure after intravenous infusion of angiotensin I. ACE13/13 mice have a normal renal structure, but they are not able to concentrate urine after dehydration as effectively as wild-type mice. This study shows that the C-domain of ACE is the predominant site of angiotensin I cleavage in vivo. Although mice lacking C-domain activity have normal physiology under laboratory conditions, they respond less well to the stress of dehydration.