Serine protease inhibitors decrease metastasis in prostate, breast, and ovarian cancers.

Targeted therapies for prostate, breast, and ovarian cancers are based on their activity against primary tumors rather than their anti-metastatic activity. Consequently, there is an urgent need for new agents targeting the metastatic process. Emerging evidence correlates in vitro and in vivo cancer invasion and metastasis with increased activity of the proteases mesotrypsin (prostate and breast cancer) and kallikrein 6 (KLK6; ovarian cancer). Thus, mesotrypsin and KLK6 are attractive putative targets for therapeutic intervention. As potential therapeutics for advanced metastatic prostate, breast, and ovarian cancers, we report novel mesotrypsin- and KLK6-based therapies, based on our previously developed mutants of the human amyloid ß-protein precursor Kunitz protease inhibitor domain (APPI). These mutants, designated APPI-3M (prostate and breast cancer) and APPI-4M (ovarian cancer), demonstrated significant accumulation in tumors and therapeutic efficacy in orthotopic preclinical models, with the advantages of long retention times in vivo, high affinity and favorable pharmacokinetic properties. The applicability of the APPIs, as a novel therapy and for imaging purposes, is supported by their good safety profile and their controlled and scalable manufacturability in bioreactors.

Functional binding of PD1 ligands predicts response to anti-PD1 treatment in patients with cancer.

Accurate predictive biomarkers of response to immune checkpoint inhibitors (ICIs) are required for better stratifying patients with cancer to ICI treatments. Here, we present a new concept for a bioassay to predict the response to anti-PD1 therapies, which is based on measuring the binding functionality of PDL1 and PDL2 to their receptor, PD1. In detail, we developed a cell-based reporting system, called the immuno-checkpoint artificial reporter with overexpression of PD1 (IcAR-PD1) and evaluated the functionality of PDL1 and PDL2 binding in tumor cell lines, patient-derived xenografts, and fixed-tissue tumor samples obtained from patients with cancer. In a retrospective clinical study, we found that the functionality of PDL1 and PDL2 predicts response to anti-PD1 and that the functionality of PDL1 binding is a more effective predictor than PDL1 protein expression alone. Our findings suggest that assessing the functionality of ligand binding is superior to staining of protein expression for predicting response to ICIs.

Voltage-Dependent Anion Channel 1 Expression in Oral Malignant and Premalignant Lesions.

BACKGROUND: The voltage-dependent anion channel 1 protein (VDAC1) plays a role in cellular metabolism and survival. It was found to be down or upregulated (overexpressed) in different malignancies but it was never studied in application to oral lesions. The purpose of this study was to retrospectively evaluate the expression of VDAC1 in biopsies of oral premalignant, malignant, and malignancy-neutral lesions and to examine the possible correlations to their clinicopathological parameters. MATERIALS AND METHODS: 103 biopsies including 49 oral squamous cell carcinoma, 33 epithelial dysplasia, and 21 fibrous hyperplasia samples were immunohistochemically stained with anti-VDAC1 antibodies for semi-quantitative evaluation. The antibody detection was performed with 3,3'-diaminobenzidine (DAB). The clinicopathological information was examined for possible correlations with VDAC1. RESULTS: VDAC1 expression was lower in oral squamous cell carcinoma 0.63 ± 0.40 and in oral epithelial dysplasia 0.61 ± 0.36 biopsies compared to fibrous hyperplasia biopsies 1.45 ± 0.28 (p < 0.01 for both; Kruskal-Wallis test). CONCLUSION: Oral squamous cell carcinoma and epithelial dysplasia tissues demonstrated decreased VDAC1 protein expression if compared to fibrous hyperplasia samples, but were not different from each other, suggesting that the involvement of VDAC1 in oral carcinogenesis is an early stage event, regulating cells to live or die.

Efficacy of Ultrasound Shear Wave Elastography in the Diagnosis of Salivary Gland Tumors.

PURPOSE: Diagnostic accuracy of fine-needle aspiration cytology (FNAC) to discriminate between the target condition and health in the evaluation of salivary gland tumors is not perfected yet and thus, false-negative results are possible. The purpose of the present study was to measure and compare the diagnostic accuracy of FNAC performed with conventional B-mode ultrasound and ultrasound with shear wave elastography (SWE) FNAC navigation. METHODS: The investigators implemented a single-blind randomized study (sealed envelope method). The study population was composed of all patients presenting for evaluation and management of suspected benign or malignant tumors of the major salivary glands between July 2013 and December 2020. The involvement of SWE navigation was the primary predictor variable affecting FNA targeting. The method involved analysis of redistribution of SWE values within the affected gland expressed in kilopascals (kPa) and the four-point ES1 (soft tissue) to ES4 (stiff) scoring. The primary outcome variable was the success in obtaining diagnostic tissue resulting in a histologically confirmed FNAC diagnosis and coded as yes/no. Age and sex of the patients and topographical locations of lesions were covariates. Descriptive and bivariate statistics were computed and the P value was set at .05. RESULTS: The sample included 132 subjects (male/female 59/73; mean age 54 ± 11 years; 144 tumors). SWE + Group (n = 66) consisted of patients presurgically diagnosed with salivary tumors SWE-guided FNAC and SWE- Group (n = 66) was diagnosed with tumors by conventional ultrasound-(B-mode)-guided FNAC. The SWE-guided FNAC statistically significantly reduced the incidence of false-negative results (n = 0; P = .001) and nondiagnostic cases (n = 3 SWE FNAC vs n = 7 B-mode US FNAC; P = .04). For SWE + Group, the FNAC diagnosis was confirmed by postsurgical histology in 95.5% with 91.0% sensitivity (confidence interval [CI] 0.62 to 0.97) and 84.4% specificity (CI 0.58 to 0.96). For SWE- Group, 81.8% confirmation was obtained (P = .05) with 82.3% sensitivity (CI 0.54 to 0.90) and 74.0% specificity. CONCLUSION: SWE can increase success in obtaining diagnostic tissue when used for FNAC navigation purposes. We suggest combining both SWE and standard B-mode ultrasonography methods when the FNAC procedure is performed.

Mutated HRAS Activates YAP1-AXL Signaling to Drive Metastasis of Head and Neck Cancer.

The survival rate for patients with head and neck cancer (HNC) diagnosed with cervical lymph node (cLN) or distant metastasis is low. Genomic alterations in the HRAS oncogene are associated with advanced tumor stage and metastasis in HNC. Elucidation of the molecular mechanisms by which mutated HRAS (HRASmut) facilitates HNC metastasis could lead to improved treatment options for patients. Here, we examined metastasis driven by mutant HRAS in vitro and in vivo using HRASmut human HNC cell lines, patient-derived xenografts, and a novel HRASmut syngeneic model. Genetic and pharmacological manipulations indicated that HRASmut was sufficient to drive invasion in vitro and metastasis in vivo. Targeted proteomic analysis showed that HRASmut promoted AXL expression via suppressing the Hippo pathway and stabilizing YAP1 activity. Pharmacological blockade of HRAS signaling with the farnesyltransferase inhibitor tipifarnib activated the Hippo pathway and reduced the nuclear export of YAP1, thus suppressing YAP1-mediated AXL expression and metastasis. AXL was required for HRASmut cells to migrate and invade in vitro and to form regional cLN and lung metastases in vivo. In addition, AXL-depleted HRASmut tumors displayed reduced lymphatic and vascular angiogenesis in the primary tumor. Tipifarnib treatment also regulated AXL expression and attenuated VEGFA and VEGFC expression, thus regulating tumor-induced vascular formation and metastasis. Our results indicate that YAP1 and AXL are crucial factors for HRASmut-induced metastasis and that tipifarnib treatment can limit the metastasis of HNC tumors with HRAS mutations by enhancing YAP1 cytoplasmic sequestration and downregulating AXL expression. SIGNIFICANCE: Mutant HRAS drives metastasis of head and neck cancer by switching off the Hippo pathway to activate the YAP1-AXL axis and to stimulate lymphovascular angiogenesis.

MEK1/2 inhibition transiently alters the tumor immune microenvironment to enhance immunotherapy efficacy against head and neck cancer.

BACKGROUND: Although the mitogen-activated protein kinases (MAPK) pathway is hyperactive in head and neck cancer (HNC), inhibition of MEK1/2 in HNC patients has not shown clinically meaningful activity. Therefore, we aimed to characterize the effect of MEK1/2 inhibition on the tumor microenvironment (TME) of MAPK-driven HNC, elucidate tumor-host interaction mechanisms facilitating immune escape on treatment, and apply rationale-based therapy combination immunotherapy and MEK1/2 inhibitor to induce tumor clearance. METHODS: Mouse syngeneic tumors and xenografts experiments were used to analyze tumor growth in vivo. Single-cell cytometry by time of flight, flow cytometry, and tissue stainings were used to profile the TME in response to trametinib (MEK1/2 inhibitor). Co-culture of myeloid-derived suppressor cells (MDSC) with CD8+ T cells was used to measure immune suppression. Overexpression of colony-stimulating factor-1 (CSF-1) in tumor cells was used to show the effect of tumor-derived CSF-1 on sensitivity to trametinib and anti-programmed death- 1 (αPD-1) in mice. In HNC patients, the ratio between CSF-1 and CD8A was measured to test the association with clinical benefit to αPD-1 and αPD-L1 treatment. RESULTS: Using preclinical HNC models, we demonstrated that treatment with trametinib delays HNC initiation and progression by reducing tumor cell proliferation and enhancing the antitumor immunity of CD8+ T cells. Activation of CD8+ T cells by supplementation with αPD-1 antibody eliminated tumors and induced an immune memory in the cured mice. Mechanistically, an early response to trametinib treatment sensitized tumors to αPD-1-supplementation by attenuating the expression of tumor-derived CSF-1, which reduced the abundance of two CSF-1R+CD11c+ MDSC populations in the TME. In contrast, prolonged treatment with trametinib abolished the antitumor activity of αPD-1, because tumor cells undergoing the epithelial to mesenchymal transition in response to trametinib restored CSF-1 expression and recreated an immune-suppressive TME. CONCLUSION: Our findings provide the rationale for testing the trametinib/αPD-1 combination in HNC and highlight the importance of sensitizing tumors to αPD-1 by using MEK1/2 to interfere with the tumor-host interaction. Moreover, we describe the concept that treatment of cancer with a targeted therapy transiently induces an immune-active microenvironment, and supplementation of immunotherapy during this time further activates the antitumor machinery to cause tumor elimination.

Intra-oral Acantholytic Squamous Cell Carcinoma: 55 Cases. Is this Variant more Aggressive?

We aimed to collect and analyze available cases of intraoral acantholytic squamous cell carcinoma (aSCC), that consisted of the authors' cases and cases derived from the existing literature, with an emphasis on the pathological staging and patient outcome. Our research question was whether aSCC is more aggressive than conventional SCC. The literature was searched for documented cases of aSCC involving the intra-oral mucosa, excluding those from the lips and tonsils, and seven new cases were added from our files. The authors compared the obtained aSCC data to existing data for conventional SCC. Fisher Exact or Pearson's χ2 tests were used for categorical variables. Fifty-five cases of intraoral aSCC were reviewed, of which 48 were retrieved from the literature. Analysis of the published cases was reinforced by contacting the authors of all the papers with incomplete data for further clarifications. The most common sites of aSCC were the tongue (24/55) and the maxilla/maxillary gingiva and/or palate (11/55). The overall survival rate was 36/53 (67.9%) with a mean follow-up period of 22 months against 62.5% for conventional SCC (p = 0.6). No statistically significant difference between the two variants of the tumor with respect to the oral cavity was detected. The differences in age, sex, survival rate, staging, and locations were not statistically significant. Based on the available data from 55 cases, there is no evidence to suggest that aSCC is more aggressive than conventional SCC in intraoral cases.

Diffusion Reflection Method for Early Detection of Oral Squamous Cell Carcinoma Specifically Targeted by Circulating Gold-Nanorods Bio-Conjugated to Anti-Epidermal Growth Factor Receptor.

BACKGROUND: Translation of nanomedical developments into clinical application is receiving an increasing interest. However, its use for oral squamous cell carcinoma (OSCC) diagnosis remains limited. We present an advanced nanophotonic method for oral cancer detection, based on diffusion reflection (DR) measurements of gold-nanorods bio-conjugated to anti-epidermal growth factor receptor (C-GNRs) specifically attached to OSCC cells. OBJECTIVE: To investigate in a rat model of oral carcinogenesis the targeting potential of C-GNRs to OSCC by using the DR optical method. MATERIALS AND METHODS: OSCC was induced by the carcinogen 4-nitroquinoline-N-oxide (4NQO). C-GNRs were introduced locally and systemically and DR measurements were recorded from the surface of the rat tongue following illumination with red laser beam. Rats were divided into experimental and control groups. The results were compared with the histologic diagnosis. RESULTS: A total of 75 Wistar-derived rats were enrolled in the study. Local application did not reveal any statistical results. DR measurements following intravenous injection of C-GNRs revealed a significant increase in light absorption in rats with OSCC compare with rats without cancer (p<0.02, sensitivity 100%, specificity 89%). In addition, absorption of light increased significantly in cases of severe dysplasia and cancer (high risk) compared to rats without cancer and rats with mild dysplasia (low risk) (86% sensitivity and 89% specificity, AUC=0.79). CONCLUSION: Combining nanotechnology and nanophotonics for in vivo diagnosis of OSCC serves as additional tier in the translation of advanced nanomedical developments into clinical applications. The presented method shows a promising potential of nanophotonics for oral cancer identification, and provides support for the use of C-GNRs as a selective drug delivery.

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System.

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Oral variant of acantholytic squamous cell carcinoma-Histochemical and immunohistochemical features.

Acantholytic squamous cell carcinoma (ASCC) is an uncommon variant of squamous cell carcinoma (SCC). It is characterized by a combination of typical SCC and pseudoglandular structures, dyskeratotic cells and prominent acantholysis. The purpose of this study was to analyze the histochemical and immunohistochemical characteristics of the intraoral variant of ASCC. Cases of intraoral ASCC were retrieved from the English language literature. Four new cases from our files were added. In total, 35 cases were included and analyzed in this study. The mean age of the patients was 61.5 + 13 years (age range 38-92 years), with a male-to-female ratio of 1.7:1. According to the available data, histochemical and immunohistochemical stains for mucins were found to be consistently negative. E- cadherin, a marker of adherens junctions, was usually reported to be expressed in areas of "typical" (non acantholytic) SCC, but reduced in the acantholytic areas. We examined for the first time the expression of claudin 1, a marker of tight junctions, and found it to be reduced in the acantholytic areas, similar to E-cadherin. Several cases of oral ASCC also expressed vimentin and cytokeratin (CK) 19, markers associated with epithelial-mesenchymal transition. A wide range of non-epithelial markers yielded negative immunoreactions. In conclusion, ASCC is an uncommon variant of squamous cell carcinoma. The acantholytic process appears to involve reduced expression of molecular components of both adherens junctions and tight junctions. These findings could suggest a relation to the epithelial mesenchymal transition process and therefore further studies are needed in order to establish such a link and the subsequent possible impact on the clinical outcome of the patients.

Rare variants of head and neck squamous cell carcinoma -differential immunohistochemical profiles.

We aimed to immunohistochemically characterize the pattern of expression of epithelial markers in rare head and neck squamous cell carcinoma (HNSCC) variants: carcinoma cuniculatum (CC) and adenosquamous carcinoma (ASC). We also present an additional variant of HNSCC with concomitant basaloid and squamous components that has overlapping morphological features with odontogenic and non-odontogenic tumors, which we termed basalo-squamous carcinoma (BSC). The selected markers included CK5/6, p40, CK19, BerEP4, p16 and SOX10. All tumors were CK5/6 and p40 positive. CK19 and BerEP4 were positive in BSC and focally in ASC but negative in CC. p16 was positive in 3 (60%) of the CCs, focally positive in ASC and negative in BSC. SOX10 was negative in all three variants. Our results highlight the plasticity of the lining epithelium revealing differential profiles of immuno-expression of the selected molecular markers, possibly reflecting their diverse histopathogenesis.

Tongue Lumps and Bumps: Histopathological Dilemmas and Clues for Diagnosis.

Exophytic lesions of the tongue encompass a diverse spectrum of entities. These are most commonly reactive, arising in response to local trauma but can also be neoplastic of epithelial, mesenchymal or miscellaneous origin. In most cases, the microscopic examination is likely to provide a straightforward diagnosis. However, some cases can still raise microscopic diagnostic dilemmas, such as conditions that mimic malignancies, benign tumors with overlapping features and anecdotal lesions. A series of "lumps and bumps" of the tongue are presented together with suggested clues that can assist in reaching a correct diagnosis, emphasizing the importance of the clinico-pathological correlations.

Gold nanorods reflectance discriminate benign from malignant oral lesions.

Nanoparticle-based contrast agents have been used as an imaging tool for selectively detecting cancerous processes. We aimed to evaluate the detection sensitivity of reflection measurements of gold nanorods (GNRs) bio-conjugated to anti-epidermal growth factor receptor (GNRs-EGFR) monoclonal antibodies in discriminating benign from premalignant and malignant human oral lesions. Tissue sections incubated with GNRs-EGFR and the reflectance spectrum was measured using hyperspectral microscopy. Reflectance intensity increased with the progression of the disease, lowest in the control group and increasing as the dysplastic changes increase (P<0.001 for linear trend of grade). Intensity was significantly higher in the moderate and severe dysplasias and cancer patients than in the controls and mild dysplasia (t test P=0.0003, Mann-Whitney P<0.0001). The GNRs reflection measurements can discriminate benign and mild dysplastic lesions from the more severe dysplasia and invasive cancer, suggesting an objective, not dependent on the qualification of a technician and with less interpretation errors.

Ethical issues in nanomedicine: Tempest in a teapot?

Nanomedicine offers remarkable options for new therapeutic avenues. As methods in nanomedicine advance, ethical questions conjunctly arise. Nanomedicine is an exceptional niche in several aspects as it reflects risks and uncertainties not encountered in other areas of medical research or practice. Nanomedicine partially overlaps, partially interlocks and partially exceeds other medical disciplines. Some interpreters agree that advances in nanotechnology may pose varied ethical challenges, whilst others argue that these challenges are not new and that nanotechnology basically echoes recurrent bioethical dilemmas. The purpose of this article is to discuss some of the ethical issues related to nanomedicine and to reflect on the question whether nanomedicine generates ethical challenges of new and unique nature. Such a determination should have implications on regulatory processes and professional conducts and protocols in the future.

Localized juvenile spongiotic gingival hyperplasia possibly originates from the junctional gingival epithelium-an immunohistochemical study.

AIMS: To immunohistochemically evaluate the cytokeratin (CK) pattern of expression in localized juvenile spongiotic gingival hyperplasia (LJSGH) as compared with the gingival epithelium (GE). METHODS AND RESULTS: Ten cases of LJSGH were semiquantitatively evaluated for the immunohistochemical pattern of CK1/10, CK4, CK8/18, and CK19. GE controls were taken from 10 cases of reactive gingival fibroepithelial hyperplasia. GEs showed mean positivity rates of 80% for both CK1/10 and CK4, and 5% for both CK8/18 and CK19. LJSGHs showed mean positivity rates of 65% for CK19, 60% for CK8/18, 30% for CK4, and 5% for CK1/10. The differences between LJSGHs and GEs were statistically significant (P < 0.01). CONCLUSIONS: The LJSGH pattern of CK expression is reminiscent of the profile described in the literature for the junctional epithelium (JE). Possibly, JE exteriorized from the gingival sulcus would be more prone to irritation from a variety of sources, resulting in inflammation and hyperplasia, with the subsequent development of LJSGH.

Scrolls: novel microparticulate systems for enhanced delivery to/across the skin.

We describe the scroll system as a new microparticulate structured delivery system for enhanced delivery to/across the skin. The basic components of the scroll system are non-ionic surface active of the type of alkyl polyglycol ethers and a glycol. The unique structures are preserved with addition of various ingredients such as polymers, vegetable oils, pharmaceuticals, and permeation enhancers but are dismissed when amphiphile is withdrawn. The microparticles have a unique scroll structure with multiple "wrapping." Besides enabling superior permeation of drugs into/across the skin, the drugs delivered by scroll systems were more effective in vitro and in vivo compared to controls. Model drugs presented high entrapment capacity in scroll systems. The systems are stable over time and are safe for skin application. In order to form, they require a small number of ingredients, simple preparation method, and are environment friendly. The scroll systems may be new potential tools in the dermal/transdermal pharmaceutical and cosmetic industry.

Decompression as a treatment of odontogenic cystic lesions in children.

PURPOSE: To evaluate the efficiency of decompression in treating odontogenic cystic lesions of the jaws in children. MATERIALS AND METHODS: All consecutive odontogenic cysts occurring in children and treated by decompression from 1994 to 2009 at 1 maxillofacial center were included in the present study. Clinical data included age, gender, jaw, histopathologic diagnosis, and decompression time. Radiologic data from panoramic radiographs before and after decompression included tooth involvement, locularity, location, involvement of adjacent vital anatomic structures, and cyst area. RESULTS: Thirty-two odontogenic cystic lesions from 26 children (14 boys [53.8%] and 12 girls [46.2%]) treated with decompression were included. The average age at the time of presentation was 11.6 ± 3.3 years (range, 7 to 18 yr). The mandible was involved in 13 cases (40.6%) and the maxilla in 19 (59.4%). All cysts were unilocular at presentation. Twenty-seven cysts (84.4%) showed tooth involvement. The diagnoses consisted of dentigerous cysts (20 [62.5%]), keratocysts (9 [28.1%]), and radicular cysts (3 [9.4%]). The mean decompression period was 7.45 ± 2.6 months (2 to 14 months). The mean standard lesion area index changed from 12.7 ± 0.9 mm(2) (3.6 to 44 mm(2)) before compression to 2.3 ± 4.3 mm(2) (0 to 22.3 mm(2)) after decompression. The mean percentage of reduction (POR) was 82 ± 16% (49 to 100%). The POR was ranked as good in 22 lesions (69%), moderate in 9 lesions (28%), and poor in 1 lesion (3%). Surgery was performed for 15 lesions (47%). CONCLUSION: Decompression results in good regeneration potential of the bone in the developing craniofacial skeleton of children. Children might benefit from a less invasive surgical protocol.

Metastatic tumors to the jaws and mouth.

Metastatic dissemination to the oral cavity is rare and is usually the evidence of a wide spread disease with an average survival rate of 7 months. In almost a quarter of the cases, oral metastasis was found to be the first indication of an occult malignancy at a distant site. Metastatic lesions can be found anywhere in the oral cavity, however, the jaw bones with the molar area is the most frequently involved site. In the oral soft tissues, the gingiva is the most common site, suggesting the possible role of inflammation in the attraction of metastatic deposits. The most common primary malignancies presenting oral metastases were the lung, kidney, liver, and prostate for men, and breast, female genital organs, kidney, and colo-rectum for women. Most patients with jawbone metastasis complain of swelling, pain, and paresthesia. An exophytic lesion is the most common clinical presentation of metastatic lesions in the oral soft tissues. Early lesions, mainly those located in the gingiva, may resemble a hyperplastic or reactive lesion. Once a lesion is recognized as metastasis, the primary tumor site should be identified following clinical, radiological and histopathological investigations. If standardized diagnostic workup fails to detect the site of origin, then the term carcinoma of unknown primary is applied. Personalized medicine tools such as tissue-of-origin assays should be applied, either by immunohistochemical testing or by molecular-profiling methods as these may lead to a more favorable outcome.