Evaluation Criteria for Chromosome Instability Detection by FISH to Predict Malignant Progression in Premalignant Glottic Laryngeal Lesions.

Background: The definition of objective, clinically applicable evaluation criteria for FISH 1c/7c in laryngeal precursor lesions for the detection of chromosome instability (CI). Copy Number Variations (CNV) for chromosomes 1 and 7 reflect the general ploidy status of premalignant head and neck lesions and can therefore be used as a marker for CI. Methods: We performed dual-target FISH for chromosomes 1 and 7 centromeres on 4 µm formalin-fixed, paraffin-embedded tissue sections of 87 laryngeal premalignancies to detect CNVs. Thirty-five normal head and neck squamous cell samples were used as a control. First, the chromosome 7:1 ratio (CR) was evaluated per lesion. The normal range of CRs (≥0.84 ≤ 1.16) was based on the mean CR +/− 3 x SD found in the normal population. Second, the percentage of aberrant nuclei, harboring > 2 chromosomes of chromosome 1 and/or 7 (PAN), was established (cut-off value for abnormal PAN ≥ 10%). Results: PAN showed a stronger correlation with malignant progression than CR (resp. OR 5.6, p = 0.001 and OR 3.8, p = 0.009). PAN combined with histopathology resulted in a prognostic model with an area under the ROC curve (AUC) of 0.75 (s.e. 0.061, sensitivity 71%, specificity 70%). Conclusions: evaluation criteria for FISH 1c/7c based on PAN ≥ 10% provide the best prognostic information on the risk of malignant progression of premalignant laryngeal lesions as compared with criteria based on the CR. FISH 1c/7c detection can be applied in combination with histopathological assessment.

EGFRvIII expression triggers a metabolic dependency and therapeutic vulnerability sensitive to autophagy inhibition.

Expression of EGFRvIII is frequently observed in glioblastoma and is associated with increased cellular proliferation, enhanced tolerance to metabolic stresses, accelerated tumor growth, therapy resistance and poor prognosis. We observed that expression of EGFRvIII elevates the activation of macroautophagy/autophagy during starvation and hypoxia and explored the underlying mechanism and consequence. Autophagy was inhibited (genetically or pharmacologically) and its consequence for tolerance to metabolic stress and its therapeutic potential in (EGFRvIII+) glioblastoma was assessed in cellular systems, (patient derived) tumor xenopgrafts and glioblastoma patients. Autophagy inhibition abrogated the enhanced proliferation and survival advantage of EGFRvIII+ cells during stress conditions, decreased tumor hypoxia and delayed tumor growth in EGFRvIII+ tumors. These effects can be attributed to the supporting role of autophagy in meeting the high metabolic demand of EGFRvIII+ cells. As hypoxic tumor cells greatly contribute to therapy resistance, autophagy inhibition revokes the radioresistant phenotype of EGFRvIII+ tumors in (patient derived) xenograft tumors. In line with these findings, retrospective analysis of glioblastoma patients indicated that chloroquine treatment improves survival of all glioblastoma patients, but patients with EGFRvIII+ glioblastoma benefited most. Our findings disclose the unique autophagy dependency of EGFRvIII+ glioblastoma as a therapeutic opportunity. Chloroquine treatment may therefore be considered as an additional treatment strategy for glioblastoma patients and can reverse the worse prognosis of patients with EGFRvIII+ glioblastoma.

A phase I-II study on the combination of rapamycin and short course radiotherapy in rectal cancer.

PURPOSE: This phase I/II study sought to determine the safety and maximum tolerated dose (MTD) of the combination of rapamycin, an mTOR inhibitor, with short-course radiotherapy in rectal cancer patients. Antitumor activity, changes in metabolic activity and perfusion on imaging, and changes in phosphorylation status of the mTOR pathway were also assessed. MATERIALS AND METHODS: Patients with primary resectable rectal cancer were treated with short-course hypofractionated radiotherapy (5×5 Gy) combined with oral rapamycin 1 week before and during radiotherapy, followed by surgical resection. RESULTS: Thirteen patients were entered in phase I. One patient developed a dose-limiting toxicity, consisting of a grade 4 leak and grade 4 bleeding. Because of an unexpected high rate of grade 3 postoperative toxicity, it was decided to treat patients with delayed surgery in phase II. Primary endpoint for phase II was tumor blood flow (K(trans)) assessed by perfusion CT. Thirty-one patients were treated with the MTD of 6 mg rapamycin daily. One patient (3%) developed a pathological complete response (pCR) and 3 patients (10%) had a ypT1N0 tumor at the time of resection. No change in tumor perfusion was observed on perfusion CT, but a significant decrease of metabolic activity was found on PET-scan. CONCLUSIONS: The combination of short-course radiotherapy and rapamycin turned out to be feasible, provided that the interval between neo-adjuvant treatment and surgical resection is at least 6 weeks. Although from this cohort no clear increase in pCR could be observed, a clear metabolic response after rapamycin run-in was observed, indicating a biological activity of this drug in rectal cancer.

BRAFV600E immunopositive melanomas show low frequency of heterogeneity and association with epithelioid tumor cells: a STROBE-compliant article.

Treatment of BRAFV600E-mutant melanoma by small molecule inhibitors that target BRAFV600E or MEK kinases is increasingly used in clinical practice and significantly improve patient outcome. However, patients eventually become resistant and therapeutic improvement is required. Molecular diversity within individual tumors (intratumor heterogeneity) and between tumors within a single patient (intrapatient heterogeneity) poses a significant challenge to precision medicine. Using immunohistochemistry, we determined the extent of BRAFV600E intratumor and intrapatient heterogeneity and the influence of morphological heterogeneity in a large series of 171 melanomas of 81 patients. The BRAFV600E mutation rate found in our melanoma series is 44%, with none of 22 (0%) melanoma in situ, 23 of 56 (41%) primary tumors, 28 of 59 (48%) regional metastases, and 24 of 34 (71%) distant metastases harboring the mutation. In general, a diffuse homogeneous immunostaining was seen, even in tumors consisting of more than one cell type, that is, epithelioid, spindle, and/or small cell types. Nevertheless, BRAFV600E-mutant melanomas more often had a purely epithelioid cell population (P=0.063), that is more evident among distant metastases (P=0.014). Only two of 75 (3%) mutated specimens (one primary and one metastasis) displayed heterogeneous BRAFV600E expression. The primary tumor was also morphologically heterogeneous and exclusively displayed BRAFV600E in the epithelioid component, confirming an association between BRAFV600E and epithelioid cells. Twenty-eight of 30 patients (93%) had concordant BRAFV600E mutation status between their tumors. Taken together, BRAFV600E intratumor and intrapatient heterogeneity in melanoma is diminutive, nevertheless, the identified exceptions will have important implications for the clinical management of this disease.

Interobserver reproducibility of diffusion-weighted MRI in monitoring tumor response to neoadjuvant therapy in esophageal cancer.

OBJECTIVE: To investigate the reproducibility of diffusion-weighted magnetic resonance imaging (DW-MRI) in assessing tumor response early in the course of neoadjuvant chemoradiotherapy in patients with operable esophageal cancer. METHODS: Eleven male patients (mean age 54.8 years) with newly diagnosed esophageal cancer underwent DW-MRI before and 10 days after start of chemoradiotherapy. Reproducibility of apparent diffusion coefficient (ADC) measurements by manual (freehand) and semi-automated volumetric methods was assessed. RESULTS: Interobserver reproducibility for the assessment of mean tumor ADC by the manual measurement method was good, with an ICC of 0.69 (95% CI, 0.36 to 0.85; P = 0.001). Interobserver reproducibility for the assessment of mean tumor ADC by the semi-automated volumetric measurement method was very good, with an ICC of 0.96 (95% CI, 0.91 to 0.98; P<0.001). CONCLUSION: Semi-automated volumetric ADC measurements have higher reproducibility than manual ADC measurements in assessing tumor response to chemoradiotherapy in patients with esophageal adenocarcinoma.

The evaluation of colon biopsies using virtual microscopy is reliable.

AIMS: Virtual microscopy offers major advantages for pathology practice, separating slide evaluation from slide production. The aim of this study was to investigate the reliability of using whole slide images as compared with routine glass slides for diagnostic purposes. METHODS AND RESULTS: Colon biopsies (n = 295) were assessed using both glass slides and whole slide images by four pathologists and two residents. Two pathologists scored the digital images of biopsies in a primary diagnostic setting. For each case, the consensus diagnosis was defined as the majority diagnosis on the study's glass slides. All diagnoses were grouped into seven main diagnostic categories, and further divided into subgroups. The overall concordance rates were 89.6% for whole slide images and 91.6% for light microscopy. The concordance rates of the subgroups 'adenoma' and 'adenocarcinoma' between whole slide images and conventional microscopy showed only small variability. The intraobserver (whole slide images versus glass slide) agreement, including subgroups, was substantial, with a mean κ-value of 0.78, and was higher than the interobserver agreement for glass slides (interobserver κ-value of 0.69). CONCLUSIONS: This study shows good diagnostic accuracy and reproducibility for virtual microscopy, indicating that this technology can reliably be used for pathological evaluation of colon biopsies in a primary clinical setting.

Whole slide images as a platform for initial diagnostics in histopathology in a medium-sized routine laboratory.

INTRODUCTION: Whole slide imaging is the process of digitizing glass slides and the creation of Whole Slide Images (WSI), which enable the examination of pathology samples on a computer screen in a manner comparable to light microscopy. WSI have been used for different applications in pathology but their use for primary diagnostics is still limited. Implementing WSI for primary diagnostics would be a turning point necessitating extensive validation to unravel pitfalls and difficulties that could be encountered within the routine workflow. This article is aimed to describe the gradual integration of WSI into routine pathology diagnostics in a medium-sized routine pathology laboratory. METHODS: This project was started with optimizing the digital work environment including the setting up of validation studies, scanning preferences, storing WSI and the implemented adjustments to the workflow for the laboratory and the pathologist. Afterwards scanning glass slides was initiated in the department of pathology at the Atrium Medical Center, Heerlen, The Netherlands, for performing primary diagnostics of breast biopsies. Later this was extended to other specimen types including resections. RESULTS: The validation studies yielded a high concordance rate between WSI and conventional diagnoses. Routine primary WSI based diagnosis was possible in 82.1% of cases. Failure of digital diagnosis was mainly related to poor image quality and logistic problems. CONCLUSION: The quality of the currently produced WSI is sufficient for primary diagnostics in 82.1% of the cases. Improving image quality, adequate retrieval and controlling scanning error will definitely encourage the wide adaptation in routine diagnostics.

Functional and morphologic analysis of the fluid-conducting meshwork in xenografted cutaneous and primary uveal melanoma.

PURPOSE: In primary uveal and cutaneous melanoma lesions, extracellular matrix (ECM) is often deposited in arcs, loops, and network patterns. Based on prognostic relevance, these patterns appear to play a significant role in facilitating metastasis. It has been demonstrated that these patterns were capable of transmitting fluid. The current study was undertaken to elucidate further the functional role of these patterns in tumor perfusion and to examine the composition of the patterns by immunohistochemistry. METHODS: To study the role of these patterns in perfusion, fluorochrome-labeled bovine serum albumin, bovine insulin, and dextrans of different molecular sizes were injected intravenously into nude mice bearing subcutaneous human cutaneous melanoma xenografts. Distribution of the human melanoma cells and murine host cells was analyzed by DNA in situ hybridization. To elucidate the composition of these patterns, human uveal melanoma tissues were analyzed for expression of ECM components by immunohistochemistry. RESULTS: Small molecules (Stokes' radius <4.4 nm) crossed the vessel wall and spread along the ECM patterns within 2 to 10 minutes, whereas larger molecules (Stokes' radius approximately 5.8 nm) required 30 to 45 minutes to enter. Murine host cells were found exclusively in the ECM pattern compartment. In primary uveal melanoma, different types of collagen, ECM-associated heparan sulfate proteoglycans, and different types of cells were present in the patterns. CONCLUSIONS: The data suggest that the ECM deposited as arcs, loops, and network patterns, accommodate the transport of plasma-derived molecules, (e.g., nutrients), to the tumor lesion, thus enhancing tumor growth and progression, and facilitating infiltration of tumor tissue by host-derived cells.

Elevated levels of vascular endothelial growth factor in serum of patients with D+ HUS.

The pathogenesis of hemolytic uremic syndrome (D+ HUS) is characterized by endothelial damage of glomeruli and tubules within the kidney. In several other diseases in which glomerular endothelial damage occurs, elevated serum levels of vascular endothelial growth factor (VEGF) have been reported. VEGF is involved in angiogenesis, permeabilization of blood vessel endothelium, and wound repair. In this study we evaluated VEGF levels in the serum of 40 D+ HUS patients in the acute phase and during the course of the disease. VEGF levels were measured using a double-sandwich ELISA. Indirect immunohistochemistry was performed for the detection of VEGF in renal biopsy material of 3 HUS patients. Significantly elevated VEGF levels were found in HUS patients compared with controls in both serum ( P<0.001) and plasma ( P<0.05). A significant relationship was found between VEGF levels and severity of the disease according to the classification of Gianantonio ( P<0.05). Levels of VEGF in blood increased during the 2nd and 3rd week after HUS was diagnosed. Immunohistochemistry of renal biopsy material showed increased levels of the receptors for VEGF in the glomeruli. During the course of HUS, plasma VEGF levels increase and the increase is dependent on the severity of the disease. This is probably associated with the repair process.

Localization and characterization of melanoma-associated glycosaminoglycans: differential expression of chondroitin and heparan sulfate epitopes in melanoma.

Glycosaminoglycans (GAGs) are anionic polysaccharides present on cells and in the extracellular matrix (ECM). They likely play a role in tumor formation because of their capacity to bind and modulate a variety of proteins including growth factors, cytokines, and proteases. Using a panel of (human) phage display-derived anti-GAG antibodies, the location and expression of GAG epitopes in human cutaneous melanocytic lesions was studied. Antibodies EW4E1 and EW4G2 identified a melanoma-associated chondroitin sulfate/heparan sulfate epitope, whereas antibody EW4B7 recognized a melanoma-associated heparan sulfate epitope. These antibodies showed a high reactivity with blood vessels and ECM in cutaneous melanoma tumors, whereas their reactivity with nevi was very low. Using a set of defined oligosaccharides it was established that sulfate groups are of main importance in the binding to the antibodies and that glycomimetics can mimic natural oligosaccharides. In xenografts of melanoma cell line MeL57, a strong association of GAG epitopes with an injected fluorescent fluid flow tracer was observed. In uveal melanoma antibody, EW4E1 proved to be a sensitive probe for the detection of the geometry of ECM structures, known to have prognostic value. Taken together, data indicate that in melanoma a defined set and location of GAG epitopes are present with possible functional significance.

EMAP-II expression is associated with macrophage accumulation in primary uveal melanoma.

PURPOSE: Primary uveal melanoma may contain arcs, loops, and networks of periodic acid-Schiff (PAS)-positive patterns, along which numerous macrophages are present. Their recruitment into tumor tissue is mediated by chemotactic cytokines, for which vascular endothelial growth factor (VEGF)-C and endothelial monocyte-activating polypeptide ((EMAP)-II are candidates. In this study, the extent of VEGF-C and EMAP-II immunoreaction was related to infiltration of macrophages. METHODS: Serial sections of 25 primary uveal melanoma lesions were analyzed by immunohistochemistry. RESULTS: The analysis showed no correlation of VEGF-C immunoreaction and localization of macrophages. However, accumulation of macrophages occurred at sites of EMAP-II expression, especially in areas containing nests of tumor cells, surrounded by arcs, loops, and network patterns. In tumors with a strong EMAP-II immunoreaction, the adhesion molecule intracellular adhesion molecule (ICAM)-1 was strongly expressed on endothelial cells. EMAP-II-positive endothelial cells did not express VEGF receptor-2. However, extensive release of von Willebrand factor was observed. Signs of apoptosis were found neither in tumor cells nor endothelial cells. CONCLUSIONS: In uveal melanoma, macrophages accumulate at sites of EMAP-II expression. Based on the results, it may be hypothesized that this process of chemotaxis is facilitated by EMAP-II-dependent expression of ICAM-1 on vascular endothelial cells and concomitantly leads to localized vascular damage, as indicated by release of von Willebrand factor.

Pathophysiological implications of stroma pattern formation in uveal melanoma.

Clinical outcome of cancer patients is mainly determined by the rate of metastasis and, also by primary tumor growth. Formation of extracellular matrix and interactions of neoplastic and non-neoplastic (host) cells in solid tumors have been shown to be essential for these processes. One result of such interactions is the outgrowth of new blood vessels from existing ones, angiogenesis, to provide the tumor tissue with oxygen and nutrients. It is assumed that the neovascular bed also facilitates the escape of metastatic cells from the primary lesions. In addition, recent reports suggested the existence of blood-conducting channels lined by melanoma cells (so-called "vascular channels") accompanied by depositions of extracellular matrix patterns in cutaneous and uveal melanoma. Since the presence of these matrix structures has been negatively associated with prognosis, we hypothesize that they play a role in melanoma outgrowth or metastasis. In this review, we will discuss the morphological and functional properties of the extracellular matrix patterns in that may underlie these clinical phenomena.

Induction of vascular endothelial growth factor receptor-3 expression on tumor microvasculature as a new progression marker in human cutaneous melanoma.

The anatomical relation between a malignant tumor and its vascular and lymphatic bed is an important factor influencing metastasis. Lack of specific markers for the lymphatic endothelium has long hampered a reliable detection of lymphatics. Here, we demonstrate that lymphatic endothelium can reliably be identified in a panel of different normal tissues and of benign and malignant tumors. Application of the previously described PAL-E/CD31 double staining protocol differentiates between blood capillaries and veins on one hand and lymphatic vessels on the other. Blood vessel marker CD34, absent from lymphatics, was used additionally to classify arteries. We found that the lymphatic vascular endothelial growth factor receptor-3 (VEGFR-3, also known as Flt-4) was present on both lymphatic and blood vessels in 76 of 113 malignant tumors [adenocarcinoma of kidney (n = 3), colon (n = 3) and liver (n = 3), breast (n = 9) and squamous cell carcinoma (n = 5), primary (n = 81), and metastatic (n = 9) melanoma]. No evident signs of tumor-induced lymphangiogenesis were observed. Evaluation of a series of 110 melanocytic skin lesions indicated that VEGFR-3 expression is confined to the lymphatic vasculature in benign lesions. However, its expression emerges on the blood neovasculature in malignant lesions as soon as metastatic potential develops. We conclude that induction of VEGFR-3 expression on tumor blood vessels may be a general phenomenon that would make VEGFR-3 a marker for tumor endothelium. In addition, we propose VEGFR-3 expression as a new microvascular progression marker in cutaneous melanoma.

Presence of a fluid-conducting meshwork in xenografted cutaneous and primary human uveal melanoma.

PURPOSE: Recently, it was reported that tumor cells themselves generate channels and networks in three-dimensional culture and can be found lining channels (some containing red blood cells [RBCs]) in vivo, and they express endothelial or vascular genes in aggressive uveal melanoma. The implications of these data for current insights in the involvement of angiogenesis in tumor growth, metastasis and therapeutic intervention are considerable. Therefore, this possibility was investigated in the current study. METHODS: Thirty human uveal melanomas and 20 xenografts of human cutaneous melanoma were analyzed by Azan histochemistry and immunostaining of endothelial markers. Additionally, in xenografted tumors a tracer study was performed with confocal microscopy and immunoelectron microscopy. RESULTS: Lumina or spaces without endothelial lining containing RBCs were not detected in any lesion. Functional evaluation of the vasculature in xenografts demonstrated rapid tracer appearance both inside and outside blood vessels. Outside blood vessels it spread along matrix networks of arcs and back-to-back loops. Confocal microscopy showed that this extracellular matrix was deposited as stromal sheets around nests of tumor cells. Laminin immunostaining revealed that between sheets surrounding adjacent nests, spaces were present. These spaces were filled, however, with collagen and different types of cells, including cells stained for macrophage markers. CONCLUSIONS: Although no evident endothelium-free and RBC-containing channels were present in the tissues examined, there are fluid-conducting spaces in the form of stromal sheets between nests of tumor cells. In this stromal network, blood vessels are embedded. The authors postulate that this extracellular matrix tissue represents a fluid-conducting meshwork.