Haematotoxicity in IMRT/VMAT curatively treated anal cancer.

INTRODUCTION: Curative chemoradiotherapy of squamous cell carcinoma achieves long-term complete remissions in most patients and minimizing treatment toxicity becomes crucial issue. The aim of the retrospective analysis was to determine an acceptable dose to the bone marrow for radiotherapy planning not leading to increased haematological toxicity. PATIENTS AND METHODS: In the period 2013-2019, 40 patients with squamous cell carcinoma were curatively treated at the Department of Oncology of the University Hospital Motol using intensity modulated radiotherapy (IMRT) /volumetric modulated arc radiotherapy (VMAT) technique. Women make up 90% of the group, the average age at the time of dia-gnosis was 65 years (47-81). Chemotherapy mitomycin C and 5-fluorouracil was given to 68% of patients. The bone marrow was contoured in the Varian Eclipse planning system, version 15.6. RESULTS: Acute hematotoxicity (G3, 4, 5 according to Common Terminology Criteria for Adverse Events - CTCAE) was significantly associated with the concomitant chemoradiotherapy (P = 0.002) and the average dose to the bone marrow 27 Gy (P = 0.011). Late haematological toxicity was mild (maximum grade 1), asymptomatic, and no dependence of late haematotoxicity on any risk factor (age, gender, WHO performance status, bone marrow dose, CHT, BMI, smoking, stage) was proved. The overall survival at 5 years was 100% in stage I, 83% in stage II, 61% in stage III and 0% in stage IV. Local control at 5 years is 100% in stage I, 92% in stage II, 87% in stage III and 0% in stage IV. Local recurrence developed in 5% of radically treated patients. Distant metastases occurred in 8% of radically treated patients. Local recurrences or metastases occurred only during the first 2 years after the treatment. CONCLUSION: Radical chemoradiotherapy in the treatment of squamous cell anal carcinoma is highly effective. IMRT/VMAT enabled to apply a sufficiently effective dose to the tumor and elective areas and reduced not only acute skin, GI and GU toxicity, but also acute haematological toxicity in cases with the dose Dmean to bone marrow lower than 27 Gy. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical, papers.

Improved survival in patients with FDG-PET/CT-based radiotherapy treatment planning for squamous cell anal cancer.

The aim of this retrospective analysis was to evaluate the impact of FDG-PET/CT-based target volume definition on locoregional control and survival, compared to conventional CT-based target volume definition and dose prescription. One hundred and twenty-two patients with squamous cell anal cancer were treated with curative radiotherapy (RT) alone (27%) or with RT with concurrent chemotherapy (73%) and analyzed. Forty-six percent had the early disease (stage I+II) and 54% were stage III. FDG-PET/CT-based staging was performed in 21% of the patients. The mean follow-up time was 60 months. Other risk factors affecting survival were investigated. According to initial staging in both groups (FDG-PET/CT and conventional CT) were 10% of stage IV disease, and they were excluded from radical radiotherapy and treated with palliative intent. Ninety-two percent of the patients achieved complete remission. Significant favorable factors in univariate analysis associated with disease-free survival (DFS) were PET/CT staging, T1/2 and N0 stage, and clinical stage I and II. Locoregional control (LRC) correlated with the T1/2 stage and initial performance status (PS) 0. There were no significant factors affecting overall survival (neither in univariate nor multivariate analysis). In multivariate analysis, the factor associated with better DFS was PET/CT staging and for LRC, PS 0 and concomitant chemoradiation. Acute toxicity was increased in the concurrent chemo-radiotherapy group. Two-, five- and ten-year overall survival rates were 83%, 69%, and 60%; disease-free survival rates were 76%, 73%, 73%; local control rates were 91%, 90%, and 90% and colostomy-free survival was 89%, 86%, and 81%, respectively. PET/CT staging allowed targeted dose escalation to the primary tumor and nodal metastases while decreasing dose to uninvolved regions, resulting in significantly improved DFS without compromising locoregional control.

Three-dimensional conformal radiotherapy versus intensity modulated radiotherapy with simultaneous integrated boost in the treatment of locally advanced head and neck carcinoma.

The treatment of locally advanced head and neck cancer (LAHNC) requires a multimodality approach. Radiotherapy with combination of chemotherapy are demonstrated to be effective, however, the treatment intensification leads to increased toxicity at the same time. The implementation of three-dimensional conformal radiotherapy (3D-CRT) allowed to irradiate the treatment volume more precisely with better surrounding healthy tissue sparing. Intensity modulated radiotherapy (IMRT) facilitated higher conformity in dose shaping to target volume. IMRT with simultaneous integrated boost (IMRT-SIB) offered the possibility to deliver individualized dose levels within one fraction and enabled escalation of the dose per fraction and radiotherapy acceleration. The aim of our study was to compare the technique of 3D-CRT and IMRT-SIB in the treatment of LAHNC and evaluate the treatment outcome and the treatment-related toxicity. 262 patients in 3D-CRT group and 263 patients in IMRT-SIB group underwent the radical treatment for LAHNC between 1/1998 and 12/2016. No statistically significant differences in locoregional control (LCR) and overall survival (OS) were found between the two groups. Acute toxicity and xerostomia were significantly reduced in the patients treated by IMRT-SIB. IMRT-SIB is a safe radiotherapy method where less toxicity was proven without compromising local control and overall survival.

DNA from microdissected tissues may be extracted and stored on microscopic slides.

With regard to complex structure of tissues, laser capture microdissection represents an important step in analytical workflow streaming to proper molecular characterization of different cell types in examined samples. Therefore the simple method for simultaneous processing of higher numbers of microdissected tissues leading not only to rapid and efficient DNA isolation but allowing also the repeated sampling and easy storage may be useful in the practice of histopathological laboratories. We elaborated such a methodology applicable downstream after the microdissection from formalin-fixed paraffin embedded tissues.The tissues for examination are microdissected directly into the circular areas having the diameter 2 mm and marked on the microscopic slide. In this way, one slide is able to accommodate multiple samples. The DNA extraction is performed in low volume of buffer with Proteinase K in a droplet covered by mineral oil just on the slide. Mineral oil in the quality for molecular biology not only avoids evaporation during DNA extraction, but it helps to position the microdisssected tissue, to control the level of cell lysis microscopically and to protect the DNA sample during subsequent manipulations. We provided the evidence that DNA isolated by our methodology remains in the positions on microscopic slide for months without any changes in the lengths of available fragments and that it may be removed from each position repetitively for different kinds of analysis. The new methodological approach presented by us can be practically applied in broad spectrum of laboratories performing routinely genetic analysis on microdissected tissues.

The branching pattern of villous capillaries and structural changes of placental terminal villi in type 1 diabetes mellitus.

Maternal diabetes is associated with changes of the placental structure. These changes include great variability of vascularity manifested by strikingly hypovascular as well as hypervascular terminal villi. In this paper, normal placental terminal villi and pathological villi of type 1 diabetic placentas were compared concerning the structure of villous stroma, spatial arrangement of villous capillary bed and quantitative assessment of capillary branching pattern. Formalin fixed and paraffin embedded specimens of 14 normal and 17 Type 1 diabetic term placentas were used for picrosirius staining, vimentin and desmin immunohistochemistry and confocal microscopy. 3D models of villi and villous capillaries were constructed from stacks of confocal optical sections. Hypervascular as well as hypovascular villi of diabetic placenta displayed changed structure of villous stroma, i.e. the collagen envelope around capillaries looked thinner and the network of collagen fibers seemed less dense. The desmin immunocytochemistry has shown that stromal cells of hypervascular as well as hypovascular villi appeared nearly or completely void of desmin filaments. In comparison with normal villi, capillaries of hypovascular villi had a smaller diameter and displayed a markedly wavy course whereas in hypervascular villi numerous capillaries occurred in reduced stroma and often had a large diameter. The quantitative assessment of capillary branching has shown that villous capillaries are more branched in diabetic placentas. It is concluded that type 1 maternal diabetes enhances the surface area of the capillary wall by elongation, enlargement of diameter and higher branching of villous capillaries and disrupts the stromal structure of terminal villi.

IFPA Meeting 2011 workshop report II: Angiogenic signaling and regulation of fetal endothelial function; placental and fetal circulation and growth; spiral artery remodeling.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2011 there were twelve themed workshops, three of which are summarized in this report. These workshops related to vascular systems and circulation in the mother, placenta and fetus, and were divided in to 1) angiogenic signaling and regulation of fetal endothelial function; 2) placental and fetal circulation and growth; 3) spiral artery remodeling.

Pericyte coverage of fetoplacental vessels in pregnancies complicated by Type 1 diabetes mellitus.

Placentas from pregnancies complicated by Type 1 diabetes mellitus (DM 1) display altered vascular morphology and function. Here we studied the extent of pericyte coverage in microvessels of normal pregnancies and pregnancies complicated by DM 1. We used smooth muscle actin (SMA) as a marker for quantitation of pericyte coverage in placental capillaries. The extent of pericyte coverage around the vessel circumference was 38 ± 11% in normal vs. 33 ± 10% in DM 1 pregnancies. We found that there is no statistically significant difference in the extent of pericyte coverage around the capillary circumference between DM 1 and normal pregnancies.

Histochemical detection of GM1 ganglioside using cholera toxin-B subunit. Evaluation of critical factors optimal for in situ detection with special emphasis to acetone pre-extraction.

A comparison of histochemical detection of GM1 ganglioside in cryostat sections using cholera toxin B-subunit after fixation with 4% formaldehyde and dry acetone gave tissue-dependent results. In the liver no pre-treatment showed detectable differences related to GM1 reaction products, while studies in the brain showed the superiority of acetone pre-extraction (followed by formaldehyde), which yielded sharper images compared with the diffuse, blurred staining pattern associated with formaldehyde. Therefore, the aim of our study was to define the optimal conditions for the GM1 detection using cholera toxin B-subunit. Ganglioside extractability with acetone, the ever neglected topic, was tested comparing anhydrous acetone with acetone containing admixture of water. TLC analysis of acetone extractable GM1 ganglioside from liver sections did not exceed 2% of the total GM1 ganglioside content using anhydrous acetone at -20 degrees C, and 4% at room temperature. The loss increased to 30.5% using 9:1 acetone/water. Similarly, photometric analysis of lipid sialic acid, extracted from dried liver homogenates with anhydrous acetone, showed the loss of gangliosides into acetone 3.0 +/- 0.3% only. The loss from dried brain homogenate was 9.5 +/- 1.1%. Thus, anhydrous conditions (dry tissue samples and anhydrous acetone) are crucial factors for optimal in situ ganglioside detection using acetone pre-treatment. This ensures effective physical fixation, especially in tissues rich in polar lipids (precipitation, prevention of in situ diffusion), and removal of cholesterol, which can act as a hydrophobic blocking barrier.

Is maternal progesterone actually independent of the fetal steroids?

Progesterone and estradiol are the foremost steroid hormones in human pregnancy. However, the origin of maternal progesterone has still not been satisfactorily explained, despite the generally accepted opinion that maternal LDL-cholesterol is a single substrate for placental synthesis of maternal progesterone. The question remains why the levels of progesterone are substantially higher in fetal as opposed to maternal blood. Hence, the role of the fetal zone of fetal adrenal (FZFA) in the synthesis of progesterone precursors was addressed. The FZFA may be directly regulated by placental CRH inducing an excessive production of sulfated 3beta-hydroxy-5-ene steroids such as sulfates of dehydroepiandrosterone (DHEAS) and pregnenolone (PregS). Due to their excellent solubility in plasma these conjugates are easily transported in excessive amounts to the placenta for further conversion to the sex hormones. While the significance of C19 3beta-hydroxy-5-ene steroid sulfates originating in FZFA for placental estrogen formation is mostly recognized, the question "Which maternal and/or fetal functions may be served by excessive production of PregS in the FZFA?" - still remains open. Our hypothesis is that, besides the necessity to synthesize de novo all the maternal progesterone from cholesterol, it may be more convenient to utilize the fetal PregS. The activities of sulfatase and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) are substantially higher than the activity of cytochrome P450scc, which is rate-limiting for the placental progesterone synthesis from LDL-cholesterol. However, as in the case of progesterone synthesis from maternal LDL-cholesterol, the relative independence of progesterone levels on FZFA activity may be a consequence of substrate saturation of enzymes converting PregS to progesterone. Some of the literature along with our current data (showing no correlation between fetal and maternal progesterone but significant partial correlations between fetal and maternal 20alpha-dihydroprogesterone (Prog20alpha) and between Prog20alpha and progesterone within the maternal blood) indicate that the localization of individual types of 17beta-hydroxysteroid dehydrogenase is responsible for a higher proportion of estrone and progesterone in the fetus, but also a higher proportion of estradiol and Prog20alpha in maternal blood. Type 2 17beta-hydroxysteroid dehydrogenase (17HSD2), which oxidizes estradiol to estrone and Prog20alpha to progesterone, is highly expressed in placental endothelial cells lining the fetal compartment. Alternatively, syncytium, which is directly in contact with maternal blood, produces high amounts of estradiol and Prog20alpha due to the effects of type 1, 5 and 7 17?-hydroxysteroid dehydrogenases (17HSD1, 17HSD5, and 17HSD7, respectively). The proposed mechanisms may serve the following functions: 1) providing substances which may influence the placental production of progesterone and synthesis of neuroprotective steroids in the fetus; and 2) creating hormonal milieu enabling control of the onset of labor.

Stimulation of protective and cross-protective immunity against influenza B virus after adjuvant mucosal immunization of mice.

Intratracheal immunization of mice with inactivated influenza B virus and delipidated Bacillus firmus as adjuvant increases protection of mice against infection with the homologous virus strain and induces cross-protection: mice immunized by influenza virus B/Yamanashi 166/98 were protected even against phylogenetically distant virus drift variant B/Lee/40 lethal for mice.

Three-dimensional arrangement of the capillary bed and its relationship to microrheology in the terminal villi of normal term placenta.

Spatial arrangement of the capillary bed, manifestations of its growth and symmetry of capillary branching were studied in peripheral villi of normal human placenta at term using confocal microscopy and image analysis. Unlike the model that has been accepted so far, it was shown that the arrangement of the capillary bed in terminal villi varied from simple, U-like loops to a richly branched network. Three different categories of terminal villi (TV) were recognised: Signs of capillary elongation and sprouting were observed in the villous capillary bed. Based on the assessment of the mean cross-sectional areas of capillaries constituting simple, Y-like capillary bifurcation in terminal villi, the capillary branching was found to be asymmetric. Therefore, we conclude that the conditions for the "plasma skimming" effect are met in human placenta.

Immune response after adjuvant mucosal immunization of mice with inactivated influenza virus.

Satisfactory mucosal immunity in the respiratory tract is very important for protection against influenza. It can be achieved only by mucosal immunization. Mucosal vaccination with inactivated influenza virus may not be sufficiently effective and suitable adjuvants are therefore sought. We tested intratracheal immunization of mice with inactivate B type influenza virus in a mixture with formolized G+ bacterium Bacillus firmus, whose adjuvant effects have previously been documented in another system. The treatment resulted in a marked increase of both systemic and mucosal antibody response in IgG and IgA classes. Stimulation of T lymphocytes after adjuvant immunization was very mild, no proliferation taking place after specific stimulation with antigen in vitro. However, slightly increased systemic (spleen) and local (lungs) production of cytokines without perceptible Th1/Th2 polarization was determined. B. firmus is an efficient adjuvant in respiratory tract immunization while with subcutaneous immunization it lowers the antibody response.

Quantitative analysis of embryonic kidney impairment by confocal microscopy and stereology: effect of 1,2-dibromoethane in the chick mesonephros.

1. Chick embryos in ovo were treated with a teratogenic dose of 1,2-dibromoethane (DBE) on embryonic day (ED) 3. On ED 6 and 10, histological sections of whole embryos were prepared for confocal microscopy. In parallel, mesonephroi of 10-d-old embryos were dissected for in situ staining with acridine orange (AO), a fluorescence probe for lysosomes. 2. DBE impaired differentiation of renal vessels which manifested as a delay in rearrangement of primitive renal vascular architecture on ED 6 and a significant reduction of the mesonephric vascularisation on ED 10. This was accompanied by delayed functional maturation of embryonic kidney, as suggested by staining with AO. 3. Renal vessels appeared to be more susceptible to DBE than tubules. Unequal growth of these renal components might be a cause of DBE-induced spatial disorganisation of tubular apparatus. 4. Nephrotoxic effects of DBE during the embryonic period are associated primarily with damage to the renal blood supply. 5. Confocal microscopy, stereological methods and three-dimensional reconstruction of developing tissues are useful tools to investigate pathogenic processes during embryonic development.

Intercellular adhesion molecule-1 (ICAM-1) deficiency protects mice against severe forms of experimentally induced colitis.

ICAM-1 (CD54), the ligand for LFA-1 and Mac-1, is up-regulated during inflammatory reaction on the activated vascular endothelium. To determine its role in intestinal inflammation, we induced acute experimental colitis in mice with a deleted ICAM-1 gene, by feeding them with 3% dextran sodium sulphate (DSS) in drinking water for 7 days. Chronic colitis was elicited by DSS similarly, followed by 2 weeks with water. In the acute phase of inflammation, ICAM-1-deficient mice exhibited a significantly lower mortality rate (5%) than control C57Bl/6J mice (35%). Control animals, but not the ICAM-1-deficient mice, exhibited diarrhoea and rectal bleeding. Histological examination of large-bowel samples evaluated the intensity of inflammatory changes, and type and extent of mucosal lesions. In the acute phase, 33.3% of samples from ICAM-1-deficient mice exhibited mucosal defects (flat and fissural ulcers), predominantly mild to moderate inflammatory infiltrate within the lamina propria mucosae and lower grades of mucosal lesions. Much stronger inflammatory changes were present in control animals, flat ulcers (sometimes multiple) and fissural ulcers being observed in 62.5% of samples. Mucosal inflammatory infiltrate was moderate to severe, typically with higher grades of mucosal lesions. In chronic colitis, smaller inflammatory changes were found in the large bowel. The two mouse strains differed, the chronic colitis being accompanied by an increased serum level of anti-epithelial IgA autoantibodies in C57Bl/6 control mice but not in ICAM-1-deficient mice. These findings provide direct evidence of the participation of ICAM-1 molecule in the development of experimentally induced intestinal inflammation.

Detection of ICAM-1 in experimentally induced colitis of ICAM-1-deficient and wild-type mice: an immunohistochemical study.

Adhesion molecules (e.g. ICAM-1, CD 54) are known to be upregulated on activated vascular endothelial cells during inflammatory reactions. To study the role of ICAM-1 in intestinal inflammation in vivo, we induced acute experimental colitis in wild-type (C57BL/6) mice and ICAM-1-deficient mice, by feeding the animals with 3% dextran sodium sulphate (DSS) in drinking water for 7 days. In the control strain the immunohistochemical staining showed a very pronounced endothelial upregulation of ICAM-1 after the DSS treatment observed in areas of inflammatory infiltrate, especially in venules or arterioles of the propria and submucosa, and partly in the mesocolon. DSS-fed ICAM-1-deficient mice showed no endothelial enhancement and only faint staining of venules or capillaries approaching that encountered in the control ICAM-1-deficient animals. Our data indicate that ICAM-1 may play a crucial role in the development of acute intestinal inflammation, consistent with our finding that ICAM-1 deficiency can obviate severe forms of experimentally induced colitis in mice.

Spatial arrangement of fetal placental capillaries in terminal villi: a study using confocal microscopy.

The spatial arrangement of capillaries was studied in terminal villi of placentas at term by using confocal microscopy and methods for different types of 3-D reconstructions. Fixed specimens embedded in paraffin or glycol methacrylate resin were cut and stained with eosin. Digitized images of serial optical sections (approximately 0.5 microm) of individual terminal villi lying completely inside physical sections (100 microm) were captured by confocal laser scanning microscopy and analyzed. Topological reconstruction of the capillary bed and measurements of its Euler number, surface and volume rendering and wire-frame visualization were performed. Our findings showed that villous capillaries are arranged either in a single loop or in a more or less complicated anastomosing system. The results suggest that the combination of confocal microscope capture, methods for 3-D rendering and characterization of topological features reveals valuable information about spatial relationships of tissues in placental villi and the arrangement of the villous microcirculation, e.g. about the branching pattern of capillaries.

Comparison of the thickness of the capillary basement membrane of the human placenta under normal conditions and in type 1 diabetes.

Using a morphometric method, the author measured the thickness of the capillary basement membrane in the placentas of healthy women in the first two trimesters and at the end of pregnancy, and in the placentas of diabetic mothers at the end of pregnancy. The mean thickness of the basement membrane of the placental capillaries was found to increase with the progress of pregnancy and to attain the maximum at the end of pregnancy. The placenta of diabetic patients has a significantly thinner capillary basement membrane than the placenta of healthy women. The findings are discussed.

Changes in the proportion of free and bound ribosomes in the syncytiotrophoblast of normal human placenta during pregnancy.

Samples of normal human placental tissue from the first and second trimester and the end of pregnancy were processed for electron microscopy. The ratio of free to bound ribosomes in the syncytiotrophoblast was determined by the point-counting method in photographs giving a 45,000-fold final magnification. The ratio of free to bound ribosomes was found to be significantly higher at 9-11 weeks than at 6-8 weeks and to be significantly lower at 12-14 weeks than at 9-11 weeks. No significant differences were found between the 12th to 14th week and the 20th to 25th week. These data were compared with the ratio of free to bound ribosomes in different regions of the placenta at the end of pregnancy.

Determination of local variation of the thickness of the capillary basement membrane of the human placenta at term by a morphometric method.

Material was taken from exactly determined sites on five placentas to determine local variation of the thickness of the capillary basement membrane in the normal human placenta at term. The thickness of the capillary basement membrane was determined in electron micrographs by a morphometric method and the resultant values were processed statistically. The results and the method are both discussed.