Showing papers by "Alexandra Giatromanolaki published in 2013"

Postmastectomy Hypofractionated and Accelerated Radiation Therapy With (and Without) Subcutaneous Amifostine Cytoprotection

Abstract: Purpose Postmastectomy radiation therapy (PMRT) provides major local control and survival benefits. More aggressive radiation therapy schemes may, however, be necessary in specific subgroups, provided they are safely administered. We report the tolerance and efficacy of a highly accelerated and hypofractionated regimen (HypoARC). Methods and Materials One hundred twelve high-risk patients who had undergone mastectomy received 10 consecutive fractions of 3.5 Gy in 12 days (thoracic wall and axillary/supraclavicular areas). Two consecutive additional fractions of 4 Gy were given to the surgical scar area (electrons 8-10 MeV) and 1 3.5-Gy fraction to the axilla (in cases with extensive nodal involvement). A minimum follow-up of 24 months (median, 44 months) was allowed before analysis. Of 112 patients, 21 (18.7%) refused to receive amifostine, the remaining receiving tolerance-based individualized doses (500-1000 mg/day subcutaneously). Results By use of a dose individualization algorithm, 68.1%, 11%, and 18.7% of patients received 1000 mg, 750 mg, and 500 mg/day of amifostine. Patchy moist skin desquamation outside and inside the booster fields was noted in 14 of 112 (12.5%) and 26 of 112 (23.2%) patients, respectively. No case of acute pneumonitis was recorded. High amifostine dose offered a significant skin protection. Within a median follow-up time of 44 months, moderate subcutaneous edema outside and within the booster thoracic area was noted in 5 of 112 (4.4%) and 8 of 112 (7.1%) cases, respectively. Intense asymptomatic radiographic findings of in field lung fibrosis were noted in 4 of 112 (3.6%) patients. Amifostine showed a significant protection against lung and soft tissue fibrosis. A 97% projected 5-year local relapse free survival and 84% 5-year disease-specific survival were recorded. Lack of steroid receptor expression, simple human epidermal growth factor 2 positivity, or triple negative phenotype defined higher metastasis rates but had no effect on local control. Conclusions PMRT with HypoARC showed an excellent early and short-term late toxicity profile, and amifostine further reduced early and late radiation sequelae. Encouraging local control rates are obtained in high-risk subgroups.

Autophagy and Bcl‐2/BNIP3 death regulatory pathway in non‐small cell lung carcinomas

Abstract: We recently showed that non-small cell lung carcinomas (NSCLCs) are of dismal prognosis when encompassing accelerated autophagic activity. The regulation of this abnormally functioning degradation system and its association with hypoxia and apoptosis in lung carcinoma patients is unexplored. In this study we used 115 NSCLC tissues to examine the immunohistochemical expression of four distinct molecules - the major regulator of autophagy Beclin 1, the anti-apoptotic and anti-autophagic protein Bcl-2, the pro-apoptotic and pro-autophagic protein BNIP3, and a marker of hypoxia and glucolysis, the glucose transporter Glut 1. Most cases showed reduced reactivity for Beclin 1 (62%) and Bcl-2 (82%) proteins, almost half of our sample revealed strong BNIP3 expression (57%), whereas most of the carcinomas strongly expressed Glut 1 antigen (71%). Beclin 1 expression showed no association with survival. Bcl-2 positivity was a marker of good prognosis (p = 0.04), whereas BNIP3 (p = 0.0004) and Glut 1 (p = 0.03) expression correlated with poor outcome in Stage I disease. Autophagic status was negatively associated with Bcl-2 (p = 0.0006), but positively with Glut 1 expression (p = 0.001). In conclusion, the accelerated autophagic status in NSCLC is unrelated to Beclin 1 and BNIP3 expression, but does show significant association with Bcl-2 reactivity. Furthermore, we showed important correlations between glucolysis and autophagy, guiding new pathways in future lung carcinoma research.

Autophagy and hypoxia in colonic adenomas related to aggressive features.

Abstract: Aim The study investigated whether autophagic activity and hypoxia parallel the adenoma–carcinoma sequence. Method The study comprised 120 tubular adenomas with high-grade dysplasia, including 22 with questionable evidence of invasion, 37 with definite stromal invasion and 29 with severely dysplastic adenoma, 10 traditional serrated adenomas and 22 classical tubular adenomas lacking aggressive features. The samples were stained immunohistochemically for autophagy (LC3A and Beclin-1) and hypoxia-inducible factor1-alpha (HIF1α) markers. Results LC3A was detected as diffuse cytoplasmic staining and as dense “stone-like” structures (SLS) within cytoplasmic vacuoles. Beclin-1 reactivity was purely cytoplasmic, whereas that of HIF1α was both cytoplasmic and nuclear. SLS counts in noninvasive, nontransformed areas of tubular adenomas were consistently low (median SLS = 0.5; 200× magnification), whereas a progressive increase was noted from areas of equivocal invasion (median SLS = 1.3; 200× magnification) and intramucosal carcinoma (median SLS = 1.4; 200× magnification) to unequivocal invasive foci (median SLS = 2.1; 200× magnification) (P < 0.0001). A similar association was shown for Beclin-1 and HIF1α expression (P < 0.05). Traditional serrated adenomas yielded low SLS counts and weak HIF1α reactivity, but high cytoplasmic LC3A and Beclin-1 expression (P < 0.01). Conclusion A hypoxia-driven autophagy in adenomatous polyps, when particularly intense and localized, is commonly associated with early invasion or severely dysplastic adenoma.

Pulmonary Mucus Gland Adenomas: Are They Always of Endobronchial Localization?

Abstract: Mucus gland adenoma is an extremely rare benign lung tumor, presumed to arise from the bronchial mucus glands; it is a TTF-1 negative tumor, centrally located, causing the clinical manifestations of obstruction. We report a TTF-1 negative mucus gland adenoma, arising into the medial bronchopulmonary segment, lacking any relation to a bronchus.

High DLL4 expression in tumour-associated vessels predicts for favorable radiotherapy outcome in locally advanced squamous cell head-neck cancer (HNSCC).

Abstract: Expression of the DLL4 (a notch pathway ligand) by tumor-associated endothelium is a postulated marker of vascular maturity and functionality. As vascular functionality is an important parameter defining chemotherapy and oxygen intra-tumoral distribution, we investigated the role of DLL4 expression in tumour vasculature in the efficacy of radio-chemotherapy for HNSCC patients. Sixty-five biopsy specimens from HNSCC patients with inoperable disease were immunohistochemically examined using anti-CD31 (pan-endothelial cell marker) and anti-DLL4 antibodies and the vascular density (VD) was recorded. Patients were treated with platinum based hypofractionated accelerated conformal radiotherapy. The median follow-up period was 24 months (4–80 months). Using the 33rd and 66th percentiles cases were grouped in three categories of low, medium and high CD31+ or DLL4+ VD. The percentage of vessels expressing DLL4 (DLL4-ratio) ranged from 17 to 100 % (mean 71 %), showing substantial variation among cases. In accordance with previous published studies, a biphasic pattern of association of CD31+ VD with poor outcome was noted. Cases with a medium VD had a significantly better local relapse free survival (LRFS) compared to cases of high VD (p = 0.0005, HR 0.15) and of low VD (p = 0.02, HR 0.28). High DLL4/CD31 ratio defined improved LRFS in both these subgroups of poor prognosis. The expression of DLL4 is associated with reduced radio-resistance, presumably by reducing hypoxia and improving chemotherapy accessibility. Using the combination of CD31 and DLL4 staining, a classification is suggested so that HNSCCs are categorized in sub-groups to be targeted by different anti-angiogenic and hypoxia targeting agents.

Mismatch repair hMSH2, hMLH1, hMSH6 and hPMS2 mRNA expression profiles in precancerous and cancerous urothelium

Abstract: Changes in the expression of the mismatch repair (MMR) genes hMSH2, hMLH1, hMSH6 and hPMS2 reflect dysfunction of the DNA repair system that may allow the malignant transformation of tissue cells. The aim of the present study was to address the mRNA expression profiles of the mismatch DNA repair system in cancerous and precancerous urothelium. This is the first study to quantify MMR mRNA expression by applying quantitative real-time PCR (qPCR) and translate the results to mRNA phenotypic profiles (r, reduced; R, regular or elevated) in bladder tumors [24 urothelial cell carcinomas (UCCs) and 1 papillary urothelial neoplasm of low malignant potential (PUNLMP)] paired with their adjacent normal tissues (ANTs). Genetic instability analysis was applied at polymorphic sites distal or close to the hMSH2 and hMLH1 locus. Presenting our data, reduced hMSH2, hMSH6 and hPMS2 mRNA expression profiles were observed in cancerous and precancerous urothelia. Significantly, the ANTs of UCCs revealed the highest percentages of reduced hMSH2 (r2), hMSH6 (r6) and hPMS2 (p2) mRNA phenotypes relative to their tumors (P<0.03). In particular, combined r2r6 (P<0.02) presented a greater difference between ANTs of low-grade UCCs vs. their tumors compared with ANTs of high-grade UCCs (P= 0.000). Reduced hMLH1 (r1) phenotype was not expressed in precancerous or cancerous urothelia. The hMSH6 mRNA was the most changed in UCCs (47.8%), while hMSH2, hMLH1 and hPMS2 showed overexpression (47.8, 35 and 30%, respectively) that was associated with gender and histological tumor grading or staging. Genetic instability was rare in polymorphic regions distal to hMLH1. Our data reveal a previously unrecognized hMSH2 and hMSH6 mRNA combined phenotype (r2r6) correlated with a precancerous urothelium and show that hMLH1 is transcriptionally activated in precancerous or cancerous urothelium. In the present study, it is demonstrated that reduction of hMSH6 mRNA is a frequent event in bladder tumorigenesis and reflects a common mechanism of suppression with hMSH2, while alterations of hMSH2 or hMLH1 mRNA expression in UCCs does not correlate with the allelic imbalance of polymorphic regions harboring the genes.

Patterns of autophagy in urothelial cell carcinomas--the significance of "stone-like" structures (SLS) in transurethral resection biopsies.

Abstract: Objectives To investigate the microtubule-associated protein LC3A, presumed to reflect autophagic activity, in urothelial cell carcinomas (UCC) for its relevance with muscle invasion in transurethral resection (TUR) biopsies. The LC3A antibody is specific for identifying the autophagy-related protein Atg8 and, hence, autophagy—a self-degradation mechanism by which cells recycle their own cytoplasmic constituents, providing with additional energy the rapidly proliferating cells. Methods The study comprised 210 TUR specimens of UCC of the urinary bladder: 70 low-grade non-muscle-invasive (NMI, group A), 70 high-grade NMI (group B), and 70 high-grade muscle invasive tumors (group C). These, together with 40 controls, were stained for Atg8/LC3 using an automated immunohistochemical technique. Results The LC3A was detected as diffuse cytoplasmic staining, and as dense, spheroidal, “stone-like” structures (SLS) of variable size (1.2–12.0 μm in diameter), typically enclosed within cytoplasmic vacuoles. The LC3A reactivity, whether expressed in the form of SLS or as diffuse cytoplasmic staining, was higher in high-grade UCC than in low-grade disease and, more importantly, it was associated with muscle invasion. The median number of SLS per optical field, per section was 17.0, 19.0, and 37.0 for groups A, B, and C, respectively (A, B vs. C P 0.0001; A vs. B P = 0.27). The median SLS diameter was 4.9, 5.3, and 9.3 μm for groups A, B, and C respectively (A, B, vs. C P P = 0.03). Conclusion It appears that the LC3A protein is closely connected with muscle invasion, but whether this finding is of clinical value in TUR specimens lacking muscularis propria remains to be proven.

Long-term angiogenic activity of free grafts and pedicle flap in a rabbit urethroplasty model.

Abstract: We studied the late angiogenic activity of free grafts and a pedicle flap in a rabbit urethroplasty model to determine whether angiogenic activity plays a role in late outcomes of urethral reconstruction in rabbits. Twenty-eight rabbits were randomly divided into five groups according to the method used to bridge a urethral defect as an onlay patch: Control, simple closure of urethral defect (Group O1); free penile skin graft (FPSG, Group A1); buccal mucosal graft (BuMG, Group B1); bladder mucosal graft (BlMG, Group C1); and pedicle penile skin flap (PPSF, Group D1). Angiogenic activity of the patch on postoperative day 84 was assessed by immunohistochemistry. The angiogenic activity in Groups O1, A1, B1, C1, and D1 was 23.33 ± 4.92 (means ± SD), 42.89 ± 6.52, 55.78 ± 3.46, 53.61 ± 6.17, and 24.11 ± 9.07 vessels per optical field, respectively. There were statistically significant differences (p < .001) between Group O1 and A1 B1, C1, Group A1 and B1, C1, D1, Groups B1 and D1 and Groups C1 and D1, but not between Groups O1 and D1 (p = 1.000) and Groups B1 and C1 (p = .872). The long-term angiogenic activity of all the groups was significantly lower (p < .001) than in the corresponding early groups. Although the angiogenic activity of all the groups decreased in the late assessment, the buccal mucosal graft continued to exhibit elevated angiogenesis above bladder or skin (free or pedicle) graft. Therefore, buccal mucosal patch graft might be preferable because of its easier harvesting.

LC3 immunostaining pitfalls.

Abstract: Sir: We read with interest the article by Choi et al. on the expression of the autophagosome-related proteins LC3A and LC3B in a large series of breast cancer cases. Although the authors failed to find any overall association with prognosis, LC3A and LC3B were directly related to high-grade tumours and a triple-negative pattern. The authors are to be congratulated on producing such a thorough investigation, and their findings are, by and large, in accordance with a previously published study of ours, in which cytoplasmic LC3A overexpression was associated with nodal involvement, but not with patient survival. However, a tumour-specific pattern, so-called ‘stone-like’ structures, which was identified in a variety of epithelial carcinomas by our group, including breast, endometrial, colon, lung, skin and bladder carcinomas, has not been recognized in the study of Choi et al. The LC3A-positive stone-like structures, which we consider to be a hallmark of epithelial cancer tissues, are readily recognized as large, rounded, densely stained material, typically enclosed within an LC3A-expressing cytoplasmic vacuole (Figure 1A,B). Choi et al. have offered an explanation for their negative result by proposing probable differences in the specificity of the LC3A antibody used in their study. Indeed, the choice of a suitable antibody is of crucial importance in autophagy research, particularly in demonstrating immunohistochemically the stonelike autophagic pattern. Our experience suggests that the anti-LC3A antibody AP1805a (Abgent, San Diego, CA, USA) provides more clear and specific immunostaining than other tested commercially available antibodies, including the anti-LC3A antibody EP1528Y (Abcam, Cambridge, UK) used by Choi’s group. In a pilot study, this latter antibody produced specific staining in only a minority of breast cancer cases, and the staining was invariably of weak intensity. This finding is in accordance with that reported by Choi et al., who found strong and extensive (>30% of cells) staining in only 6% of the cases examined; a strikingly high proportion of tumours, up to 93.7%, were LC3A-negative. In a subsequent study, and in an attempt to increase the proportion of LC3A-positive cells, the authors considered as positive even cases with weak staining intensity and a very low percentage of positive cells. These staining patterns obtained with the EP1528Y antibody are in direct contrast to those obtained with the AP1805a antibody, which showed strong and extensive (>50% of cells) positivity in >30% of breast carcinomas. By use of the AP1805a antibody, the LC3A-positive ‘stonelike’ structures were readily recognized (Figure 1A,B) and they were related to an unfavourable prognosis. Similar results have been reported recently by Spowart et al., who, after using the AP1805a antibody, not only demonstrated stone-like structures in ovarian carcinomas but also associated their increased number with a poor outcome. It is therefore mandatory for authors to pay extra attention when they choose LC3 antibodies for autophagy research. Vigorous validation is necessary, because there is uncertainty regarding which form of

Low-Grade Uterine Epithelioid Hemangioendothelioma Presented as a Submucosal Leiomyoma during Labor

Abstract: With the exception of leiomyomas, soft tissue tumors of the uterine corpus are not common. This is particularly true for vascular neoplasms, with the epithelioid hemangioendothelioma being a curiosity; not more than twenty-two cases of malignant hemangioendotheliomas have been reported in the literature so far, all of which were high-grade hemangioendotheliomas (hemangiosarcomas). We present herewith a unique case of low-grade epithelioid hemangioendothelioma of the uterus in a pregnant woman aged 29 years. The clinical, histological, and immunohistochemical characteristics of this entity, together with its differential diagnosis, are discussed.

Endometrial Stromal Hyperplasia: An Underrecognized Condition

Abstract: Hyperplasia of the endometrial stroma is a poorly recognized lesion, lacking widespread recognition with most, if not all, such cases sequestrated in the literature as endometrial stromal nodules or low-grade endometrial stromal sarcomas. In this paper, we describe three examples of “endometrial stromal hyperplasia” which have a remarkable morphological similarity with the normally proliferating endometrial stroma and the endometrial stromal neoplasms, but which also possess subtle, but sufficient, differences to justify their taxonomic separation.

Immunohistochemical studies of wound healing after monopolar electrocautery and ultrasound submucosal inferior nasal turbinate reduction in sheep.

Abstract: BACKGROUND: Extracellular matrix (ECM) proteins such as fibronectin and collagen III, enzymes such as matrix metalloproteinases and macrophages have been demonstrated to intervene in nasal and paranasal sinuses wound healing AIM OF THE STUDY: To compare concentration of ECM proteins, enzymes and the recruitment of macrophages during wound repair after monopolar electrocautery in contrast with ultrasound submucosal surgical tissue reduction of inferior nasal turbinate (INT) tested in sheep MATERIALS AND METHODS: Prospective controlled study in sheep Immunostaining for collagen III, fibronectin, CD68 and matrix metalloproteinase-9 (MMP9) was applied in tissue specimens of INT mucosa after monopolar electrocoagulation (MEC) and ultrasound tissue reduction (UTR) Twelve INTs were studied 1, 3 and 8 weeks post-operatively in each interventional group (MEC and UTR) and 5 INTs were studied in animals of the control group (without surgery) The immunoreactivity was quantitatively graded between 0% to 100% immunoreactivity by a blinded senior pathologist RESULTS: At the end of the study period collagen III, fibronectin and MMP9 were increased in both groups compared to the levels of the control group When compared to control group, CD68 immunoreactivity was found higher in MEC group but not in UTR group Fibronectin subepithelial immunoreactivity exhibited a substantial negative correlation with mucosal epithelial cell necrosis, a substantial positive correlation with fibrosis in MEC-treated specimens and a significant positive correlation with sinusoid engorgement in UTR-treated specimens Collagen III tissue immunoreactivity showed a particularly significant negative correlation with sinusoid engorgement in MEC-treated specimens CONCLUSION: Correlation of fibronectin and collagen III immunoreactivity to histopathologic findings suggests different ECM repair processes between MEC and UTR turbinate tissue reduction The use of CD68 and MMP9 provides additional clues to the mode of actions of these techniques and to the molecular and cellular events of the nasal mucosa wound healing process