ROMANIAN JOURNAL of MORPHOLOGY and EMBRYOLOGY

Vol. 47 No. 1, January-March 2006

1. Tumor angiogenesis in gastric cancer

Daniela Lazăr, M. Raica, I. Sporea, Sorina Tăban, A. Goldiş, Marioara Cornianu

Growth of solid tumors, including gastric cancer, and the formation of metastasis depend on the induction of new blood vessels; in tumors, angiogenesis is uncontrolled and immature. This is a complex process, depending on a great variety of angiogenic factors, one of the most important being the vascular endothelial growth factor. In order to suppress the tumor development and the occurrence of metastasis, clinical trials have been developed with angiogenesis inhibitors, many of them with encouraging results. Further research is needed in regard to the idea of combined antiangiogenic therapy with conventional chemotherapy, or even immune or genetic therapies, in order to increase treatment efficiency and to suppress side effects.

Corresponding author: Daniela Lazăr, MD, PhD, e-mail: gene_lazar@yahoo.com

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2. The HOPE technique opens up a multitude of new possibilities in pathology

E. Vollmer, J. Galle, D. S. Lang, S. Loeschke, H. Schultz, T. Goldmann

Fixation of tissues with formalin results in well-preserved morphology but to a high degree leads to degradation of nucleic acids, which substantially constricts the spectrum of applicable molecular techniques. The novel HOPE-fixative with subsequent paraffin embedding, as an alternative to formalin, has been shown to result in a morphological preservation comparable to formalin-fixed, paraffin-embedded specimens. Due to a similar workflow like in formalin-fixation and paraffin embedding, the HOPE technique can be successfully established within any pathological institute. We have shown that DNA, RNA and proteins are protected in HOPE-fixed, paraffin-embedded tissues for at least eight years. Moreover, we described procedures which permit successful application of all common molecular techniques such as in situ hybridization targeting either DNA or RNA, immunohistochemistry without antigen retrieval and for formalin-refractory antigens, PCR, RT-PCR, Western blot, Northern blot, and transcription microarrays to HOPE-fixed, paraffin-embedded tissues. Furthermore, HOPE-fixed tissues can be used for the construction of tissue microarrays for enhanced high-throughput analyses on the molecular level. Using the HOPE technique as its crucial methodological base, ex vivo model systems could be established, e.g. for the simulation of early events in human infections and detection of chemotherapy resistances in human cancer. In addition to tissues, cell-culture preparations have been prepared utilizing the HOPE technique, which were then successfully applied to in situ hybridization targeting mRNA or immunocytochemistry with excellent preservation of morphological details. Taken together, the HOPE technique to date represents an alternative fixation that is, in contrary to other procedures, scientifically broadly analyzed. Therefore, new possibilities are opened up especially within the rapidly growing field of molecular pathology.

Corresponding author: Ekkehard Vollmer, MD, PhD, e-mail: evollmer@fz-borstel.de

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3. Digitized pathology: theory and experiences in automated tissue-based virtual diagnosis

K. Kayser, D. Radziszowski, P. Bzdyl, R. Sommer, G. Kayser

Aims. To describe the theory and develop an automated virtual slide screening system. Theoretical considerations. Tissue-based diagnosis separates into (a) sampling procedure to allocate the slide area containing diagnostic information, and (b) evaluation of diagnosis from the selected area. Nyquist's theorem broadly applied in acoustics, serves to presetting the sampling accuracy. Tissue-based diagnosis relies on two different information systems: (a) texture, and (b) object information. Texture information can be derived by recursive formulas without image segmentation. Object information requires image segmentation and feature extraction. Both algorithms complete another to a 'self-learning' classification system. Methods. Non-overlapping compartments of the original virtual slide (image) are chosen at random with predefined error-rate (Nyquist's theorem). The standardized image compartments are subject for texture and object analysis. The recursive formula of texture analysis computes median gray values and local noise distribution. Object analysis includes automated measurements of immunohistochemically stained slides. The computations performed at different magnifications (x2, x4.5, x10, x20, x40) are subject to multivariate statistically analysis and diagnosis classification. Results. A total of 808 lung cancer cases of diagnoses groups: cohort (1) normal lung (318 cases) - cancer (490 cases); cancer subdivided: cohort (2) small cell lung cancer (10 cases) - non-small cell lung cancer (480 cases); non-small cell lung cancer subdivided: cohort (3) squamous cell carcinoma (318 cases) - adenocarcinoma (194 cases)- large cell carcinoma (70 cases) was analyzed. Cohorts (1) and (2) were classified correctly in 100%, cohort (3) in more than 95%. The selected area can be limited to 10% of the original image without increased error rate. A second approach included 233 breast tissue cases (105 normal, 128 breast carcinomas) and 88 lung tissue cases (58 normal, 38 cancer). Texture analysis revealed a correct classification with only 10 training set cases in >92% for both, breast and lung tissue. Conclusions. The developed system is a fast and reliable procedure to fulfill all requirements for an automated 'pre-screening' of virtual slides in tissue-based diagnosis.

Corresponding author: Klaus Kayser, MD, PhD, e-mail: klaus.kayser@charite.de

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4. Evaluation and quantification of morphological characteristics associated to hepatitis C virus infection: comparative study with hepatitis B

Marioara Cornianu, Alis Dema, Sorina Tăban, Daniela Lazăr, Elena Lazăr, Corina Vernic

Hepatic histological features described in hepatitis C virus (HCV) infection include bile duct damage, lymphoid follicles and/or aggregates in portal tracts, large- and small-droplet fat, Mallory body-like material in hepatocytes, liver cell dysplasia and multinucleation, and activation of sinusoidal inflammatory cells. We have examined the frequency of these lesions in 189 liver biopsy specimens, to distinguish HCV from hepatitis B infection. We have analyzed a set of three features of tures more likely to be seen in HCV than in HBV infection: lymphoid follicles and/or aggregates (68%/20%), bile duct damage (44%/22%) and large-droplet fat (77%/64%). A fourth lesion, Mallory body-like material, was seen more frequently in HCV than HBV (25%/5%). These four histological lesions are useful pathological parameters in the diagnosis of liver disease caused by HCV.

Corresponding author: Marioara Cornianu, MD, PhD, e-mail: sorinataban@yahoo.com

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5. Vascular convolutes in the brain: a peculiar entity

D. Arsene, Carmen Ardeleanu

The present study intends to systematically assess a lesion with potential clinical expression - the vascular convolutes of the brain parenchyma. Those, firstly described by Gertz and Frydl in 1987, are frequently reported in various studies as being associated to aging. Their mechanism seems to be an arteriolar tortuosity. The association with pathologically important manifestations, as brain infarct or hemorrhage could be of clinical importance. The study was done on 70 consequently autopsied cases, on paraffin-embedded material, using the classical histological staining methods: Hematoxylin-Eosin and Masson's Trichrome, as well as the Gomori silver method for reticulum. In our series, only two cases had specific features suggesting vascular convolutes. More frequently were encountered vascular tortuosities, which seems unrelated to the former. We conclude that vascular convolutes and vascular tortuosities are dissimilar lesions. Both are less represented in reality than reported in the literature. Possible mechanisms of the two changes and their pathogenic significance are discussed.

Corresponding author: Dorel Arsene, MD, PhD, e-mail: dorelarsene@yahoo.com, dorelarsene890@hotmail.com

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6. Histopathological and immunohistochemical aspects in both basaloid and spindle cell variant of cervical carcinoma

Daniela Drîgnei-Veselu, Cristiana Simionescu, Claudia Valentina Georgescu, Fl. Bogdan

The study was performed by using a number of eight cases of cervical carcinomas suiting to basaloid carcinomas and spindle cell carcinomas under the circumstances of usual histopathological staining. Immunohistochemically we used anticytokeratin monoclonal antibody (AE1/AE3) to confirm the epithelial differentiation and also PCNA to evaluate the cell proliferation rate. Tumor positiveness for AE1/AE3 cytokeratin cocktail confirmed its epithelial nature. Immunoexpression for PCNA indicated a high index of cell proliferation for all the cases with a more increased value for the basaloid carcinomas.

Corresponding author: Daniela Drîgnei-Veselu, MD, PhD, e-mail: dana_drignei@yahoo.com

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7. Correlated histological and morphometric study of kidney and adrenal gland from guinea pig exposed to hyperbaric-hyperoxic environment

Mihaela Hîncu, L. C. Petcu, Rodica Mehedinți, T. Mehedinți

Our study revealed morphological changes in the two organs of Guinea pig kidney and suprarenal gland exposed to hyperbaric-hyperoxic environment. Proceeding from these data and knowing that in hyperbaric-hyperoxic environment the production of free oxygen radicals is increased, while the afferent arterioles undergo vasoconstriction, with direct implications on the cellular metabolism, our study puts forth the survey of the influence of hyperoxic environment on kidneys and adrenal gland - complex organs which are exposed to stress.

Corresponding author: Mihaela Hîncu, MD, PhD, e-mail: abgavrila@yahoo.com

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8. Histopatological aspects of endometroid carcinoma in correlation to the state of tumoral progression in women patients during their premenopausal period

Mihaela Doina Morărescu, Cristiana Simionescu, I. Roşculescu, Serenada Bălă, N. Cernea, Emilia Pătru

The performed study has comprised a number of 60 endometrial carcinomas, prevailed from women aged in the premenstrual period, in whose cases we have followed also the appreciation of the differentiating grading as well as the various aspects in neoplasies progression. The appreciation of the differentiating grading has allowed their placing as it follows: 39 cases of endometroid carcinoma well differentiated, seven cases of endometroid carcinoma mildly differentiated and five cases of endometroid carcinoma poorly differentiated. The correlation of the differentiating grading with the stage of tumoral progression has allowed the observation of the fact that while well invasive differentiated endometroid adenocarcinoma have been limited to the level of the uterine body, mildly and poorly differentiated invasive endometroid carcinoma besides profound myometrial invasion have associated cervical invasion and metastasis in structures situated at a real distance.

Corresponding author: Cristiana Simionescu, MD, PhD, e-mail: csimionescu2004@yahoo.com

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9. Considerations on the sinus node microangioarchitecture

Codruța Ileana Petrescu, V. Niculescu, N. Ionescu, M. Vlad, M. C. Rusu

The importance of the sinus node as the cardiac pacemaker is well known. The aim of the present study was to investigate the microangioarchitecture at the level of the sinus node. Ten human adult hearts were injected with India ink in the initial segments of the coronary arteries. Pieces were drawn and diaphanized. The results of the study can be summarized: (1) the sinus node is rather an irregularly shaped structure, with peripheral strands intermingling with strands of the atrial myocardium; at this level two vascular patterns can be recognized: (a) the myocardial capillary networks that parallels the muscular bundles, and (b) the peripheral nodal networks built upon dichotomizing arterioles; (2) it seems that while the thick and large sinus node artery does not branch in the nodal tissue, the blood supply of this tissue is ensured by the peripheral nodal networks; (3) characteristically, in the periphery of the nodal tissue are largely present glomeruli made by capillaries with pericellular dispositions. The results strongly suggest that the nodal tissue is mainly supplied from its periphery and the sinus node artery is rather a scaffold than a supplier of that tissue.

Corresponding author: Mugurel Constantin Rusu, MD, PhD, e-mail: anatomon@gmail.com

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10. Considerations on the left papillary muscles microangioarchitecture

M. C. Rusu, Codruța Ileana Petrescu, V. Niculescu, N. Ionescu, M. Vlad

The aim of the present study was to bring macroscopic and microscopic evidence on the left papillary muscles blood supply in human hearts. For the study were used human adult hearts from patients without clinically known cardiac ischemic history. Ten hearts were used for injecting China ink in the coronary arteries and other twenty hearts were dissected to evidence the characteristics of the main arteries of the left papillary muscles. Pieces - left papillary muscles - were drawn from the injected hearts and diaphanised. In all dissected hearts, the left anterolateral papillary muscles were supplied by the left coronary system: anterior interventricular artery, second diagonal branch and left (obtuse) marginal artery. In 70%, the left posteromedial papillary muscles were supplied by the right coronary system (posterior interventricular artery, left retroventricular artery) and in 30% by the left coronary system (circumflex artery). The left papillary muscles were supplied each by one or two main arteries that penetrated the muscles longitudinally. The ventricular wall attaching the papillary muscles was supplied by the subepicardial vessel sending the main arteries of the papillary muscles but also by neighbor subepicardial vessels distributed in that wall. The mural vessels were finer than the papillary muscles main arteries. Injected papillary muscles presented each with two systems of blood perfusion: one represented by segmental centers of arterial branching and distribution of the main arteries of the muscle and other represented by capillary extensions of the mural networks at that level. From the segmental branching centers were perfused the neighbor segments of the papillary muscles and intrasegmental anastomoses were recognized. The microvascular study of the left papillary muscles proves the usual overlapping of sources for segmental supply; this overlapping is reinforced by the high capillary density to ensure the vascularisation of the papillary muscles.

Corresponding author: Mugurel Constantin Rusu, MD, PhD, e-mail: anatomon@gmail.com

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11. Modifications of the dental pulp in marginal periodontitis. Electronomicroscopical contributions

V. Deva, Maria Vătăman, H. Manolea

Histological studies on teeth with marginal periodontitis, but without cavity lesions have shown a frequent apparition of modifications in the pulp tissue structure. In this study, by using the electron microscopy method, we have shown a series of interesting aspects of the intimate modifications that appear on the level of all components of the pulp tissue. The observations show that the degree of affecting can be correlated to the type of marginal periodontitis, and at the same time, they contribute to a right evaluation of the defensive abilities of the pulp of the teeth with marginal periodontitis.

Corresponding author: Virgil Deva, MD, PhD, e-mail: virgil_dv@yahoo.com

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12. Morphological considerations about middle ear cholesteatoma

Nicoleta Măru, Fl. Pop

The aim of this study is to present various morphologic aspects of the middle ear cholesteatoma, concerning both container (tympanic cavity) and content (cholesteatoma). There are two different aspects of the study: a mezoscopic study of the tympanic walls and the elements within the middle ear (ossicular chain, folds, ligaments, middle ear clefts) in order to evidentiate the critical areas (sinus tympani, facial sinus, anterior attic) and the pathways of spread for the cholesteatomas; a classical histological study of the middle ear cholesteatomas, in order to present the structural parts and the modifications in the neighboring structures.

Corresponding author: Nicoleta Măru, MD, PhD, e-mail: nicoletamaru@yahoo.com

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13. The value of radio-imaging methods in the diagnosis of chest wall metastases

S. Bolintineanu, S. Brad, Viorica Brad, A. Motoc

The aim of the study was to reveal the value of radio-imaging methods in the diagnostic assessment of chest wall metastases. We retrospectively reviewed 48 cases of chest wall metastases examined clinically, radiologically, by ultrasound, CT-scan and MRI. All cases were histologically assessed by surgical biopsies. Plain chest radiography (X-ray) was in all cases a prerequisite for any evaluation of the chest wall being of great value in demonstration of the displaced rib fractures, localized bony destruction and the detection of most soft tissue lesions. The ultrasound examination (US) allowed real time analysis and thus the functional imaging of the chest wall metastases with special interest in vascular impairment being limited by relatively small field of view with more difficulties in anatomical orientation. With involvement of bone marrow and spinal cord, MRI became the most sensitive and the most specific imaging method, successfully diagnosing intramedullar lesions in three cases. In the detection of chest wall infiltration by primary malignancies from the lung, pleura, pericardium, breast and spinal cord, we preferred the CT-scan, which revealed the presence of the lesions in 29 cases with typically obtuse angles against the adjacent chest wall and pleura. In the imaging evaluation of malignant lymphoma, the CT-scan demonstrated chest wall extension in five cases. CT-scan and MRI revealed chest wall localization for distant metastases in 14 cases. The imaging methods clearly showed the number, the extent, the anatomic reports and the aggressive characteristics of chest wall metastases being of great value in the decision of adequate therapy.

Corresponding author: Sorin Bolintineanu, MD, PhD, e-mail: amotoc@mail.dnttm.ro

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14. Ectopic osteogenesis in the rectus sheath

C. Meşină, Mihaela-Iustina Meşină-Botoran, G. S. Drăgoi, M. Paşalega, D. Vîlcea, I. Vasile

Although was published many cases of ectopic osteogenesis of traumatic, neurogenic cause or hereditable form, the etiology of ectopic osteogenesis remaining unknown. We present ectopic osteogenesis in the rectus abdominal sheath. The study material was represented from fragments of ectopic bones discovered in rectus sheath of four patients suffering iterative surgical abdominal interventions. The pieces of ectopic bone were decalcified and then were made to the standard techniques (paraffin inclusion, general techniques dyeing). The process of ectopic osteogenesis was analyzed through microscopically study to seriated sections of discovered piece, finding the presence of the hematopoesis foci. We conclude that is important identifying and characterizing the osteoinductor agents because these allowed the study of osteogenesis to the cellular level and make an estimation of the abnormally bone developing mechanisms. A possible osteoinductor factor has been considerate the non-absorbable wound closure material.

Corresponding author: Cristian Meşină, MD, PhD, e-mail: mesina.cristian@doctor.com

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