Chondrosarcoma with Target-Like Chondrocytes: Update on Molecular Profiling and Specific Morphological Features.

This is the first histological and molecular analysis of two chondrosarcomas with target-like chondrocytes that were compared with a group of conventional chondrosarcomas and enchondromas. The unique histological feature of target-like chondrocytes is the presence of unusual hypertrophic eosinophilic APAS-positive perichondrocytic rings (baskets). In the sections stained with Safranin O/Fast green, the outer part of the ring was blue and the material in the lacunar space stained orange, similarly to intercellular regions. Immunohistochemical examination showed strong positivity for vimentin, factor XIIIa, cyclin D1, osteonectin, B-cell lymphoma 2 apoptosis regulator (Bcl-2), p53 and p16. The S-100 protein was positive in 25 % of neoplastic cells. Antibodies against GFAP, D2-40 (podoplanin), CD99, CKAE1.3 and CD10 exhibited weak focal positivity. Pericellular rings/baskets contained type VI collagen in their peripheral part, in contrast to the type II collagen in intercellular interterritorial spaces. Ultrastructural examination revealed that pericellular rings contained an intralacunar component composed of microfibrils with abundant admixture of aggregates of dense amorphous non-fibrillar material. The outer extralacunar zone was made up of a layer of condensed thin collagen fibrils with admixture of non-fibrillar dense material. NGS sequencing identified a fusion transcript involving fibronectin 1 (FN1) and fibroblast growth factor receptor 2 (FGFR2) at the RNA level. At the DNA level, no significant variant was revealed except for the presumably germline variant in the SPTA1 gene.

Chondrosarcoma with Target-Like Chondrocytes: Update on Molecular Profiling and Specific Morphological Features.

The original article was published in Folia Biologica (Praha) Volume 68, No. 3 (2022), 112-124.

New Developments in Understanding the Histological Structure of Human Ear Cartilage.

Histological, immunohistochemical and molecular examination of bioptic samples of 30 normal adult auricular cartilages and small samples from 6 ear cartilages from aborted foetuses was performed. The adult cartilage was the tissue with minimal proliferative activity, which we were able to confirm with antibodies against Ki67 in contrast to a high proliferative activity in the auricular cartilage of foetal tissues. It may therefore be presumed that the process of foetal tissue maturation is undoubtedly associated with a significant reduction in proliferative activity. The mature lamella of the adult auricular cartilage has a histological tri-lamellar structure. There are a great number of elastic fibres in the intercellular matrix of the central zone, which are conversely present in only small amounts in both peripheral layers. While the external layer of the concave surface of the cartilage contains a fewer number of oval elements, the external layer of the convex side is composed of numerous fusiform chondrocytes. Antibodies against various subtypes of S-100 protein showed that auricular chondrocyte activity is modified depending on the configuration of individual distinct zones (isoforms A1, A6, B2 and P were positive in all layers, isoforms A2 and A2 in peripheral zones). The most active cells metabolically are most likely chondrocytes in both external layers adjacent to the perichondrium. We have also demonstrated α-smooth muscle actin (SMA)-positive chondrocytes in both peripheral layers of the auricular cartilage adjacent to the perichondrium. In addition, we found definite differences in the distribution of actin-positive cells depending on the external shape of the pinna. The majority of these fusiform cells were localised primarily in the areas of great curvature of the pinna, especially the convex side, as mentioned above. On the basis of these unique structural features we assume that the ear cartilage may embody an example of the socalled intelligent biological material, which has its internal structure made in such a way as to more easily develop and yet still maintain all the shape characteristics of the human auricle. The knowledge of these specific structural characteristics is important especially for use of auricular cartilage in auricular reconstruction.

[Structural Damage to the Hamstring Graft due to Interaction with Fixation Material and its Effect on Biomechanical Properties of ACL Reconstruction]. / Strukturální zmeny stepu z hamstringu zpusobené fixacním materiálem a jejich vliv na biomechanické vlastnosti náhrady LCA.

PURPOSE OF THE STUDY Hamstring grafts are commonly used for ACL reconstruction. The purpose of our study is to determine the effects of the suspension fixation compared to graft cross-pinning transfixation, and the effect(s) of structural damage during the preparation of the graft on biomechanical properties of the graft. MATERIAL AND METHODS The design of the study is a cadaveric biomechanical laboratory study. 38 fresh-frozen human hamstring specimens from 19 cadaveric donors were used. The grafts were tested for their loading properties. One half of each specimen was suspended over a 3.3mm pin, the other half was cross-pinned by a 3.3mm pin to simulate the graft cross-pinning technique. Single impact testing was performed and the failure force, elongation and acceleration/deceleration of each graft was recorded and the loading force vs. elongation of the graft specimens was calculated. Results for suspended and cross-pinned grafts were analysed using ANOVA method, comparing the grafts from each donor. RESULTS The ultimate strength of a double-strand gracilis graft was 1287 ± 134 N when suspended over a pin, the strength of a cross-pinned graft was 833 ± 111 N. For double-strand semitendinosus grafts the strengths were 1883 ± 198 and 997 ± 234 N, respectively. Thus, the failure load for the cross-pinning method is only 64.7% or 52.9% for the suspension method. DISCUSSION Structural damage to the graft significantly reduces the graft strength. Also, extensive suturing during preparation of the graft reduces its strength. CONCLUSIONS Fixation methods that do not interfere with the graft's structure should be used to reduce the risk of graft failure. Key words: ACL reconstruction, hamstring graft, biomechanical testing.

Iron Overload Causes Alterations of E-Cadherin in the Liver.

Iron overload causes tissue damage in the liver, but its initial effects at the molecular and cellular level are not well understood. Epithelial cadherin (E-cad) is a major adhesion protein in adherens junctions and is associated with several signal transduction pathways. Dysfunction of E-cad causes instability of adherens junctions, which leads to cell invasion, cell migration, and carcinogenesis. We found in liver samples from iron-overloaded mice that the apparent molecular mass of E-cad was reduced from 125 to 115 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions and immunoblotting, and that the cellular expression of E-cad was decreased in immunohistochemistry. The mRNA level of E-cad, however, did not change significantly, suggesting that the alterations are posttranslational. Interestingly, incubation of control liver extracts with Fe2+ alone also produced the same mobility shift. Neither an oxidant nor an antioxidant influenced this shift in vitro, suggesting that reactive oxygen species, which are generated by iron and known to cause damage to macromolecules, are not involved. Treatment of the 115 kDa E-cad with deferoxamine, an iron chelator, thus removing Fe2+, shifted the molecular mass back to 125 kDa, demonstrating that the shift is reversible. The observation also implies that the alteration that causes the mobility shift is not due to transcriptional control, deglycosylation, and proteolysis. This reversible mobility shift of E-cad has not been previously known. The alteration of E-cad that causes the mobility shift might be an initial step to liver diseases by iron overload.

Comparison of the cellular composition of two different chondrocyte-seeded biomaterials and the results of their transplantation in humans.

Our study compares the histological and immunohistochemical cellular composition of two different chondrocyte-seeded biomaterials and the results of their transplantation. Our study cohort included 21 patients, comprising 19 men and two women with a mean age of 32 years, who were affected by single chondral lesions of the femoral condyles. These patients were enrolled in our study and treated with arthroscopic implantation of the tissue Hyalograft C and/or Brno culture. Brno culture bioengineered with a fibrin-based scaffold contains round cells showing features of differentiated chondrocytes expressing S-100 protein and α-smooth muscle actin. In contrast, in the case of Hyalograft C, the scaffold was made up of a fibrillar network composed of biomaterial fibres of the esters of hyaluronic acid and cells resembling fibroblasts and myofibroblasts and expressing only α-smooth muscle actin. The average size of the defects was 2.5 cm2. Patients were evaluated using the standardized guidelines of the International Knee Documentation Committee. During the comparison of bioptic samples obtained from both patient cohorts, we did not observe any important differences in the histological makeup of the newly formed cartilage. The histological analysis of these two groups of homogeneous patients shows that this bioengineered approach, under proper indications, may offer favourable and stable clinical results over time, in spite of the different matrix and cellular composition of the two transplants used.

Podoplanin (D2-40) is a reliable marker of urinary bladder myofibroblasts (telocytes).

Podoplanin, D2-40, has been described in a variety of normal and neoplastic tissues. It is often used for highlighting lymphatics. We evaluated the expression of podoplanin in α-smooth muscle actinpositive myofibroblasts producing the suburothelial layer in tunica propria of the urinary bladder that have some similar features with telocytes. Our results showed that these cells demonstrate distinct D2-40 immunoreactivity from telocytes occurring in the renal pelvis and ureter. We observed positive reaction not only in bioptic specimens from women with interstitial cystitis, but also in a control group of women and men treated for pathological bladder lesion different from interstitial cystitis. It is interesting that identical staining reaction was observed in the ureters only exceptionally. In addition, we examined samples from myofibroblastic tumoriform lesions of soft tissue such as nodular fascitis and fibromatosis (desmoid) and we obtained negative results. It means that the so-called myofibroblasts of urinary bladder tunica propria have a unique immunophenotype that has probably not been described until now. Our findings suggest that D2-40 can be used as a complementary immunostainer to α-smooth muscle actin on urinary bladder biopsies from patients with interstitial cystitis. The role of D2-40 as an immunohistochemical marker is still being investigated.

[Giant-cell lesions of bone and their differential diagnosis]. / Obrovskobunecné léze kostí a jejich diferenciální diagnostika.

UNLABELLED: Giant-cell lesions of bone-neoplastic and reactive growths form a group of clinicopathological entities that differ in their behaviour and may present substantial problems in differential diagnosis. The presence of multinucleated giant cells of the osteoclast type, reactive osteoplasia and the formation of secondary aneurysmal cysts in many unrelated lesions of bone further complicates their classification. Clinicopathologic correlation of all findings, including the radiologic features and laboratory tests, is of paramount importance in reaching a correct diagnosis in this group of histologically overlapping entities. Malignant features can be identified in the primary and secondary conventional malignant giant cell tumours and in some types of osteosarcoma with a giant cell component. KEYWORDS: giant-cell lesions of bone - giant-cell tumor - osteoclast - osteosarcoma.

Primary vaginal squamous cell carcinoma arising in a squamous inclusion cyst: Case report.

UNLABELLED: We report the case of a 39-year-old female with primary vaginal squamous cell carcinoma arising from a squamous inclusion cyst of the posterior wall. The tumor was located in the vaginal wall and extended into the rectovaginal septum. The overlying mucosa was intact. Histologically, there was invagination of the surface squamous vaginal epithelium forming a cystic lesion. In some areas of this invagination, the squamous epithelium showed dysplastic changes (VAIN3) transitioning into invasive squamous cell carcinoma. To the best of our knowledge, we have documented the first case of primary squamous cell carcinoma arising in a vaginal cyst in a patient without having undergone a previous hysterectomy. KEYWORDS: squamous cell carcinoma - vagina - squamous inclusion cyst - embryonal remnants - rectovaginal septum.

Histopathological autoptic findings in 8 patients with pandemic influenza A (H1N1) pneumonia.

UNLABELLED: In the years 2009 and 2010 a novel influenza A (H1N1) caused the first influenza pandemic after 41 years. In the Czech Republic it culminated in November and December 2009 and there were 101 laboratory-confirmed deaths. Another few cases occurred later in the year 2010 and at the beginning of 2011. Here we report 8 autoptic cases of patients who died between 2009 and 2011 with confirmed H1N1 influenza and underwent a post mortem examination at the Institute of Pathology, General University Hospital in Prague, Czech Republic. This group differs from the others reported in literature by having a higher age as well as a higher percentage of patients with pre-existing severe comorbidities including malignant diseases. All 8 patients developed atypical pneumonia with subsequent respiratory failure. In this article we present these cases with related clinical data and findings in other organs, but we focus primarily on the findings in the respiratory tract which were shown to be approximately similar to those in the other studies and case reports. Nevertheless there were also some noteworthy variations. The most prominent feature observed was diffuse alveolar damage accompanied by intraalveolar haemorrhage and inflammatory infiltrate of variable extent. Less frequent features included cytopathic changes of pneumocytes and their desquamation, reactive changes of bronchial epithelium, intraalveolar fibrinous exudate, minor necroses, residual necrotizing bronchitis, focal granulation tissue and incipient fibrosis. In one case we found an extraordinary vascular change of uncertain origin. In conclusion, this group of patients is slightly atypical and differ in some features from those in other published studies and case reports concerning novel pandemic influenza. By reporting them we wish to extend the number of described cases, which may contribute to a better understanding of the pathogenesis of novel influenza infection. KEYWORDS: influenza A virus, subtype H1N1 - viral pneumonia - diffuse alveolar damage.

Myxoid mixed low-grade endometrial stromal sarcoma and smooth muscle tumor of the uterus. Case report.

We report the case of a 73-year-old female with myxoid mixed low-grade endometrial stromal sarcoma and smooth muscle tumor of the uterus. Grossly, the tumor sized 130 x 130 x 100 mm involved the uterine corpus almost in its entirety. Histologically, the tumor consisted of two cell types. In some areas, the tumor cells showed typical features of endometrial stromal tumors and resembled stromal cells of proliferative endometrium. In other areas, however, the tumor showed smooth muscle features and consisted of larger mostly epitheloid cells with a moderate amount of cytoplasm. In all areas, myxoid changes and multiple hyalinizing giant rosettes were present. The tumor infiltrated the myometrium in a pattern typical of low-grade endometrial stromal sarcoma. Immunohistochemically, the tumor cells showed expression of vimentin, estrogen and progesterone receptors and variable expression of CD10, α-smooth muscle actin, desmin, h-caldesmon, and cytokeratin AE1/AE3. Other markers examined including CD99, α-inhibin, cytokeratin CAM5.2, S-100 protein, and HMB45 were negative. To the best of our knowledge, mixed low-grade endometrial stromal and smooth muscle tumor with myxoid changes has not been described to date.

Comparison of the IHC, FISH, SISH and qPCR methods for the molecular diagnosis of breast cancer.

Her2 proto-oncogene amplification and protein overexpression is observed in 20-40% of patients with breast cancer and plays a crucial role in invasive breast cancer and its treatment. In the present study, we investigated samples from 131 patients with invasive breast carcinoma. In all cases, the overexpression/amplification level of Her2 was determined using manual immunohistochemistry (IHC) and/or automatic IHC, fluorescence in situ hybridization (FISH), silver in situ hybridization (SISH) and quantitative polymerase chain reaction (qPCR). Using various methods, we demonstrated candidate methods for Her2 detection and their dependability. Our results demonstrate that these methods are highly comparable for the detection of Her2 overexpression/amplification. It was also revealed that qPCR is a valuable tool for the evaluation of Her2 gene overexpression/amplification. The results from pPCR analysis positively correlated with the results from IHC and FISH analysis. Moreover, in contrast to IHC or SISH/FISH, the results obtained by qPCR were not encumbered with any subjective error on the part of the evaluator.

Intrapericardial teratoma as a cause of fetal death--a case report.

Intrapericardial teratoma is a rare congenital tumor that without treatment leads to cardiac failure in either the prenatal or postnatal period. Early diagnosis and recent surgical advances can, in some cases, delay development of intrauterine symptoms and allow final treatment through a tumor resection. However, a large number of intrapericardial tumors go undetected during prenatal diagnostics, until they are found as a cause of intrauterine death or postnatal cardiorespiratory insufficiency, as in our case report. An abortion was induced in the 23rd gestational week because there was no cardiac activity detected during a routine ultrasound scan in a 35-year old woman. The tumor was found during the postmortem of the fetus.

Study of the effect of neoadjuvant chemotherapy on the status of Her2/neu.

Her2/neu proto-oncogene amplification and protein over-expression is observed in 20-40 % of patients with breast cancer and plays a crucial role in invasive breast cancer and its treatment. A number of studies postulated the stability of Her2/neu gene expression, showing that in most patients the status of expression had not significantly changed after the neoadjuvant treatment. In the present study, we investigated samples from 20 patients with invasive breast carcinoma who had undergone neoadjuvant chemotherapy and subsequent surgery. In all cases, the expression level of Her2/neu was evaluated in both pre-therapeutically obtained tumour tissue by core needle biopsy and from specimens obtained during final surgery using immunohistochemistry. Fluorescence in situ hybridization and quantitative reverse transcription polymerase chain reaction methods were used for verifying the results obtained by immunohistochemistry. Her2/neu status determined by immunohistochemistry remained unchanged in 12 of 20 (60%) patients after neoadjuvant treatment. In six cases (30%) minor changes were observed after the treatment. However, in two cases (10%) we found altered Her2/neu expression from strongly positive in the pre-treatment biopsy to negative in the post-treatment surgery specimen. Moreover, this is the first report describing the changes in Her2/neu status at all protein, RNA and DNA levels by using immunohistochemistry, quantitative reverse transcription polymerase chain reaction and fluorescence in situ hybridization, respectively. By using variable methods we demonstrated possible new ways for Her2/neu detection and their dependability. Improvement in specific molecule detection can prevent the use of tailored targeted therapy in an untargeted manner.

Glomus tumor of the stomach: a case report and review of the literature.

Glomus tumor is a benign soft tissue neoplasm which commonly affects the subungual region of the fingers. But the tumors can also arise in the other sites such as the antrum of the stomach. We are reporting a case of a glomus tumor of the stomach in a 71-year-old female patient who presented with dyspepsia. The tumor was confined to the lamina muscularis propria, it consisted of round cells with small uniform nuclei, which surrounded thin walled blood vessels. Immunohistochemistry revealed the tumor to be positive for smooth muscle actin, vimentin, calponin, h-caldesmon and negative for c-KIT, S-100, CD34, CD99, synaptophysin, chromogranin, desmin and EMA. The proliferation marker Ki-67 was positive in less than 5% of tumor cell nuclei. Glomus tumors are usually benign but malignant cases have been published. Criteria for the malignant potential of gastric glomus tumors remain poorly defined.

[Current options and principles of pathomorphology-based tumour diagnostics]. / Soucasne moznosti a principy patomorfologické diagnostiky nádoru.

Modern pathomorphology tumour diagnostics involve comprehensive cytology cell examination and, importantly, tissue biopsy examination using the most up-to-date assessment techniques. To correctly classify a tumour process, it is essential not only to define its histological typology and identify its biological character but, at the same time, using immunohistochemistry and diagnostic molecular pathology, to evaluate prognostic and predictive indicators. As regards the prognostic indicators, it is mainly grading and staging as part of the TNM system, the sentinel lymph node assessment, if it is assessed, and other nodes extracted in this context. Histological specimens of invasive carcinomas and skin melanomas are assessed with respect to the depth of the invasion. Endocrine oestrogen and progesterone receptors are assessed in endocrine-dependent breast carcinomas. Predictive pathology uses biomarkers; biomarkers confirm the presence of target molecules following their reaction with the administered small molecule-like drugs, usually protein kinases or antibodies. HER2 gene amplification is estimated using the FISH method and predicts effectiveness of breast cancer therapies. In patients with a colon carcinoma, HER1 oncogene is identified immunohistochemically and the presence of a mutated K-ras is subsequently tested using the RT-PCR technique. CD 117 (c-kit) expression is determined in gastrointestinal stromal tumour and CD 20, 33 and 52 in malignant lymphomas and leukaemias. The most difficult task for a pathologist is defining the primary focus of the tumour in cases where only a fraction of a metastasis is available for examination. Nevertheless, at present, it is feasible to track the tumour differentiation down and, using an antibody battery, particularly against specific cytokeratines and vimentine and including organ-specific markers, to identify the primary source or at least to establish an assumption about the most likely organ affected by the tumour process.

[Systemic amyloidoses in renal biopsy samples]. / Systémové amyloidózy v biopsiích ledvin.

UNLABELLED: The kidneys are one of the most frequent sites of amyloid deposition during systemic AL, AA, and several hereditary amyloidoses. Distinguishing between different forms of amyloids is clinically important because of their different treatment. MATERIAL AND METHODS: We present a 5-year retrospective study of amyloidoses diagnosed in renal biopsy samples. The classification of amyloidosis was made by immunofluorescence and immunohistochemical staining with antibodies to kappa and lambda immunoglobulin light chains, and for serum amyloid A protein. RESULTS: From January 2003 to December 2007, 996 renal biopsy samples from one centre were evaluated. Amyloidosis was diagnosed in 62 samples (6.2%); 33 (53.2%) were classified as AL and 25 (40.3%) as AA amyloidosis. Four cases have remained unclassified. We did not identify any difference in the distribution of deposits among cases with AL and AA amyloidosis, respectively. The majority of patients underwent the renal biopsy due to severe proteinuria or nephrotic syndrome. Three patients had very low proteinuria, less than 0.5 g/day. Diagnosis of amyloidosis was suspected by nephrologists in 48 patients (77.4%). CONCLUSION: Diagnosis of amyloidosis involves detection of amyloid deposits and classification of the amyloid form, which represent the basic step for appropriate therapy. Altogether it is not an easy task for pathologists, and with the emergence of markedly different treatments depending on the specific type of amyloid, the precise typing is of increasing clinical importance and should be performed with great care. Immunofluorescence can be very useful in daily practice for classification of the type of amyloidosis.

Statistical analysis of symptoms, endoscopy and urothelial morphology in 58 female bladder pain syndrome/interstitial cystitis patients.

INTRODUCTION: The goal of the study was to assess the course of painful syndrome in patients with bladder pain syndrome/interstitial cystitis and to assess the changes in endoscopic and histopathological findings in relation to the type of treatment. PATIENTS AND METHODS: We included a total of 58 patients with histologically diagnosed interstitial cystitis. Out of these, 31 patients were treated with oral pharmacotherapy and 27 patients were treated by intravesical application of heparin. The patients were followed from time of diagnosis for 6.9 +/- 2.5 and 6.6 +/- 2.7 years, respectively. RESULTS: Treatment - irrespective of its type - had a clear demonstrable effect on the monitored parameters; intravesical treatment was more effective than oral. Statistically significant (p < 0.05) changes could be observed in both groups (with two exceptions). When evaluating the relationship between subjective symptoms and objective criteria, and patients' age and time to diagnosis, it is clear that the higher the age and the longer the time from symptoms to diagnosis, the more severe the symptoms. CONCLUSIONS: When evaluating the monitored parameters, we found significant correlations (both positive and negative). However, these relationships cannot be used to simplify the evaluation algorithm (according to ESSIC) and the initial criteria cannot predict the course of the disease.

[S-100 protein positivity in osteogenic tumours and tumour-like bone forming lesions]. / Exprese S-100 proteinu v osteogenních nádorech a tumoriformních osteoplastických lézích.

Various osteogenic tumours and bone producing tumour-like lesions of bone were examined for S-100 protein using the immunostaining methods. The positive reaction for S-100 protein of some bone cells was detected not only in osteoblastic osteosarcomas and osteoblastomas, but also in osteoplastic reactive lesions, fibrous dysplasia, Paget's disease and in the tissue of the bony callus. The positive reaction for osteocalcin in these cells showed, that they may be of osteoblastic and osteocytic lineage. The S-100 protein positive bone cells have to be differentiated from chondrocytes persisting in new-formed bone trabecullae. On the one hand S-100 protein positive osteocytes and osteoblasts may represent transient form of osteocytes histogenetically related to chondrocytes, but we were not able to prove such suggestion. Therefore, S-100 protein positivity can be caused by polyclonal character of the used antibody, that is a mixture of three antibodies, S-100 A1, A6 and anti B2. The specificity of these three components differs depending on the histogenesis of cells and their functional status. Such explanation is supported by the results of our study, because we also observed intense positivity of bone cells in the reaction with monoclonal antibody against S-100A6. In contrast, no staining of bone cells was detected with monoclonal antibodies against S-100 A1 and B2.

A comparision of RT-PCR and FISH techniques in molecular diagnosis of Ewing's sarcoma in paraffin-embedded tissue.

Ewing's sarcoma is relatively uncommon tumor representing 6-8 percent of malignant bone tumors with variable morphology. Cytogenetically, Ewing's sarcomas are characterized by a specific reciprocal chromosomal translocation t(11;22)(q24;q12). The presence of this chromosomal translocation has been detected in approximately 85 percent of the cases. The translocation results in the fusion of EWS gene from chromosome 22 to FLI1 gene at 11q24 which is a member of ETS family of transcription factors. In this study we performed a comparison of two molecular diagnostic strategies, namely RT-PCR and FISH, in fresh, frozen and formalin-fixed paraffin-embedded tissues. We conclude that FISH is a more sensitive technique than RT-PCR for the diagnosis of Ewing's tumors in formalin-fixed paraffin-embedded tissue. In conclusion, molecular pathology techniques, using reverse transcription-polymerase chain reaction (RT-PCR) and/or fluorescence in situ hybridization (FISH) are valuable diagnostic tools for evaluation of undifferentiated small round-cell tumors like Ewing's sarcoma.