A 3-bp Deletion VK600-1E in the BRAF Gene Detected in a Young Woman with Papillary Thyroid Carcinoma.

Papillary thyroid cancer (PTC) derived from follicular cells is a frequent thyroid tumor. The incidence of this type of malignancy is still growing worldwide. Several major genetic causes are recognized to cause PTC-mutations in the BRAF and RAS genes or rearrangements with the RET proto-oncogene. The most common genetic change found in PTC is a V600E mutation in the BRAF gene presented in 36-69 % of all PTC cases. For routine purposes, several methods were developed to selectively detect only this mutation. However, these methods miss other mutations in the BRAF gene located elsewhere. We focused on the analysis of the exon 15 of the BRAF gene by next-generation sequencing. Here we report a three nucleotide deletion VK600-1E in one patient and present this finding in the context of 13 previously described PTC cases with this deletion. Our patient is the second youngest one among the reported cases. Clinical features of PTC patients with VK600-1E are summarized. For the future, it is important to evaluate genotype-phenotype characteristics of patients with rare BRAF mutations and to follow up them for years.

Post-mortem analysis of Candida albicans breakthrough infection during echinocandin treatment in haematopoietic stem cell transplant recipient.

We present case of a girl deceased due to Candida albicans breakthrough invasive infection during the echinocandin treatment after undergoing allogeneic haematopoietic stem cell transplant for relaps of acute myeloid leukaemia. Candida albicans generally susceptible to all antifungal drugs wasn't considered for potential resistance and conventional blood culture positivity was too late to reveal the resistance to echinocandins. Due to severe organ toxicities (liver, kidneys) she received echinocandin as an antifungal prophylaxis, no change was made for the treatment of Candida albicans infection. Later, the molecular analysis proved the mutation S645P known as being responsible for the echinocandin resistance. The post mortem analysis of fungal burden in autopsy samples showed very high levels of Candida DNA in gut, liver, spleen and kidneys.

[Nephroblastoma -  30-years period of its treatment in the University Hospital Motol, Prague]. / Nefroblastom -  30 let lécby ve Fakultní nemocnici v Motole.

INTRODUCTION: Nephroblastoma (Wilms tumor - WT) is the most common solid tumor of kidney in children. We present treatment development of WT at the Department of Pediatric Hematology and Oncology, Charles University in Prague, 2nd Faculty of Medicine and University Hospital Motol (KDHO) in the Czech Republic over 30 years. Patients that were treated prior to access to the International Society of Pediatric Oncology (SIOP) protocols are considered to be the historical group, then we have patients treated according to SIOP 9, SIOP 93-01 and SIOP 2001 protocols as full participants of SIOP studies. PATIENTS AND METHODS: Between January 1980 and April 2009, we treated 330 patients with WT at KDHO: 91 patients in historical group (1980-1988), 94 pts in SIOP 9 (1988-1993), 80 pts in SIOP 93-01 (1994-2001) and 65 pts in SIOP 2001 (2002-2009). Overall survival (OS) and event-free survival (EFS) were analyzed by Kaplan-Meier test. RESULTS: The overall ten-year EFS was 81.2% and OS 87.6%. Fifty-eight patients from the 330 (17.6%) had metastases at diagnosis, EFS without metastatic process was 84.6% compared to 65.4% with metastasis presented at diagnosis (p = 0.0003), OS was 70.7% compared to 91.2% (p < 0.0001). One hundred and seventy patients (51.5%) were treated with preoperative chemotherapy and/or radiotherapy, whereas 158 patients (47.5%) underwent primary nephrectomy; EFS and OS did not differ: neoadjuvant vs primary nephrectomy EFS was 81.2% vs 80.9% (p = 0.85), OS 89.4% vs 85.4% (p = 0.38). Sixty (18%) patients experienced disease recurrence; OS after relapse was 33%. In the historical group, EFS and OS were 85.7% and 91.2%. In patients treated according to the SIOP 9 protocol, EFS and OS were 68.1% and 74.5%, resp. In patients treated according to SIOP 93-01, it was 83.6% and 93.7%, resp. and in patients treated according to 87 SIOP 2001, it was 7% and 95.4% (p = 0.001 and p = 0.0008), resp. CONCLUSION: WT is a well treatable disease. The aim for the future is to maintain the current very good survival while minimizing the treatment intensity.

[Treatment results in patients treated from 1980 to 2004 for Wilms' tumour in a single centre]. / Lécebné výsledky pacientu lécených v letech 1980-2004 na jediném pracovisti pro nefroblastom.

BACKGROUNDS: The principle behind the treatment of nephroblastoma has been similar for at least 4 decades, based on vincristine and dactinomycine, radiotherapy in selected stages. The last three decades have been characterised by the aim to reduce the intensity and length of treatment. DESIGN: To retrospectively compare survival rates and treatment success in a cohort of patients aged under 19 years, treated from 1980 to 2004 at a single centre by five consecutive treatment protocols. MATERIALS AND METHODS: The outcome was evaluated in patients treated consecutively by two protocols established at the centre before 1980 and modified in 1986, and from 1988 consecutively by three accepted protocols, SIOP9, SIOP93 and SIOP2001. RESULTS: Overall survival as well as event-free survival rates were evaluated by Kaplan-Meier functions in 315 patients (52.7% women). The average age at diagnosis was 3.9 +/- 2.9 years, median 3.3, range 0.01-17.2 years. Age over 12 years in 2.2% patients. The average follow-up time was 13.1 +/- 7.8, median 13.6, range 0.2-27.8 years. The original 104 weeks of protocol KDO86 treatment had a 10-year overall survival rate of 91.9 +/- 3.2%. Overall survival significantly fell with radiotherapy reduction in lower clinical stages and treatment diversification in protocols with substantial treatment length reduction. Overall survival returned to the original value of KDO86 only in 1994, when SIOP93 was accepted with a 10-year overall survival rate of 92.47 +/- 3.0% and event-free survival 85%, with similar trends in the latest protocol, SIOP2001. In the entire cohort two coincident malignancies (tumour duplicities) were found: one B-lymphoma, one neuroblastoma. A second malignancy occurred in one patient--superficial spreading melanoma. CONCLUSION: from the retrospective view the accepted SIOP9 protocol has a significantly worse outcome in both the overall survival and in event-free survival rate compared with the original therapy. Only the SIOP93 and SIOP2001 protocols accepted after 2003 have an acceptable 10-year overall survival rate (around 92%) as well as event-free survival (85%) with substantially reduced length and intensity of treatment, lowering the risk of late effects.

BRAFV600E mutation in the pathogenesis of a large series of papillary thyroid carcinoma in Czech Republic.

BACKGROUND: Activating point mutation of the BRAF gene, the most common genetic alteration reported in papillary thyroid carcinomas (PTC), has been associated with poor prognostic characteristics. AIM: Our objective was to determine the frequency of BRAFV600E mutation in PTC tumor tissues from the period 1960-2007 and to correlate it with clinicopathological parameters. SUBJECTS AND METHODS: DNAs were extracted from 242 PTCs, 23 sporadic medullary carcinomas, one anaplastic carcinoma and 6 poorly differentiated carcinomas. The presence of BRAFV600E mutation was determined using single strand conformation polymorphism method and verified by direct sequencing. RESULTS: BRAFV600E mutation was detected in 81 of 242 PTCs (33.5%), in one of 6 poorly differentiated carcinomas (16.7%) and in anaplastic carcinoma. BRAFV600E mutation was much less frequent in the follicular variant compared to classical variant and mixed follicular- classical variant of PTCs (p=0.001). BRAFV600E mutation was significantly associated with presence of nodal metastasis (p=0.029), more advanced TNM stage (p=0.014) and recurrence of disease (p=0.008). The mutation correlated with a higher age at diagnosis (p=0.049) and with a greater tumor size (p=0.041). Multivariate analysis confirmed these findings. The prevalence of BRAFV600E mutation before 1986 was significantly lower than after it (p=0.008). CONCLUSIONS: Our data suggest that BRAFV600E mutation is associated with high-risk clinicopathological characteristics of PTC and worse prognosis of patients. The frequency of the mutation significantly varied during the observed period but rather because of the different age distribution of patients in particular periods than as a consequence of Chernobyl accident.

[Clinical relevance of chromosomal aberrations in bone and soft tissue tumors in children and young adults]. / Klinický význam chromozomálních zmen u nádoru kostí a mekkých tkání detí a mladistvých.

BACKGROUND: We present the results of a cytogenetic and molecular cytogenetic analysis of a series of patients with bone and soft tissue tumors. PATIENTS ANDMETHODS: We analyzed a cohort of 26 patients with Ewing sarcoma/PNET, 15 patients with rhabdomyosarcoma, 5 with synovial sarcoma and one patient with an undifferentiated sarcoma using the cytogenetic and molecular cytogenetic techniques M-FISH and arrayCGH. RESULTS: We found nonrandom chromosomal structural and numerical changes with diagnostic and prognostic relevance in most patients. Eight patients with ES/PNET had only a t(11;22)(q24;q12), eight patients had secondary aberrations as well and six had only secondary aberrations. In the RMS patients we detected the t(1;13)(p36;q14) once and the t(2;13)(q35;q14) four times, both of them characteristic for the alveolar subtype with poor prognosis and numerical aberrations, characteristic for the embryonal subtype, in five patients. Four patients with synovial sarcoma had the diagnostic t(X;18)(p11.2;q11.2), one of them had a complex karyotype with a complex t(X;18;21) (p11.2;q11.2;q11.2) together with t(2;5)(q24-32;p13-14) and t(12;20)(p11;q13). We correlated the karyotype of cancer cells with histopathologic morphologic analysis, clinical outcome and foreign published results. CONCLUSION: Cytogenetic and molecular cytogenetic analysis is a valuable diagnostic tool in bone and soft tissue tumors, especially in less differentiated subtypes, and as such it should be an integral part of curative care.

[Invasive zygomycosis of the large intestine in a diabetes patient]. / Invazivní zygomykóza tlustého streva u pacienta s diabetem.

Invasive mucormycosis is a rare, serious or even fatal condition. The authors diagnosed and successfully treated the disorder in a 62-year-old diabetes patient. The course of the disease, including justification of the surgical procedure are described in the case review. All treatment options were implemented and, after 121 days of hospitalization, the patient was released for homecare.

Somatic mutations in the RET proto-oncogene in sporadic medullary thyroid carcinomas.

The frequency and prognostic relevance of RET proto-oncogene somatic mutations in sporadic medullary thyroid carcinoma (MTC) remain controversial. In order to study somatic mutations in the RET proto-oncogene in sporadic MTCs found in the Czech population and to correlate these mutations with clinical and pathological characteristics, we investigated 48 truly sporadic MTCs by sequencing classical risk exons 10, 11, 13, 14, 15 and 16. From the 48 tumors studied, 23 (48%) had somatic mutation in the RET proto-oncogene in exons 10, 11, 15 or 16. The classical somatic mutation Met918Thr in exon 16 was only found in 13 tumors (27%). In five cases, multiple somatic mutations and deletions were detected. A statistically significant correlation between the presence of somatic mutation with more advanced pathological TNM stages was observed. Other clinical and pathological characteristics did not show any statistical significant association with the presence or absence of somatic mutation.

[Sentinel lymph node biopsy in oral and oropharyngeal carcinomas]. / Biopsie sentinelové uzliny u pacientu s karcinomem ústní dutiny a orofaryngu.

BACKGROUND: The aim of our study was to assess feasibility and accuracy of sentinel lymph node biopsy in patients with head and neck squamous cell carcinoma with clinically N0 neck. METHODS AND RESULTS: The sentinel lymph node was localised preoperatively by lymphoscintigraphy and intraoperatively by hand-held gamma probe after peritumoral injection of a Tc99m-labeled colloidal human serum albumin. The histology of the sentinel lymph node was compared with the histology of the nodes of the elective neck dissection performed in all patients. 27 patients with oral and oropharyngeal carcinomas accessible to injection in local anaesthesia were enrolled into a prospective trial between July 1993 and December 2005. The sentinel lymph node was localised by preoperative lymphoscintigraphy in 26 of 27 patients. Sentinel lymph node was identified perioperatively by hand-held gamma probe in all 28 necks of 27 patients. Occult metastases were found in 4 sentinel lymph nodes in 4 cases. In one case (3.6 %) the result of sentinel lymph node biopsy was false negative. The sentinel lymph node biopsy correctly predicted the positivity and negativity of the neck in 27 of 28 cases (96.4 %). CONCLUSIONS: Sentinel lymph node biopsy in patients with oral and oropharyngeal carcinomas is feasible and seems to accurately predict the status of the regional lymph nodes.

Double germline mutations in the RET Proto-oncogene in MEN 2A and MEN 2B kindreds.

Medullary thyroid carcinoma (MTC) is a rare form of thyroid cancer representing about 10% of all thyroid malignancies. It occurs mostly as a sporadic tumor or in association with autosomal dominant inherited cancer syndromes--multiple endocrine neoplasia (MEN) types 2A and 2B and familial MTC. Germline mutations in exons 8, 10, 11, 13, 14, 15 and 16 of the RET proto-oncogene are found in most of the familial cases. There are only a few published data reporting multiple germline mutations in the RET proto-oncogene. We have detected double germline mutations in 2 different exons on the same RET allele in two MEN 2 families. In the MEN 2A family, double germline mutation in exons 10 (Cys620Phe) and 13 (Tyr791Phe) was detected. In the MEN 2B family, beside the classical germline mutation in exon 16 (Met918Thr) a second germline mutation in exon 13 (Tyr791Phe) was found. This study revealed that MEN 2 syndromes can also be caused by double germline mutations in the RET proto-oncogene and these families can be added to small worldwide cohort of families with multiple germline mutations.

Congenital solitary liver cysts.

AIM: The aim of the study was to describe clinical and pathological findings in children with rare congenital solitary liver cyst. MATERIAL AND METHODS: Six children with congenital liver cyst were treated at the authors' institution from 1995 to 2002. Clinical records and operative findings of these patients were reviewed retrospectively. RESULTS: Age at presentation ranged from neonatal to 14 years, and there were four girls and two boys. Hepatomegaly was the most common symptom in neonates while the older children presented with abdominal pain. Ultrasound was conclusive for diagnosis in all children, prenatal ultrasound in three. Intrahepatic cyst was found in five children. Partially extrahepatic cyst in the porta hepatis was found in one. The cyst diameters varied from 8 to 10 cm. Cysts were excised via an open abdominal approach in all patients. Histological findings confirmed the diagnosis of congenital liver cyst. Columnar epithelium was found in newborns while atrophic changes of epithelial lining were seen in older children. In a case of a 13-year-old girl the cyst was lined by metaplastic squamous epithelium with foci of slight dysplasia. Immunohistochemical studies were available in three recent children with positive findings in all of them. The postoperative course was uneventful in all patients. The follow-up period ranged from 36-120 months and no recurrence was observed. CONCLUSION: With appropriate selection of symptomatic patients, total excision of a congenital liver cyst is a safe procedure with excellent long-term results. Given the possibility of malignant degeneration of a congenital liver cyst we recommend its surgical excision in most cases.

Aleukemic granulocytic sarcoma with AML1/ETO fusion gene expression and clonal T cell populations.

We report a unique case of aleukemic granulocytic sarcoma of the neck, originally misdiagnosed as non-Hodgkin's lymphoma (NHL), though chloroma was also suspected due to a greenish macroscopic appearance and the presence of myeloid chloroacetate esterase (CAE)+ cells. The proof of clonal T cell receptor gamma chain (TcRgamma) gene rearrangements in the recurring tumor was deemed to confirm the initial diagnosis of T cell NHL. Altogether five distinct types of clonal TcRgamma gene rearrangements were found in the tumor, bone marrow and peripheral blood. Only retrospectively, using RT-PCR, did we detect the acute myeloid leukemia subset-specific fusion gene AML1/ETO in the frozen samples of the relapsed tumor, as well as in the otherwise unaffected bone marrow and peripheral blood (representing 'minimal initial disease' in the latter two samples). Simultaneous staining verified that the neoplastic CAE+ cells and CD45RO+ T cells were different populations.

Necrobiotic xanthogranuloma presenting as a solitary tumor.

We report on a 60-year-old overweight white woman who presented with an asymptomatic flat, hard, yellow-brown subcutaneous plaque on her right hip. A total excision was performed. Histopathologic examination showed all the major features of a necrobiotic xanthogranuloma (NX) involving the entire dermis and subcutis, including areas of necrobiosis with cholesterol clefts, granulomatous infiltrate with some bizarre giant cells, numerous Touton cells, foamy cells, lymphoid follicles with germinal centers, foci of plasma cells, and "Touton cell panniculitis." A laboratory investigation revealed only slightly increased titers of cholesterol, strong positivity of anti-Borrelia antibodies, and diffuse skeletal osteoporosis with fractures of seven vertebrae. After 4 years of close follow-up, the osteoporosis was improved, and there were no signs of paraproteinemia, malignancy, or new skin lesions. The authors suggest that this case could represent a solitary clinical variant of NX without paraproteinemia.

[Progressive multifocal leukoencephalopathy: morphologic possibilities of diagnostic classification methods and in situ hybridization]. / Progresivní multifokální leukoencefalopatie (PML): Morfologické moznosti diagnostiky klasickými metodami a in situ hybridizací.

Progressive multifocal leucoencephalopathy is caused by infection with JC virus. The disease affects patients with immunodeficiencies, hematologic diseases, and patients treated with radiotherapy. The disease is characterised by foci of demyelinisation with atypical astrocytes and oligodendrocytes. Oligodendrocytes contain typical intranuclear inclusions. Progressive multifocal leucoencephalopathy and its verification is presented in three cases. Two patients died of progression of a malignant neoplasm and the leucoencephalopathy was a complication of the malignancy. The third case was a biopsy specimen taken from the brain of a patient who received a renal transplant. The material of all patients was analysed by light and electron microscopy, and in situ hybridisation with a probe specific for JC virus. In situ hybridisation proved to be the most specific and a simple method to demonstrate the infection in all cases. It is useful in instances in which the histologically detectable lesion is not characteristic, and in cells in which the conventional histologic methods fail to reveal the intranuclear inclusions of JC virus.

[Sinus histiocytosis with massive lymphadenopathy--a disseminated form of the Rosai-Dorfman syndrome]. / Sinusová histiocytóza s masivní lymfadenopatií--diseminovaná forma syndromu Rosai-Dorfman.

A 20 year-old black male presented with a generalized lymphadenopathy, skin papules with maximum involvement of the head and trunk region, and elevated temperature. Ultrasound, CT and bone scans revealed infiltration of the kidneys and bones. Surgical specimens of the cervical lymph nodes showed massive infiltration of sinuses by histiocytes with engulfed lymphocytes and granulocytes in their cytoplasm. A similar finding was found in the excised dermis and nasopharyngeal mucosa. Immunohistochemical investigations showed a strong positive reaction of sinusoidal macrophages with anti S100 protein, anti alpha-1 antitrypsin and alpha-1 antichymotrypsin antibodies, negative CD1a. The engulfed granulocytes were positive with lysozyme and MAC387 antibodies; lymphocytes reacted with antilymphocytic antibodies L26, HLADR, UCHL-1 and OPD4 only rarely, possibly due to alterations of their cell membranes in the cytoplasmic environment of histiocytes. Occasional plasma cells were also entrapped in the macrophages. The best results were obtained with CD3 antibody which showed focal predominance of phagocytosed T cells. These findings confirm a nonselective nature of the emperipolesis. In situ hybridization with probes EBER and BHLF against Epstein-Barr virus sequences was negative. The pacient was treated by multidrug chemotherapy with a moderate regression of the infiltrates. The symptoms of the disease persisted for 10 months from the treatment start but further course of the disease is uncertain. The patients is lost to follow up.

[Visceral and cutaneous leishmaniasis]. / Viscerální a kutánní leishmaniázy.

Two cases of kala azar and 5 cases of cutaneous leishmaniasis were found in bioptic files from previous 50 years in Hlava Institute of Pathology in Prague, 4 of them were cumulated in 1994 and 1995. All cases followed short stays in Mediterranean or Caribbean area. The latest kala azar was identified from bone marrow trephine biopsy and subsequently, leishmanias were also found in an inapparent hepatopathy. In cutaneous cases, a fibrinoid vasculitis was observed in addition to accumulation of leishmanias laden macrophages and tuberculoid granulomas.

Eosinophilic fasciitis in an adolescent girl with lymphadenopathy and vitiligo-like and linear scleroderma-like changes. A case report.

The patient, a 14-year-old girl, suffered from arthralgias which occurred after tonsillitis. Two months later she developed edema of the left lower extremity, finger flexion contractures and induration of the skin of the left leg, associated with hypergammaglobulinemia, peripheral hypereosinophilia, elevated ESR and a positivity of ANA and anti ds-DNA antibodies. A biopsy of the inguinal lymph node, performed because of left inguinal and retroperitoneal lymphadenopathy, showed only slight inflammatory activation and a granulomatous reaction after lymphography. A few days after the lymphography linear erythema evolving later into hyperpigmentation and corresponding to the superficial lymphatics developed on the left side of the body, very probably as a reaction to the patent-blue dye. Deep en-block skin biopsy confirmed the diagnosis of eosinophilic fasciitis (EF). After two years of therapy with prednisone and d-penicillamine the patient felt well, and her flexion contractures resolved, ANA were positive, while anti ds-DNA were negative. Linear hyperpigmentation persisted, and linear scleroderma-like changes developed on the left lower limb. A vitiligo-like lesion on the right foot which occurred after one year of therapy persisted. The possible risk of developing systemic connective tissue disease necessitates the long term follow up of this patient.

[An unusual vascular tumor of the pericardium resembling Kaposi's sarcoma]. / Neobvyklý vaskulární nádor perikardu blízký Kaposiho sarkomu.

A peculiar vascular tumor of the pericardial cavity affecting a 21-year-old man is reported. The tumor was formed by spindle cells and narrow vascular channels. It was difficult to decide between primary pericardial Kaposi's sarcoma or angiosarcoma. The patient was HIV negative and no other tumors were revealed in the course of the disease. Immunohistochemical investigations showed positive endothelial markers-FVIII, CD 31, CD 34 in the tumor cells. Due to an inconclusive diagnosis of the tumor six cases of Kaposi's sarcoma and six cases of angiosarcomas were retrieved from our files, the same spectrum of immunohistochemistry as in above case applied, and the results compared. We found no significant differences in the positivity of endothelial markers among these cases and when compared with the case of pericardial tumor. The reactivity of CD 34 was, however, strong in cases of Kaposi's sarcoma whereas in angiosarcomas it yielded a weak and focal reaction only. In spite of the latter finding we feel that in individual cases, like the described tumor of the pericardial cavity, the immunohistochemical investigation cannot discriminate angiosarcoma from Kaposi's sarcoma. The reported tumor of the pericardial cavity was considered an unusual form of a low grade angiosarcoma with immunophenotypic features of Kaposi's sarcoma.