Thrombin Generation Testing in Patients with Myelofibrosis.

BACKGROUND: Primary myelofibrosis (PMF) is a chronic clonal myeloid disorder. Together with essential thrombocythemia (ET) and polycythemia vera (PV), it belongs to a group of Philadelphia chromosome-negative myeloproliferative neoplasms. Thrombotic events are serious complications negatively influencing the quality and length of these patients' lives. The confirmed risk factors for venous thromboembolism are age over 60 years, a positive history of thromboembolism, presence of common cardiovascular risks, JAK2 V617F mutation and, according to some authors, leukocytosis. Various opinions on the role of thrombocythemia have been published. The present study was undertaken to evaluate the benefit of thrombin generation test and its potential use in predicting the risk of thrombosis in MF patients. METHODS: The analysis included plasma samples obtained from 36 patients diagnosed with MF in our center from 2004 to 2016 (JAK2 V617-positive 53%; CALR-positive 31%; MPL-positive 14%; triple negative 2%) and a control group comprising 20 healthy volunteer blood donors. Thrombin generation was measured in platelet-rich plasma using the TECHNOTHROMBIN® TGA kit (Technoclone, Austria) and the fully automated system Ceveron® Alpha (Technoclone). The results were correlated with clinical and laboratory parameters of the patients. RESULTS: There were differences in thrombin generation as expressed by endogenous thrombin potential (ETP) between patients and healthy controls, with ETP being lower in the patient group (p = 0.0003). Analysis confirmed a significant correlation between thrombin generation and platelet counts, with higher thrombin generation in patients with thrombocythemia > 400 x 109/L (p = 0.04). ETP values were consistently higher in earlier disease stages and lower in CALR-mutated myelofibrosis. CONCLUSIONS: In MF patients, thrombin generation is mainly influenced by platelet counts and, to a lesser extent, by mutation status, activity, and progression of the disease. Thrombin generation test results have confirmed that thrombocythemia is a potential risk factor for thrombotic complications.

Effect of CALR and JAK2 mutations on the clinical and hematological phenotypes of the disease in patients with myelofibrosis - long-term experience from a single center.

Primary myelofibrosis (PMF) is a chronic clonal myeloid disorder. Together with essential thrombocythemia (ET) and polycythemia vera (PV), it belongs to a group of Philadelphia chromosome-negative myeloproliferative neoplasms. An integral part of laboratory tests carried out in this disease group is detecting the presence of mutations in the Janus kinase 2 gene at position 617 (JAK2 V617F) and in the gene encoding for the receptor for thrombopoietin (myeloproliferative leukemia virus oncogene, MPL) found in approximately 60% of PMF patients. The discovery of mutations affecting exon 9 of the calreticulin (CALR) gene was of great benefit to the diagnosis of the diseases in JAK2 V617F and MPL unmutated cases. This is a study of the effect of a mutation in the CALR gene on the clinical course in patients with primary, post-ET and post-PV myelofibrosis. Analysis of 66 patients (54.5% JAK2 V617F; 34.8% CALR; 6.1% MPL; 3.0% triple negative; 1.5% coincidence of CALR and JAK2 V617F) confirmed a different phenotype of the disease in CALR-mutated patients as compared with CALR-unmutated individuals. Those with CALR mutation were significantly younger and had borderline higher platelet counts, less pronounced splenomegaly and less frequent B symptoms at diagnosis. The study suggests that the driver mutation types define variations in the biological basis, clinical manifestations and course of the disease. The presence of CALR mutation has been shown to be an independent prognostic favorable factor. Careful risk stratification of these patients is of great importance to adequate therapeutic decision-making and aids in selecting high-risk patients eligible  for allogeneic hematopoietic stem cell transplantation which continues to be the only treatment modality for myelofibrosis having curative potential.

CD34⁺ cell content in unrelated allogeneic peripheral blood stem cell grafts transported internationally. Does the inter-laboratory variability affect comparability of graft quality data?

BACKGROUND: Increasing numbers of unrelated hematopoietic stem cell grafts are transported internationally and evaluated concurrently in different laboratories. The graft quality assessment using the CD34(+) enumeration could be influenced by inter-laboratory variability. METHODS: We retrospectively analyzed the content of CD34(+) cells in 154 consecutive collections being performed in different transplant centers during two periods (2003-2004, 2007-2010). All samples were tested twice in our own and partner laboratories. CD34(+) percentage and absolute number were compared. RESULTS: The percentage and the total CD34(+) content correlated well in both observed periods (CD34(+)%: r=0.899 and r=0.922; CD34(+)×10(8)/kg: r=0.966 and r=0.880; p<0.0001). Median CD34(+) percentages obtained in our centre in comparison with other laboratories were 0.54% vs. 0.46% in 2003-2004 and 0.69% vs. 0.70% in 2007-2010 period. The degree of laboratory compliance was affected by the laboratory identity. CD34(+) percentage reported by one laboratory and CD34(+)×10(8)/kg reported by three from twelve laboratories lacked statistically significant correlation with our own data. CONCLUSIONS: The study documented that results of CD34(+) cell dose assessment of the same grafts reported by different transplant centers are comparable. The graft quality data and the CD34(+) enumeration possess a limited level of inter-laboratory variability.

[Patient with B-CLL with a history of unrelated hematopoietic cells donation--retrospective analysis of CLL development and implication for the recipient]. / Pacient s B-CLL s anamnézou darování krvetvorných bunek nepríbuznému pacientovi - retrospektivní sledování vývoje nemoci a dusledky pro príjemce.

BACKGROUND: Donor cell leukemia (DCL) is a relatively rare but well documented complication of hematopoietic stem cell transplantation. So far, publications described only DCL arising de novo in the recipient. OBSERVATION: In this study, we describe a case of chronic lymphocytic leukemia (B-CLL) developing in a volunteer unrelated donor from the Czech National Marrow Donors Registry (CNMDR) several years after donation. From archival DNA sample, we have retrospectively found that subclinical CLL clone was already present at the time of donation but early death of recipient prevented eventual development of DCL. This case documents well the long period between detection of B-CLL clone and full development of clinical-laboratory symptomatology. The medical and ethical questions posed by an isolated case of detection of hematological malignancy present either only in the donor or only in the recipient are discussed. CONCLUSION: The case demonstrates the increasing risk of development of various forms of DCL and thus highlights the need for long-term monitoring of stem cell donor, not only in terms of health of donor but also in terms of potential risks for the recipient.

[The incidence of malignancies and surveillance of hematopoietic stem cells donors--the results of the Haemato-Oncology Department University Hospital in Plzen (Pilsen) and Czech National Marrow Donors Registry observation]. / Výskyt malignit a dispenzarizace dárcu krvetvorných bunek--výsledky sledování Hematologicko-onkologického oddelení FN Plzen a Ceského národního registru dárcu drene

BACKGROUNDS: Granulopoesis colony-stimulating factor filgrastim is used to mobilize peripheral stem cells but there are concerns regarding an elevated risk of haematological malignancies. We analyzed the incidence of malignancies and the system of haematopoietic stem cells donor surveillance. PATIENTS AND METHODS: prospective observation of sibling donors of the Haemato-Oncology Department University Hospital in Plzen (Pilsen) and of unrelated donors of the Czech National Marrow Donors Registry (CNMDR) in 2001-2010. RESULTS: No malignancy was observed in a group of 344 unrelated CNMDR donors, providing 753 person-years; one case of chronic lymphocytic leukaemia manifested 6 years after bone marrow donation, with leukaemia clone retrospectively detected by DNA analysis in blood samples taken prior to the marrow donation. Acute myeloid leukaemia, non-Hodgkin lymphoma, renal and colorectal carcinoma were observed in a group of 84 peripheral stem cells sibling donors, providing 337 person-years observation. The respective incidence of the two haematologic malignancies was 593 cases and the expected incidence rate was 143 per 100,000. The sibling (related) donors age was significantly higher: 48 (16-75) vs. 31 (20-42) years, (p<0.0001). Significantly more lost-to-follow-up donors were among the related donors (32% vs. 3%, p<0.0001), even though active surveillance system was implemented. CONCLUSION: The development of malignancies in hematopoietic stem cells donors can naturally be expected. Related (sibling) donors are at higher risk because of their generally older age, and higher susceptibility to haematological malignancies developed within the family. The contribution of filgrastim exposure needs to be further investigated. The follow-up cooperation with related (sibling) donors is limited.

Prognosis of pediatric patients transplanted for Ph+ chronic myeloid leukemia in the period from 1989 to 2006 in the Czech Republic.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by clonal proliferation of primitive hematopoietic stem cell. The median age at diagnosis is 55 to 60 years with less than 10% of patients younger 20 years. Incidence of CML in children in the Czech Republic is 0.106 cases/100 thousands per year. Here we report outcome of 38 pediatric patients (median age 12.5 years; range 1.8 - 17.3) with Ph-positive CML diagnosed between years 1989 to 2006. Primarily chronic phase of the disease was diagnosed in 32 (84%) patients. 32 (84.2%) patients underwent hematopoietic stem cell transplantation (HSCT) with the median age at transplantation of 14.9 years (range 6.9 - 20.5 years). Out of transplanted patients 16 (50%) obtained graft from unrelated donor, 13 (41%) from matched sibling donor, 2 from haploidentical family donor and autologous transplantation has been performed in one case. 6 patients were not transplanted, 4 of them died (median 1.2 years from diagnosis), 2 are alive 0.6 and 17.8 years from the diagnosis. Overall survival (OS) in 25 patients after HSCT at our department during the whole period is 66.7% with 15/16 being in stable continuous molecular-genetic remission (94%). During the period of time results of transplantations have been significantly improved (p=0.0071). OS after HSCT until year 1997 is 25% while from year 1998 until now is 87.5%. All centers OS of patients after HSCT is 71%. Results of HSCT in children with CML obtained from the year 1998 at our center are fully comparable with results achieved in large and experienced centers. HSCT remains the only proven and effective method for the treatment of CML. Clinical studies assessing the role of tyrosine kinase inhibitors in children instead of early HSCT should be planned carefully in order to avoid sub-optimal outcomes.

Cytokine response of porcine cell lines to Salmonella enterica serovar typhimurium and its hilA and ssrA mutants.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular bacterium which can infect and colonize pigs. After contact with enterocytes and macrophages, S. Typhimurium induces production of cytokines thus triggering the innate immune response. In this study we evaluated the cytokine response of two porcine cell lines, IPI-2I and 3D4/31, of epithelial or macrophage origins, respectively, to the wild-type S. Typhimurium and its hilA and ssrA mutants. We observed that the 3D4/31 cell line essentially did not respond to S. Typhimurium infection when a medium with foetal calf serum was used. However when the 3D4 cell line was incubated overnight in the presence of porcine serum, it efficiently responded to the wild-type strain and the ssrA mutant but not to the noninvasive hilA mutant as measured by mRNA quantification of TNF-alpha, IL-8 and GM-CSF by the real-time RT-PCR. In IPI-2I, all the cytokines were also induced by the wild-type S. Typhimurium and the ssrA mutant although the induction of TNF-alpha was lower than that induced by the wild-type strain. The hilA mutant was unable to induce any of the cytokines tested. The ssrA mutant can therefore be considered as more suitable for further vaccine development as the stimulation of innate immune response is important for animal protection against a challenge with virulent strains.

Degradation of 5-nitroguaiacol by soil bacteria of the genus Rhodococcus.

Two bacterial strains were isolated from forest soil by selective enrichment of the mineral medium containing 4-nitropyrocatechol as the sole carbon and energy source. Both strains could utilize 4-nitropyrocatechol and 5-nitroguaiacol. Degradation of 5-nitroguaiacol and stoichiometric release of nitrites was measured during its degradation both in growing culture and for resting cells. Both strains were unable to degrade other nitroaromatic compounds such as 4-nitroguaiacol, 2-nitrophenol, 3-nitrophenol, 4-nitrophenol, 2,4-dinitrobenzoic acid, 4,5-dimethoxy-2-nitrobenzoic acid and 2,3-difluoro-6-nitrophenol. One strain was identified as Rhodococcus opacus and the second one as Rhodococcus sp.

[Asynchronous occurrence of three neoplastic diseases: chronic B-cell lymphatic leukemia, renal carcinoma and prostatic adenocarcinoma]. / Asynchronní triplicitní výskyt nádorového onemocnení: chronické B lymfatické leukemie, karcinomu ledviny a adenokarcinomu prostaty.

Authors present a case of three asynchronous malignancies in man working life-long in chemical industry. Course of diseases and their treatment are described: B chronic lymphatic leukaemia (B-CLL) (age of 55), conventional clear cell renal carcinoma (CRCC) (age of 61) and adenocarcinoma of prostate (CaP) (age of 72). B-CLL treatment was chlorambucil for 2 years and follow-up subsequently. CRCC treatment was radical transperitoneal nephrectomy. Due to locally advanced CaP bilateral orchiectomy and radical external beam radiotherapy were performed. Patients is alive 30 months without any signs of any malignity. Incidence of multiple malignancies and particularly triplicities are discussed in this article. Authors point out pertinence of radical therapeutic approach also in multiple malignancies and oncological screening not only in patients with heamatological malignancy, but considering it in all tumours.

[Bilateral renal tumors]. / Bilaterální nádory ledvin.

At the Urological Clinic in Hradec Králové in 1990-1994 320 patients with renal tumours were admitted and treated. In 12 the tumour was bilateral (3.7%), incl. 8 patients where it was synchronous and four where it was asynchronous. Only in one female patient bilateral nephrectomy was performed, in 8 patients unilateral nephrectomy and on the other side enucleation of the tumour, in two patients bilateral enucleation of the tumour and in one patient with a malignant lymphoblastic lymphoma only unilateral enucleation of the tumour and epinephrectomy. In 10 patients histological examination confirmed a renal carcinoma, in one patient a malignant lymphoblastic lymphoma, in one female patient a bilateral multifocal oncocytoma. The patients survival was not evaluated in the report.

[Renal angiomyolipoma]. / Angiomyolipom ledviny.

The authors present their own experience as well as data from the literature concerning the diagnosis and treatment of renal angiomyolipomas. In a group of 320 patients hospitalised at the Urological Clinic in 1990-1994 these tumours were diagnosed before surgery and confirmed by histological examination in 7 patients (2.2%). The group comprised three men and four women, mean age 51.7 years. Only one female patient had classical manifestations of tuberous sclerosis, the remaining patients did not have manifestations of tuberous sclerosis. Two patients were admitted with symptoms of haemorrhage into the retroperitoneum, three suffered from pain on pressure in the side and in two patients the tumour was revealed incidentally during US examination. Four patients were subjected to nephrectomy, in two enucleation of the tumour was performed and in one female patient with classical manifestations of tuberous sclerosis with a bilateral tumour conservative treatment was applied.

[Conservative surgical treatment of kidney tumors: indications, techniques, results]. / Konzervativní chirurgická lécba nádoru ledviny: indikace, technika, výsledky.

At the Urological Department in Hradec Králové the authors operated in 1989-1993 287 patients with renal tumours, in 45 of them a conservative procedure was used (15.6%). The group comprises 29 men and 16 women, mean age 56.5 years. The contralateral kidney was normal in 29 patients (64%), in 5 patients nephrectomy was performed on account of carcinoma and in 9 patients (20%) the second kidney was hypofunctional (hydronephrosis, cysts). In two patients a conservative procedure was used on account of malignity of the blood. In 33 patients the tumour was enucleated (73.3%), in 12 the upper or lower pole of the kidney was resected. In 6 patients the tumour was in the first clinical stage, but in four already in T 3a (imperative indication). Twenty-seven tumours were evaluated by the histologist as medium differentiated, 2 of 35 as poorly differentiated. Thirty-five tumours were carcinomas (77.7%), 8 were adenomas, one was angiomyolipoma and in one instance the scar after PEC was removed which was not reliably diagnosed before surgery. The preoperative examination comprised also immunological examination which, however, was not made in two patients. The immunologist evaluated the criteria as favourable in 21 patients (60%), as risky in 12, incl. 6 were immunotherapy was administered. One patient developed a urinous fistula which healed on a stent. During the investigation period no local relapse was observed.

Bleeding to adrenal gland in adults.

At the Department of Urology in Hradec Králové five patients were treated in 1991 to 1993 with bleeding to the adrenal glands. We present our reports of these patients and stress the difficulty of preoperative diagnosis.

Bleeding to adrenal glands in adults.

At the Department of Urology in Hradec Králové five patients were treated in 1991 to 1993 with bleeding to the adrenal glands. We present our reports of these patients and stress the difficulty of preoperative diagnosis.

[Blunt injury of the kidney--personal experience and present views on its therapy]. / Tupá poranení ledvin--vlastní zkusenosti a soucasné názory na jejich lécbu.

Between 1972 to 1994 there were admitted 105 patients with blunt trauma of the kidney, 89 men and 16 women with median age 32 years. In 53 patients the left kidney was affected and in 52 of them the right side was injured. According to the grade of trauma we evaluated in 77 patients a contusion (73.3%), by 16 patients a laceration (15.2%), in 11 patients a rupture of the kidney with fragmentation of the parenchyma and in one the renal artery was injured. The mechanism leading to the blunt trauma of the kidney was following: traffic accident in 52 patients (49.5%), during sport in 20 patients (19.5%). 16 patients were injured in own home (9.5%) and 7 patients in work (6.7%). In 28 patients the trauma of the kidney was serious, by 5 of them we have done nephrectomy (17.8%), in two suture of the parenchyma (7.1%) and in further two patients heminephrectomy. After considering clinical status, grade of hypovolemic shock and finding on CT scan we prefer lately conservative steps in the treatment serious trauma of the kidney.

[Adrenal gland hemorrhage in adulthood]. / Krvácení do nadledviny v dospelém veku.

The author presents five case-histories of haemorrhage into the adrenal in adult age. He emphasizes specially the difficulty of preoperative diagnosis.

Conservative surgery for renal cell carcinoma--indications, technique and results.

At the Department of urology in Hradec Králové between 1989-1993 287 patients with a tumor of the kidney were operated on, in 45 of them (15.6%) conservatively. In this set there are 29 men and 16 women with the mean age 56.5 years. In 29 patients the contralateral kidney was normal (64%), 5 patients underwent nephrectomy for renal cell carcinoma, in 9 patients the opposite kidney was hypofunctionalised (hydronephrosis, cysts). In two patients the conservative approach was used for reasons of another type of malignancy (haematological). In 33 (73.3%) patients enucleation, in 12 patients resection of the lower or upper pole were done. The tumor was classified as T 1 stage in 6 patients, but in 4 patients already as T 3a (in patients with imperative indication). In 35 patients the tumor was moderately differentiated, in two ones with histologically confirmed renal cell carcinomas the tumors were poorly differentiated, in 8 patients there were histologically adenomas, in one angiomyolipoma and in another case scar after PCN (percutaneous nephrolithotomy). Preoperative examination included immunological examination, this was not done in two patients. Good results were obtained in 21 patients (60%), 12 patients were in high risk and 6 of them received immunotherapy. As an early complication one patient had urinary fistula, which resolved after introduction of stent. Relapse of malignancy in the screening period was not noticed.

[Conn's syndrome in tumors of the adrenal cortex]. / Connuv syndrom v sestave nádoru kory nadledvin.

During the period from Jan. 1 1989 to June 30 1992 at the Urological Clinic in Hradec Králové three women with cortical adenomas were treated, the tumour being the cause of primary hyperaldosteronism. The age of the patients was 25-40 years, in one woman the tumour was on the right side, in two on the left side. All patients complained of marked muscular weakness, fatigue, one woman developed cramps of the hands when working and another patient was repeatedly hospitalized at the neurological department on account of attacks of weakness. In one patient the interval from the onset of complaints to assessment of the diagnosis was 23 years. All patients had complete examinations at specialized departments before operation, in all the diagnosis was established by biochemical examinations and CT. In all instances minor tumours--diameter 2-3 cm--were involved which were easily removed by surgery. The postoperative course in all patients was without complications.

[Tumor thrombosis of the inferior vena cava--personal experience with surgical therapy]. / Nádorová trombóza dolní duté zíly--vlastní zkusenosti s operacní lécbou.

Patients treated at the Urological Clinic in Hradec Králové on account of carcinoma of the kidney are systematically followed up since 1962. In the submitted paper the authors evaluate patients treated during 1985-1991. During the mentioned period at the Urological Clinic in Hradec Králové 365 patients with carcinoma of the kidney were treated. In 55 (15%) invasion into the renal or inferior vena cava was proved (in 43 men and 12 women). In 38 patients the tumour with a thrombus of the inferior vena cava was on the right side, in 17 a tumour of the left kidney was removed. The right to left ratio is 2.2:1. Surgery of tumourous thrombosis depends on the level of the upper margin of the tumourous thrombus. A tumourous thrombus in the subhepatic part of the inferior vena cava can be treated either by massaging into the renal vein after obliteration of the artery or by parietal resection of the inferior vena cava. In 18 patients the authors were able to remove the thrombus by massaging into the renal vein. In 32 patients the inferior vena cava was resected. In five the tumourous invasion was not resolved, either because of extensive secondaries (in the liver and lungs) or because of direct tumourous infiltration of the wall of the inferior vena cava.(ABSTRACT TRUNCATED AT 250 WORDS)