Alena Dohnalová

Prognostic impact of DNMT3A mutations in patients with intermediate cytogenetic risk profile acute myeloid leukemia

Objectives: Recently, mutations in DNMT3A gene have been described in about 25% acute myeloid leukemia (AML) cases, preferentially in monocytic AML. They were found to predict worse overall survival (OS) of mutated patients. Patients and methods: RT‐PCR followed by direct sequencing was used to test the presence of DNMT3A mutations in 226 AML patients with an intermediate‐risk (IR) cytogenetics. Results: Sixty‐seven patients of 226 (29.6%) carried a mutation in the DNMT3A gene. Occurrence of DNMT3A mutations was associated with female sex (P = 0.027) and with the presence of FLT3/ITD (P = 0.003), but not with particular FAB subtypes. Patients with DNMT3A mutation had higher initial WBC counts than those without it (P = 0.064) only because of higher incidence of FLT3/ITD within these cases. There was no difference between mutated and wild‐type groups in reaching complete remission (CR) (P = 0.380). OS was not affected by DNMT3A mutation (P = 0.251), but OS of patients who reached CR was longer in DNMT3A negative cases (P = 0.025). Patients with DNMT3A mutation had a higher relapse rate (P = 0.007). Patients carrying both the DNMT3A mutation and FLT3/ITD relapsed more often than either patients with single DNMT3A mutation (P = 0.044) or patients with FLT3/ITD only (P = 0.058). DNMT3A mutations were associated with higher relapse rate even within the FLT3/ITD‐negative group (P = 0.072). After reaching CR, these two genetic factors were independent predictors of relapse at multivariate analysis (P < 0.001). Only three of 30 ‘double‐mutated’ (FLT3/ITD+, DNMT3A+) patients are still alive, all of them having undergone hematopoietic stem cell transplant. Conclusions: We have confirmed the high incidence of DNMT3A mutations in patients with AML with IR cytogenetics. Patients with DNMT3A mutations relapse more often and have inferior OS when only patients achieving CR are analyzed. ‘Double‐mutated’ patients have a very poor prognosis.

Cell-Mediated Immunity in Cervical Cancer Evolution

Cell-mediated immunity (CMI) response to different antigens was examined in healthy women, in patients with cervical precancerous lesions, and in patients with cervical cancer. Cervical lesions were diagnosed by cytological (PAP) smears, from examination by colposcopy, and from “punch” biopsy material by histology. CMI response is related to specific processes in healthy and cancer cells. CMI was investigated by leukocyte adherence inhibition (LAI) assay using specific antigen (prepared from cervical carcinoma tissue) and non specific antigen (prepared from blood of mice infected by LDH—lactate dehydrogenase—virus). The CMI responses of healthy women and cancer patients to the antigens used are different: the majority of T lymphocytes display adherence and non adherence, respectively (but the CMI responses elicited by the antigens are not equal and small quantitative differences are observed). Regardless of the CIN (cervical intraepithelial neoplasia) grades, CMI responses correspond either to healthy women or to cervical carcinoma patients (at about similar ratio of cases in all the CIN groups). Effect of non specific antigen suggests that cervical carcinoma transformation may be connected with reduction of mitochondrial activity similar to processes in LDH virus infection.

Peritoneal Washing Cytology on Fluid Hysteroscopy and After Curettage in Women with Endometrial Carcinoma

OBJECTIVE To assess the influence of fluid hysteroscopy with target biopsy of the endometrium and the influence of added curettage on the results of peritoneal washing cytology (PWC) in endometrial carcinoma. STUDY DESIGN In 42 women at risk of endometrial carcinoma, we performed fluid hysteroscopy with target biopsy of the endometrium and curettage. Evaluation of PWC of the pouch of Douglas was performed three times during the procedure: prior to hysteroscopy, after fluid hysteroscopy with target biopsy and after curettage. RESULTS On cytologic slides from peritoneal washings in 11 patients with carcinoma of the endometrium, malignant endometrial cells were found after curettage in 72.7%. There was no statistically significant difference in PWC prior to hysteroscopy (two women, 20%) or after hysteroscopy with target biopsy (three women, 30%). There was a statistically significant difference (.05 level) in positive PWC after hysteroscopy with target biopsy (three women, 33.3%) and after curettage (eight women, 88.9%). CONCLUSION Slides from carcinoma of the endometrium in PWC do not deteriorate after hysteroscopy with target biopsy of the endometrium, but tumor cells will appear in the pouch of Douglas after curettage.

Effects of sinusoidal magnetic field on adherence inhibition of leucocytes: preliminary results

The leucocyte surface properties manifest the cell-mediated immunity. The response of the cell-mediated immunity to external magnetic field was examined by observing leucocyte adherence to solid state surfaces. In the presence of antigen, leucocytes taken from cancer patients exhibit decreased adherence in contrast with adherence of leucocytes from healthy humans. After 1 h exposure to a sinusoidal magnetic field of 50 Hz and of 1 mT or 10 mT, adherence of leucocytes taken from cancer patients is strongly increased. The 1 mT magnetic field has stronger effect than the 10 mT field.

The Influence of Propionibacterium acnes (Corynebacterium parvum) Fractions on Immune Response in vivo

Bacterin of Propionibacterium acnes (Corynebacterium parvum), its cellular fractions (lipids, fractions obtained by mechanical disruption and differential centrifugation, by phenol-water and pyridine extractions), and a polysaccharide from culture filtrate were prepared and tested in mice. The activation of RES by splenomegaly and hepatomegaly, prevention of listerial infection, prevention of the lethal effect of sarcoma 180, and depression of liver microsomal cytochrome P-450 were employed. The bacterin was effective in all tests. Lipid-free cells were less active, in particular in the activation of RES and in the listerial infection model. Fractions prepared by the disruption and differential centrifugation lost their activity in all tests along with a decrease in molecular weight. Lipids extracted by ethanol caused pronounced splenomegaly and decreased the cytochrome P-450 content. The residue left after the phenol-water extraction was very active, its delipidation did not destroy the activity. Pyridine extraction provided a completely inactive extract, but a very active residue. The possibility of reducing the complexity of bacterin while preserving immunomodulatory effect is demonstrated.

Genetic and epigenetic characterization of low-grade gliomas reveals frequent methylation of the MLH3 gene.

Diffuse astrocytomas and oligodendrogliomas (WHO grade II) are the most common histological subtypes of low‐grade gliomas (LGGs). Several molecular and epigenetic markers have been identified that predict tumor progression. Our aim was in detail to investigate the genetic and epigenetic background of LGGs and to identify new markers that might play a role in tumor behavior. Twenty‐three patients with oligodendroglioma or oligoastrocytoma (LGO) and 22 patients with diffuse astrocytoma (LGA) were investigated using several molecular‐cytogenetic and molecular methods to assess their copy number variations, mutational status and level of promoter methylation. The most frequent findings were a 1p/19q codeletion in 83% of LGO and copy‐neutral loss of heterozygosity (CN‐LOH) of 17p in 72% of LGA. Somatic mutations in the isocitrate dehydrogenase 1 or 2 (IDH1/IDH2) genes were detected in 96% of LGO and 91% of LGA. The O‐6‐methylguanine‐DNA‐methyltransferase (MGMT) promoter was methylated in 83% of LGO and 59% of LGA. MutL homolog 3 (MLH3) promoter methylation was observed in 61% of LGO and 27% of LGA. Methylation of the MGMT promoter, 1p/19q codeletion, mutated IDH1, and CN‐LOH of 17p were the most frequent genetic aberrations in LGGs. The findings were more diverse in LGA than in LGO. To the best of our knowledge, this is the first time description of methylation of the MLH3 gene promoter in LGGs. Further studies are required to determine the role of the methylated MLH3 promoter and the other aberrations detected.

Karyotype at diagnosis, subsequent leukemic transformation and survival in myelodysplastic syndrome

240 patients with MDS studied cytogenetically at diagnosis between 1981 and 1990 were followed until death or until April 1992 to evaluate the prognostic significance of FAB classification, age and karyotype. 61 patients (25.4%) subsequently transformed into AML and 176 (73.3%) died during the follow-up period. Patients with blastic MDS types had a shorter survival and a higher probability of leukemic transformation. The younger age increased the probability of leukemic transformation, but was associated with a longer survival. The absence of analyzable mitoses was associated with a shorter survival. The complex chromosomal abnormalities at the initial evaluation identified a subgroup of patients with a high risk of a short survival and/or subsequent leukemia transformation. In refractory anemia the presence of complex chromosomal abnormalities was linked with a relative risk of 3.58 of leukemic transformation and shorter survival as compared with other cytogenetically defined groups.

Diseases caused by defects of energy level and loss of coherence in living cells.

Abstract Human and animal diseases are brought about by pathological alterations of production, composition, and conformation of macromolecules and structures in cells. Additional contributing factors include changes in physiological states caused by disturbances of energy supply, energy transduction, energy dissipation in moving or oscillating parts, and parasitic energy consumption. Disturbances of energy states may endanger existence of the system. The cell-mediated immunity (CMI) response of T lymphocytes correlating with their adherence properties was examined using antigen prepared from the serum of inbred laboratory mice strain C3H H2k infected with lactate dehydrogenase elevating (LDH) virus. LDH virus is a parasite on the cellular energy system. Significant CMI response was elicited in T lymphocytes prepared from the blood of patients with cancer of different phenotypes, acute myocardial infarctions, schizophrenia, and recurrent spontaneous abortions in early pregnancy from unknown reasons. The CMI response is assumed to monitor transferred information about decreased levels of energy states and decoherence in the cells caused by mitochondrial malfunction, parasitic consumption, production of lactate, and possibly other disturbances. The LDH virus infection or similar pathological processes caused by different agents might be connected with the diseases and monitored by the examined CMI response. A large amount of mitoses with chromosome defects in aborted fetuses suggest increased mutability of genomes caused by defective energy states.

Cytologic Detection of Cervical and Endometrial Carcinoma with Other Genital Tract Involvement

OBJECTIVE To investigate the possibility of a correct cytologic diagnosis of cervical and endometrial carcinoma with other genital organ involvement. STUDY DESIGN From uteri removed during hysterectomy due to cervical (33 cases) and endometrial (44 cases) cancer, samples were taken by cytobrush or spatula from the ectocervix, endocervix and endometrium of uteri opened longitudinally. Smears and cytosediments were stained by the Papanicolaou polychrome method. Moreover, acid beta-galactosidase activity was demonstrated in serial cytosediments by the indigogenic method of Lojda. From quenched tissue samples taken from the same sites as those for cytology, a series of cryostat sections was prepared and stained by hematoxylin and eosin or azure A, or subjected to the reaction for acid beta-galactosidase. RESULTS In 17 of 33 patients with cervical cancer, the same type of cancer was also found in smears of the endocervix and endometrium. In six patients the type of cancer was different. Of 44 patients with endometrial cancer, 16 had an endocervical malignancy of the same type. In seven cases the type of cancer was different. The reaction for acid beta-galactosidase helped in the differentiation between squamous (negative reaction in cancer cells) and cylindrocellular (positive reaction) cancer in cytologic preparations. CONCLUSION Before treatment, it is necessary to determine if there is involvement of the endocervix in endometrial cancer and of the endometrium in cervical cancer. Routine cytologic examination supplemented by the reaction for acid beta-galactosidase proved to be useful for this purpose.

Locally advanced laryngeal cancer: Total laryngectomy or primary non-surgical treatment?

Between January 1997 and December 2013, the Charles University 3rd Medical School and Royal Vinohrady Teaching Hospital Ear, Nose and Throat oncology team treated 185 patients with advanced laryngeal cancer, which, from a surgical perspective, required a total laryngectomy. Overall, ~70% of these patients (n=129) underwent conventional treatment (i.e., total laryngectomy with post-operative radiotherapy), and ~30% (n=56) were treated with larynx preservation protocols (including primary radiotherapy, neoadjuvant chemotherapy followed by radiotherapy or chemoradiotherapy, or primary chemoradiotherapy). Patients treated with laryngeal preservation protocols had a 5-year survival probability of 48%, whereas those treated with total laryngectomy and post-operative radiotherapy had a 5-year survival probability of 63%. This difference was not statistically significant. However, patients who underwent primary surgical treatment survived for a significantly longer period (P<0.010). The sex of the patient did not have a statistically significant impact on patient survival probability. More extensive local disease and more advanced disease stages conferred a lower survival probability, but were not statistically significant; however, a lower survival probability in patients >70 years was identified to be statistically significant (P<0.010). Local disease recurrence and recurrent cervical nodal metastases had a statistically significant impact on the 5-year survival probability (P<0.001). A step wise Cox regression analysis was used to compare the parameters of sex, patient age, tumor extent, disease stage, choice of primary surgery, local recurrence and cervical nodal recurrence. In the first step, local recurrence was selected as the parameter having the greatest effect on survival (P<0.001); patient age >70 years (P<0.001) was selected in the second step; cervical nodal recurrence (P<0.001) in the third step; and disease stage (P<0.010) in the fourth step. Other parameters did not significantly affect survival. The results of the present study confirmed that primary non-surgical treatment is an alternative approach to total laryngectomy, and that an informed patient should determine the treatment approach. The decreased overall survival observed in more extensive tumors suggests that surgical treatment may be a better selection in these cases. Due to increased overall survival, primary non-surgical treatment may be recommended for younger patients. If the patient chooses primary non-surgical treatment, concomitant chemoradiotherapy is recommended. If the patient cannot tolerate cytostatic chemotherapy, radiotherapy alone is recommended.