Gilteritinib as treatment for extra-medullary relapse of FLT3-ITD acute myeloid leukemia FLT3-ITD, after allogeneic haematopoietic stem cell transplantation.

Case of a patient with acute myeloid leukemia (AML) positive for mutations in both genes NPM1 and FLT3-ITD who underwent two allogeneic haematopoietic stem cell transplants (HSCT); the second allograft one was followed by extramedullary relapse (granulocytic sarcoma of right breast), with blast cells positive for FLT3-ITDmutation.  Treatment with Gilteritinib, a second generation selective oral type I FLT3 inhibitor, was started after the second HSCT with complete regression of breast granulocytic sarcoma in absence of hematological and extra hematologic toxicity. We conclude that Gilteritinib can represent an effective therapy for extra hematologic relapse, with acceptable toxicity and outpatient management.

'Real-life' report on the management of chronic GvHD in the Gruppo Italiano Trapianto Midollo Osseo (GITMO).

Several guidelines have been published about management of chronic GvHD (cGvHD), but the clinical practice still remains demanding. The Gruppo Italiano Trapianto di Midollo Osseo (GITMO) has planned a prospective observational study on cGvHD, supported by a dedicated software, including the updated recommendations. In view of this study, two surveys have been conducted, focusing the management of cGvHD and ancillary therapy in cGvHD, to address the current 'real life' situation. The two surveys were sent to all 57 GITMO centers, performing allografting in Italy; the response rate was 57% and 66% of the interviewed centers, respectively. The first survey showed a great disparity especially regarding steroid-refractory cGvHD, although extracorporeal photo-apheresis resulted as the most indicated treatment in this setting. Another challenging issue was the strategy for tapering steroid: our survey showed a great variance, and this disagreement could be a real bias in evaluating outcomes in prospective studies. As for the second survey, the results suggest that the ancillary treatments are not standardized in many centers. All responding centers reported a strong need to standardize management of cGvHD and to participate in prospective trials. Before starting observational and/or interventional studies, a detailed knowledge of current practice should be encouraged.

Comparative molecular pathology of sporadic hyperplastic polyps and neoplastic lesions from the same individual.

AIM: The biology of colorectal hyperplastic polyps is of considerable relevance, because recent evidence suggests that under certain circumstances hyperplastic polyps may be precursors of neoplasms. The aim of this study was to assess and compare the clinical and molecular characteristics of hyperplastic polyps and neoplastic lesions removed from patients without the hyperplastic polyposis syndrome. METHODS: One hundred and twenty six patients were identified through a series of genetic epidemiological studies. Each patient had at least one neoplastic lesion and one hyperplastic polyp; there was a total of 147 hyperplastic polyps. All lesions were evaluated for K-ras mutations, loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) gene, and microsatellite instability. RESULTS: K-ras mutation was detected in 15 (10%) hyperplastic polyps, all from the rectosigmoid colon. No hyperplastic polyp had APC LOH or microsatellite instability. Patients with adenomas or carcinomas showing K-ras mutations were not more likely to have hyperplastic polyps with K-ras mutations. The average number of adenomas did not differ between those patients with hyperplastic polyps with K-ras mutations and those without K-ras mutations. There was no association between the hyperplastic polyp and the adenoma regarding the colon segments from which the two lesions were removed. CONCLUSIONS: The sporadic hyperplastic polyp is a lesion with limited molecular change and no relation to patients' neoplastic lesions.

Molecular changes in the Ki-ras and APC genes in primary colorectal carcinoma and synchronous metastases compared with the findings in accompanying adenomas.

AIMS: To compare the molecular genetic changes in the Ki-ras and adenomatous polyposis coli (APC) genes between colorectal carcinomas and synchronous metastases, and then to compare and contrast those changes with previously reported changes in the two genes between these carcinomas and accompanying adenomas. This expanded comparison would provide greater understanding of the progression of molecular changes in neoplastic tissue during the development of malignancy from a benign adenoma to carcinoma and then to metastatic spread of the malignancy. METHODS: DNA was extracted from paraffin wax embedded tissue. This was followed by polymerase chain reaction and gel electrophoresis for mutations in the Ki-ras gene using single stranded conformational polymorphism analysis. Amplification of a CA repeat marker was used to assess loss of heterozygosity (LOH) at the APC gene. RESULTS: The findings for the Ki-ras gene in 42 paired carcinomas and synchronous metastases were identical, regardless of whether or not the carcinoma and its companion adenoma had identical Ki-ras findings. The results of APC LOH for 39 paired carcinomas and synchronous metastases were also identical, whether or not the carcinoma and its companion adenoma had identical APC LOH findings. Results were uninformative for three pairs. CONCLUSIONS: With respect to these two genes, a carcinoma may be discordant from its companion adenoma, but the metastasis remains consistent with the colonic carcinoma.

Molecular changes in the Ki-ras and APC genes in colorectal adenomas and carcinomas arising in the same patient.

The purpose of this study was to compare the molecular genetic changes in the Ki-ras and adenomatous polyposis coli (APC) genes between adenomas and carcinomas removed from the same patients. This comparison of benign and malignant tissue would enhance understanding of the progression of molecular changes during the development of colorectal malignancy and similarities between paired lesions could be indicative of a common aetiology. The basic procedures used were DNA extraction from wax blocks of removed tissue, followed by polymerase chain reaction (PCR) and gel electrophoresis for mutations in the Ki-ras gene using single strand conformational polymorphism (SSCP); amplification of a CA repeat marker was used to assess for loss of heterozygosity (LOH) of the APC gene. The main findings in 100 adenoma and carcinoma pairs for the Ki-ras gene were as follows: the frequency of Ki-ras mutation in the adenomas increased with increasing villous component, but did not vary in the paired carcinomas; the frequency of Ki-ras mutation in villous adenomas was greater than in carcinomas; and when both paired lesions had Ki-ras mutations, only 44% had the identical mutation. For the APC gene, the incidence of LOH in the adenomas did not vary by histological type; the LOH status of the adenoma was associated with that of the paired carcinoma; but when both paired lesions had LOH of the APC gene, only 50% had LOH for the same allele. In conclusion, these data on paired adenomas and carcinomas suggest that a Ki-ras mutation is not a consistent finding between the adenoma and carcinoma from the same bowel. The development of LOH of the APC gene is a slightly more consistent finding between the pair, but is not always allelic-specific.

K-ras mutation and loss of heterozygosity of the adenomatous polyposis coli gene in patients with colorectal adenomas with in situ carcinoma.

BACKGROUND: The majority of colorectal carcinomas, if not all, arise from a benign adenoma. The DNA of the carcinomatous cells frequently has mutations in several genes. However, it is not exactly clear when during the neoplastic process each mutation develops. An adenoma with an area of in situ carcinoma provides an opportunity to evaluate genetic changes within a single neoplasia whose separate areas are comprised of both the benign adenoma as well as the malignant carcinoma. METHODS: Thirty-seven neoplasms with areas of both benign adenoma and in situ carcinoma were studied. Both portions were evaluated for loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) gene and for mutations in codons 12/13 of the K-ras oncogene using the polymerase chain reaction technique. RESULTS: Twenty-eight neoplasms showed no LOH in either portion whereas both portions of 4 neoplasms revealed a loss of heterozygosity. In three lesions the APC gene was normal in the adenomatous portion but LOH was present in the carcinomatous portion. Two neoplasms were uninformative for LOH of the APC gene. Thirteen neoplasms showed the wild-type pattern for the K-ras oncogene whereas 15 contained the identical mutation in both portions. Of the remaining nine neoplasms, six had a K-ras mutation in the adenomatous portion only and three had one pattern in the adenomatous portion and a different pattern in the in situ carcinoma portion. CONCLUSIONS: LOH of the APC gene is an early and persistent feature in the evolution of a benign colorectal adenoma into an in situ carcinoma. There is less consistency regarding K-ras mutations; one in five in situ carcinomas contains a K-ras mutation different from that observed in the adenomatous portion.

Comparison of allelic ratios from paired blood and paraffin-embedded normal tissue for use in a polymerase chain reaction to assess loss of heterozygosity.

BACKGROUND: One method to assess loss of heterozygosity (LOH) of various genes is the amplification of DNA from neoplastic tissue by using microsatellite markers. LOH can best be considered on a quantitative basis as a comparison of allelic ratios of neoplastic tissue to that of the normal control. We will illustrate through quantitative methods the importance of using the appropriate controls when determining allelic loss. METHODS AND RESULTS: DNA extracted from 28 paired blood and formalin-fixed, paraffin-embedded normal mucosal tissue was amplified using the DP1 microsatellite marker, consisting of a variable number of CA repeats. This marker is located within the D5S346 (DP1) region on chromosome 5 and is linked to the adenomatous polyposis coli gene. Allelic ratios were calculated after scanning autoradiographs on a densitometer. Ratio values approaching 1 were observed when the two alleles were close in molecular weight, whereas ratios less than 1 were detected when the two alleles had very different molecular weights. This discrepancy was more pronounced in paraffin-embedded tissue than with blood samples. CONCLUSION: For LOH amplification assays, it is best to use normal control samples that are of the same tissue source as the neoplastic sample being analyzed. When assessing LOH in neoplastic tissue, a quantitative value rather than visual assessment of the alleles should be considered. The values may be normalized by dividing the ratio of the two tumor alleles by the ratio of the two normal alleles.

Conformational properties of Z-forming DNA oligomers bearing terminal unpaired bases.

Three sets of semi-self-complementary deoxyribonucleotide decamers with the sequence XX-(5meCG)4, (5meCG)4-XX, or Y-(5meCG)4-Y, where XX = AA, CC, GG, or TT and Y = A, C, G, or T, were synthesized along with the self-complementary octamer (5meCG)4. The 8-mer duplex readily undergoes a B-to-Z conformational conversion upon increasing the NaCl concentration with a transitional midpoint of approximately 1.1 M NaCl. The 10-mers should form 8-bp duplexes a with core sequence of [(5meCG)4]2 with 5'-XX overhangs, 3'-XX overhangs, or 5',3'-Y/Y mismatches. Circular dichroism was employed to determine the conformations of all oligomers. Salt titrations were performed to measure the effect of overhangs and terminal mismatches on the B-to-Z conversion. In general, the presence of 5'-XX overhangs results in a transition midpoint equal to or slightly higher than the control, whereas the presence of 3'-XX overhangs results in a transition midpoint slightly lower than the control. The 3'-CC and 5'-GG overhangs are exceptions, with transition midpoints much higher than the control. These oligomers apparently form duplexes with 5',3'-C/C or 5',3'-G/G mismatches abutting a [(G5meC)4]2 duplex core. The presence of terminal mismatches in the third set of oligomers results in transition midpoints higher than the control. Ultraviolet absorbance methods were used to evaluate the effect of the various stacking motifs of the 10-mers on the thermodynamics of melting relative to the 8-mer for both B and Z conformations. We found that in both the B and Z conformations, the presence of an overhang stabilizes the [(5meCG)4]2 duplex, with the 5' overhangs having a greater stabilizing effect relative to the 3' overhangs. The presence of 5',3'-Y/Y mismatches also imparts a stabilizing effect on the control 8-mer in both the B and Z conformations. These results are discussed in terms of stacking interactions of the terminal unpaired bases.

Sequence and environmental effects on the self-assembly of DNA oligomers possessing GxT2Gy segments.

It is well-known that DNA oligomers possessing contiguous guanine bases can assume non-Watson-Crick type structures through the formation of four-stranded species. The architecture of these four-stranded structures is highly dependent upon the sequence of the DNA and the conditions (e.g., buffer, pH, ionic strength, cations present, and temperature) under which the DNA is prepared. This lab has previously reported the self-assembly of DNA oligomers of sequence C4T4G4T1-4G4 into multistranded high molecular weight species [Dai, T.-Y., Marotta, S. P., & Sheardy, R. D. (1995) Biochemistry 34, 3655-3662]. In order to further investigate the sequence and environmental effects on the self-assembly of DNA oligomers possessing GxT2Gy (where x = 1, 3, or 4 and y = 2-5) segments, the synthesis of a number of such oligomers was undertaken. DNA samples were prepared in standard phosphate buffer (10 mM phosphate, pH 7.0) and NaCl, KCl and/or MgCl2 added to different concentrations in order to evaluate the influence of the cations and their concentrations on the self-assembly of the DNA oligomers. The self-assembly of these oligomers was monitored by nondenaturing polyacrylamide gel electrophoresis and circular dichroism studies. Electrophoresis of the oligomers in either 100 mM K+ or 50 mM Na+ with 50 mM K+ indicated the formation of one or two molecular species for these oligomers. In contrast, electrophoresis of these oligomers in the presence of both 100 mM K+ and 20 mM Mg2+ give a ladder of multiple bands of high molecular weight indicative of multistanded DNA structure formation. The results presented here indicate that self-assembly into high molecular weight species is favored by the presence of Mg2+ as well as the presence of four or more bases in the terminal Gy segment. These results also suggest that the structure of telomeric DNA, which possesses similar sequences, may be quite unusual.

Self-assembly of DNA oligomers into high molecular weight species.

Thermal denaturation, gel electrophoresis, and circular dichroism methods were used to characterize DNA oligomers possessing one or two segments of four contiguous G bases in order to investigate their environmentally dependent conformational properties. The sequences of the oligomers studied were the following: HP1-T series, C4T4G4T5-8; HP1-TG series, C4T4G4T1-4G4. In NaCl at concentrations up to 200 mM, the melting profiles of these oligomers are characterized by single inflection points whose Tm values are independent of DNA concentration. In addition, these oligomers run as single bands in polyacrylamide gels under those same conditions as well as in 100 mM K+ or 20 mM Mg2+. These data suggest that these oligomers exist as intramolecular hairpins comprised of four G:C base pairs in the stems, loops of four T bases, and 3'-overhangs of T5-8 or T1-4G4. In the presence of 100 mM K+ plus 20 mM Mg2+, however, gel electrophoresis indicates that oligomers of the HP1-T series exist as equilibria between parent hairpins and four-stranded structures (i.e., quadraplexes). Quadraplex formation for any member of the HP1-T series requires unfolding of the hairpin, exposing the G4 segment prior to quadraplexation. Members of the HP1-TG series self-assemble into multistranded species of high molecular weight in the presence of 100 mM K+ plus 20 mM Mg2+. For this series of oligomers, the data suggest that these higher order species arise from successive additions of parent oligomer to an initially formed quadraplex.(ABSTRACT TRUNCATED AT 250 WORDS)

A thermodynamic investigation of the melting of B-Z junction forming DNA oligomers.

Ultraviolet absorbance methods were used to characterize the thermodynamics of melting of a series of 16 bp deoxyoligonucleotides over a wide range of NaCl concentrations (0-4.5 M) and to obtain complete thermodynamic profiles for their melting at 0.115 and 4.5 M NaCl. The sequence of the series (one strand of duplex) was: 5'-CGCGCGCGAMNGACTG-3', where C indicates m5dC and -MN- was varied to include all combinations of Py:Py stacks (CC, TT, CT, TC). The unmethylated deoxyoligonucleotide 5'-CGCGCGCGACTGACTG-3' was used as a control sequence. All of the methylated oligonucleotides studied undergo a NaCl-induced transition to a hybrid form containing a left-handed, Z-DNA, region joined to a right-handed region by a B-Z junction. Our experiments allowed us to quantitatively evaluate the effects of NaCl, sequence, and methylation and the transition to the hybrid BZ structure on DNA thermal stability. We found that alteration of a single dinucleotide step has profound effects on the thermal stabilities of the 16 bp fragments studied. Methylation was found to destabilize the double helix, resulting in a decrease in Tm. Transition to the hybrid BZ structure, somewhat surprisingly, was found to only slightly destabilize DNA, with an observed decrease in free energy of melting of approximately 0.5 kcal/mol relative to the control, right-handed, sequence in high salt. Transition melting temperatures (Tm) were found, in agreement with previous studies on polymeric DNA, to depend upon NaCl concentration in a complicated, nonlinear fashion. m values increase to maximal values at circa 1.0 M NaCl, but decrease thereafter with further addition of salt.(ABSTRACT TRUNCATED AT 250 WORDS)

Body temperature and heart rate rhythms in acutely head-injured patients.

This descriptive pilot study investigated the circadian (24-hour) rhythms of heart rate and temperature in acutely head-injured patients and the relationships between these variables. The convenience sample consisted of 10 patients who were admitted to the hospital with the diagnosis of acute head injury. Patel's Method of Partition was used to estimate rhythmicity. Only one patient demonstrated circadian rhythms for body temperature and heart rate with the peaks occurring at appropriate times. While three other patients had significant rhythms for these variables, the rhythms had cycles of less than 24 hours and peaks occurring at inappropriate times. Although significant correlations were found between body temperature and heart rate rhythms, they were low.

Endocrine and psychologic responses of patients to cardiac pacemaker implantation.

The findings that all HPA and SAM indexes increased during the first postoperative days strongly suggest that transvenous, permanent cardiac pacemaker implantation is a stressor. Since the psychologic tests did not demonstrate marked changes in anxiety or affective mood states, and the former was only weakly related to the endocrine responses, psychologic stimuli cannot be ascribed a prominent role in causing the observed endocrine alterations. Thus, the data suggest that physiologic stressors, such as surgical trauma and the irritation of the tissue surrounding the pacemaker, were the primary stimuli that activated the HPA and SAM systems. Although the structured teaching program resulted in a marked improvement of the treatment groups's knowledge of the device and the follow-up care it requires, it did not affect the endocrine or psychologic responses of patients to cardiac pacemaker implantation.

Effects of corticosterone and metyrapone on gastrointestinal neurotransmitter enzyme activities.

Adrenal glucocorticoids have been shown to produce alterations in the enzymes which synthesize and degrade cholinergic and catecholaminergic neurotransmitters in the central and peripheral nervous system. The present study examined the impact of altering plasma corticosterone levels via corticosterone or metyrapone ingestion, via the drinking water, on the developmental profile of choline acetyltransferase (ChAT), acetylcholine esterase (AChE), tyrosine hydroxylase (TH) and monoamine oxidase (MAO) in various gastrointestinal (GI) segments of rats. Three groups were studied: non-treated, corticosterone treated and metyrapone treated. Drug intake of pregnant (i.e., beginning on the 15th day of gestation), lactating and weaned rats was monitored until the male pups were sacrificed at 1, 3, 7, 14, 21, 35 and 50 days of age. Results showed that ChAT and TH activities peaked earlier in development in corticosterone treated rats as compared to non-treated and metyrapone treated rats. During the early postnatal period plasma corticosterone levels were inversely related to AChE and MAO activities in most GI segments. These results indicate that neurotransmitter enzyme activities in various GI segments are influenced by corticosterone during a critical period of development.

Role of diets in modifying gastrointestinal neurotransmitter enzyme activity.

Manipulation of diet, by altering the composition or quantity, can produce alterations in the central nervous system as evidenced by changes in memory or behavior. The present study was designed to examine the effects of fasting/starvation or a choline deficient diet on the enzymes that synthesize and degrade acetylcholine and norepinephrine, both neurotransmitters in the gastrointestinal tract. This study was conducted on three groups of male rats: control nontreated, choline deficient diet for 14 days, and fasting/starvation (water only) for 3 days. Enzyme activities were determined throughout the length of the gut. The lack of dietary choline produced a decrease in adrenergic enzyme activity but had little effect on cholinergic enzyme activities. Starvation decreased adrenergic enzyme activities and increased activity of the acetylcholine synthesizing enzyme in most segments of the gastrointestinal tract. Starvation was also a stressor, as evidenced by elevated levels of plasma and adrenal corticosterone. The implication from these experiments is that dietary manipulation may produce functional changes (i.e., motility and secretion) in the gastrointestinal tract as a result of altering neurotransmitter enzyme activities.

Hypoxic and nycthemeral responses by the adrenal cortex of partially hepatectomized rats.

Adrenocortical nycthemeral (day-night alteration) and hypoxic stress responses of rats were evaluated 48 h following 70% hepatectomy. Morning plasma and adrenal corticosterone concentrations of hepatectomized (H) rats at 0800 hours were significantly higher than those of sham (S) operated animals. Although both groups showed significant nocturnal elevations in these parameters at 2000 hours, the nocturnal rises in plasma corticosterone levels were 75% and 223% in H and S rats, respectively, yet the absolute steroid levels did not differ between the groups. The hypothalamic-pituitary-adrenocortical (HPA) response of H animals to hypoxia was assessed by measuring plasma corticosterone levels at various times before, during and after exposure to 10% O2 at ground level. Curvilinear analysis of the plasma steroid levels revealed a significantly slower corticosterone rise during the hypoxic phase in H than S animals, as well as a much slower return to baseline levels after the hypoxic stimulus was terminated. The latter indicates a decreased hepatic capacity to inactivate steroids in H animals, while the sluggish activation of the HPA system by hypoxia may be due to altered neuroendocrine control resulting from prior exposure of feedback sites to elevated basal plasma corticosterone levels.

Comparative in vitro responses of fetal, pregnant and non-pregnant rabbits' adrenal glands to steroidogenic agents.

The in vitro secretion of aldosterone and corticosterone by the adrenal glands of fetal (day 30), pregnant and non-pregnant rabbits was examined under basal and stimulated conditions. In general, non-pregnant animals basally secreted less aldosterone than either pregnant or fetal rabbits, whereas basal corticosterone secretion by pregnant animals exceeded that of either fetal or non-pregnant animals. At similar doses of adrenocorticotropin (ACTH), fetal and pregnant adrenal glands produced comparatively more aldosterone than non-pregnant animals, while corticosterone secretion was accelerated to a greater degree in fetal rabbits than in the other groups. Angiotensin II had its greatest effect on the aldosterone secretory rates of fetal and non-pregnant animals without affecting corticosterone secretion in any group. Elevated potassium (K+) enhanced the secretory rates of aldosterone and corticosterone in fetal animals, while increasing only aldosterone secretion in non-pregnant rabbits. Serotonin accelerated aldosterone secretion in all animals, whereas it increased corticosterone secretion only in non-pregnant animals. These results suggest that (1) in fetal rabbits, the secretory rates of both aldosterone and corticosterone are regulated primarily by ACTH and to a much lesser extent by angiotensin II and K+, (2) the corticosterone secretory rates of pregnant and non-pregnant rabbits are controlled mainly by ACTH, and (3) aldosterone secretion by non-pregnant animals is regulated primarily by angiotensin II and secondarily by ACTH and K+, while in pregnant animals ACTH may be the primary regulator of aldosterone secretion as it is in the fetus.

Development of neurotransmitter enzyme activity in the rat gastrointestinal tract.

Acetylcholine and norepinephrine play important roles in determining gastrointestinal (GI) tract motility and secretion. At this time, however, little is known concerning the postnatal developmental patterns of the enzymes that synthesize and degrade these two neurotransmitters within the GI tract. The present study examined the developmental activities, expressed per gram protein per minute, of the cholinergic enzymes choline acetyltransferase (ChAT) and acetylcholine esterase (AChE) and the adrenergic enzymes tyrosine hydroxylase (TH) and monoamine oxidase (MAO) in male rats between days 1 and 50. Seven anatomic segments of the GI tract were assayed for enzyme activities. In general, all four neurotransmitter enzymes were present throughout the length of the GI tract and increased during the early postnatal period. The synthesizing enzymes ChAT and TH displayed peak activity prior to day 21, while the degrading enzymes AChE and MAO continued to increase past day 21. Each enzyme exhibited segmental differences and unique postnatal developmental patterns. Such differences in enzyme activities may be related to developmental increases in neuronal density, hormonal factors, or direct stimulation of the GI tract by liquid and/or solid diet.