A simple one-tube assay for immunophenotypical quantification of leukemic stem cells in acute myeloid leukemia.

Relapses after initial successful treatment in acute myeloid leukemia are thought to originate from the outgrowth of leukemic stem cells. Their flow cytometrically assessed frequency is of importance for relapse prediction and is therefore assumed to be implemented in future risk group profiling. Since current detection methods are complex, time- and bone marrow consuming (multiple-tubes approach), it would be advantageous to have a broadly applicable approach that enables to quantify leukemia stem cells both at diagnosis and follow-up. We compared 15 markers in 131 patients concerning their prevalence, usefulness and stability in CD34(+)CD38(-) leukemic stem cell detection in healthy controls, acute myeloid leukemia diagnosis and follow-up samples. Ultimately, we designed a single 8-color detection tube including common markers CD45, CD34 and CD38, and specific markers CD45RA, CD123, CD33, CD44 and a marker cocktail (CLL-1/TIM-3/CD7/CD11b/CD22/CD56) in one fluorescence channel. Validation analyses in 31 patients showed that the single tube approach was as good as the multiple-tube approach. Our approach requires the least possible amounts of bone marrow, and is suitable for multi-institutional studies. Moreover, it enables detection of leukemic stem cells both at time of diagnosis and follow-up, thereby including initially low-frequency populations emerging under therapy pressure.

Peripheral blood minimal residual disease may replace bone marrow minimal residual disease as an immunophenotypic biomarker for impending relapse in acute myeloid leukemia.

As relapses are common in acute myeloid leukemia (AML), early relapse prediction is of high importance. Although conventional minimal residual disease (MRD) measurement is carried out in bone marrow (BM), peripheral blood (PB) would be an advantageous alternative source. This study aims to investigate the specificity of leukemia-associated immunophenotypes used for MRD detection in blood samples. Consistency of PB MRD as compared with BM MRD was determined in flow cytometric data of 205 paired BM and PB samples of 114 AML patients. A significant correlation was found between PB and BM MRD (r=0.67, P<0.001), while median PB MRD percentage was factor 4-5 lower compared with BM MRD. Primitive blast (CD34+/CD117+/CD133+) frequency was significantly lower in PB (median factor 23.7), indicating that PB MRD detection is more specific than BM. Cumulative incidence of relapse 1 year after induction therapy was 29% for PB MRD-negative and 89% for PB MRD-positive patients (P<0.001). Three-year OS was 52% for MRD-negative and 15% for MRD-positive patients (P=0.034). Similar differences were found after consolidation therapy. As PB MRD appeared to be an independent predictor for response duration, the highly specific PB MRD assay may have a prominent role in future MRD assessment in AML.

Minimal residual disease detection defined as the malignant fraction of the total primitive stem cell compartment offers additional prognostic information in acute myeloid leukaemia.

INTRODUCTION: Immunophenotypic detection of minimal residual disease (MRD) in bone marrow (BM) of acute myeloid leukaemia (AML) patients is of high prognostic relevance. Standard MRD percentage is assessed as a percentage of total white blood cells (WBCs) and is therefore highly dependent on WBC count. Peripheral blood (PB) contains more than five times lower MRD percentages. Therefore, PB in BM aspirates cause dilution of the MRD cells, possibly leading to false-negative results for BM MRD. The latter is avoided when relating the fraction of malignant primitive cells, identified by aberrant marker expression [aberrant primitive cells (aPC)], to the total population of primitive cells. Such a fraction may in addition reflect an important biological parameter. METHODS: As this approach is thus independent of WBC count and the total size of the primitive compartment, we investigated the role of aPC fractions on overall and relapse-free survival (RFS) in 98 patients with AML under the age of 60. RESULTS: We show that this approach identifies MRD-negative (as defined by % of WBC) but aPC-positive (as defined by % of primitive cells) patients with poor outcome after both first and second induction cycle of chemotherapy. CONCLUSION: As a result, in cases with a primitive marker present, RFS is best predicted when combining standard MRD percentage with aPC fractions.

The role of minor subpopulations within the leukemic blast compartment of AML patients at initial diagnosis in the development of relapse.

The majority of pediatric and younger adult (<60 years) AML patients achieve complete remission. However, 30-40% of patients relapse and display a dismal outcome. Recently we described a frequent instability of type I/II mutations between diagnosis and relapse. Here, we explored the hypothesis that these mutational shifts originate from clonal selection during treatment/disease progression. Subfractions of blasts from initial diagnosis samples were cell sorted and their mutational profiles were compared with those of the corresponding relapse samples of 7 CD34(+) AML patients. At diagnosis, subfractions of the CD45(dim)CD34(+)CD38(dim/-) compartment were heterogeneous in the distribution of mutations, when compared to the whole CD45(dim)CD34(+) blast compartment in 6 out of 7 patients. Moreover, within CD45(dim)CD34(+)CD38(dim/-) fraction of initial samples of 5 of these 6 AML patients, we found evidence for the presence of a minor, initially undetected subpopulation with a specific mutational profile that dominated the bulk of leukemic blasts at relapse. In conclusion, our findings lend support to the AML oligoclonality concept and provide molecular evidence for selection and expansion of a chemo-resistant subpopulation towards development of relapse. These results imply that early detection of pre-existing drug-resistant leukemic subpopulations is crucial for relapse prevention by proper timing of targeted treatment.

Behavior and gastroesophageal reflux in the premature neonate.

BACKGROUND: The belief that behavioral observations assist in the clinical diagnosis of gastroesophageal reflux (GER) disease in premature neonates has not been formally tested. The purpose of this study was to determine whether esophageal acidification was associated with a recognizable pattern of behavioral changes in these infants. METHODS: The behavior of 14 healthy premature infants was recorded by a video camera while esophageal pH was simultaneously monitored. For each of 20 acid GER episodes recorded, a 10-minute video epoch, encompassing the onset of acid GER and lasting at least 4 minutes after the onset of GER, was examined. Two independent observers each scored reflux-associated epochs of "general" infant behavior and behavior previously shown to be indicative of reflux in normal term infants. RESULTS: The occurrence of esophageal acidification due to reflux did not significantly alter scores for general behavior. Infants frequently demonstrated reflux-specific behavior, including discomfort, head retraction, and mouthing; however, none of these behavioral patterns was temporally associated with the occurrence of acid GER. CONCLUSIONS: These data indicate that reflux-specific behavioral criteria, established in older term infants, may be inappropriate as diagnostic criteria for GER in premature neonates and may lead to the unnecessary use of antireflux therapy.

Measurement of upper esophageal sphincter tone and relaxation during swallowing in premature infants.

Upper esophageal sphincter (UES) motor function has not been previously evaluated in premature infants. The motor patterns associated with tonic activity and swallow-related relaxation of the UES were recorded for 1 h after completion of gavage feeding in 11 healthy preterm neonates (postmenstrual age 33-37 wk) with a micromanometric assembly, which included a sleeve sensor specifically adapted for UES recordings. A clearly defined UES high-pressure zone was observed in all premature infants studied. Resting UES pressure ranged from 2.3 to 26.2 mmHg and was higher during periods of irritability and apparent discomfort. During dry swallows, UES pressure relaxed from a resting pressure of 28.2 +/- 4.0 mmHg to a nadir of 1.1 +/- 3.3 mmHg. The mean UES relaxation interval (the time from relaxation onset to relaxation offset) was 0. 31 +/- 0.11 s. We conclude that in premature infants >/=33 wk postmenstrual age the motor mechanisms regulating UES resting pressure and the onset of UES relaxation are well developed.

Reproducibility of the 13C-octanoic acid breath test for assessment of gastric emptying in healthy preterm infants.

BACKGROUND: The 13C-octanoic acid breath test has been used to measure gastric emptying in preterm infants, but the reproducibility of the test has not been evaluated in this population. METHODS: Fifty-six paired breath test analyses were performed on 28 healthy preterm infants 1 to 5 days apart using the same food type, volume, and energy content for each paired sample. Breath samples were taken before the feeding, at 5-minute intervals after feeding for 30 minutes, then each 15 minutes for 4 hours. Samples were analyzed using an isotope-ratio mass spectrometer, and 3C recovery was used to calculate values for gastric-emptying coefficient and gastric half-emptying time. RESULTS: There was no significant difference between test results on different days in the paired samples studied. gastric-emptying coefficients for the first and subsequent samples were 2.6+/-0.1 (mean+/-SEM) and 2.7+/-0.1, respectively, and half-emptying times were 44.5+/-3.7 minutes and 41.4+/-3.2 minutes. CONCLUSION: The 13C-octanoic acid breath test is a reliable, noninvasive, and reproducible measure of gastric emptying in preterm infants that should have wide application for use in this population.

Mechanism of gastroesophageal reflux in premature infants with chronic lung disease.

BACKGROUND/PURPOSE: The aim of this study was to characterize the motor events responsible for gastroesophageal reflux (GER) and esophageal acid clearance in a cohort of preterm infants with chronic lung disease (CLD). METHODS: Esophageal motility was recorded for 2 to 3 hours postprandially in 14 preterm infants with mild-moderate or moderate-severe CLD at 33 to 39 weeks postmenstrual age using a sleeve-side hole micromanometric assembly into which a pH probe had been installed. RESULTS: Twenty-six acid GER episodes were recorded by pH probe. Of these, 22 (85%) were associated with transient lower esophageal sphincter (LES) relaxation. Transient LES relaxations were significantly longer in duration than single swallows (13.7 seconds v 4.6 seconds, P < .001) and had lower nadir pressures (1.1 mm Hg v 2.5 mm Hg, P < .001). During periods of esophageal acidification (pH below 4), peristaltic esophageal body pressure wave sequences cleared acid refluxate more effectively than nonperistaltic pressure wave sequences. CONCLUSIONS: In preterm infants with CLD, transient LES relaxations are the predominant mechanism underlying GER, and esophageal clearance mechanisms are fully functional, which is similar to that seen in healthy preterm infants.

Mechanisms of gastroesophageal reflux in healthy premature infants.

OBJECTIVES: The aim of this study was to characterize the motor events responsible for gastroesophageal reflux (GER) and esophageal acid clearance in a cohort of healthy preterm infants. STUDY DESIGN: Esophageal motility was recorded for 2 to 3 hours after a feeding in 24 preterm infants, 31 to 38 weeks' postmenstrual age, by using a sleeveside hole micromanometric assembly incorporating a pH probe. RESULTS: Sixty acid GER episodes were recorded by pH probe, and 133 non-acid GER episodes were recorded manometrically by the presence of esophageal common cavities. Of the 193 GER episodes, 159 (82%) were associated with transient lower esophageal sphincter relaxation (TLESR). TLESRs were significantly longer in duration than single swallows (15.1 seconds vs 5.6 seconds, P < .001) and had lower nadir pressures (0.8 mm Hg vs 2.3 mm Hg, P < .001). A total of 3216 esophageal body pressure waves were analyzed; 70% of swallow-induced pressure waves were peristaltic in sequence compared with 5% of swallow-unrelated pressure waves. During periods of esophageal acidification (pH below 4), peristaltic esophageal body pressure wave sequences cleared acid refluxate more effectively than nonperistaltic pressure wave sequences. CONCLUSIONS: In healthy preterm infants, TLESRs are the predominant mechanism underlying GER, and esophageal clearance mechanisms are well developed by at least 31 weeks' postmenstrual age.

Odontomicronychial ectodermal dysplasia.

This paper describes odontomicronychial dysplasia, a pure ectodermal dysplasia of the 2-3 subgroup of group A. It is characterised by precocious eruption and shedding of deciduous dentition, precocious eruption of secondary dentition with short, rhomboid roots, and short, thin, slow growing nails. This condition probably results from an autosomal recessive gene.

Diagnosis of IgE-mediated allergy in the upper respiratory tract.

A statistical analysis was made of data concerning diagnosis of IgE-mediated allergy in the upper respiratory tract in 292 patients. A study was made of: skin test, total and specific IgE (RAST), X-sinus, red blood investigation, and cytology of nasal smear. It appears that screening for the presence of an IgE-mediated allergy in the upper respiratory tract can be performed efficiently with a limited number of skin tests (rye grass, timothy, birch, house dust mite and cat). In this study a greater number of skin tests (15 instead of 5) yielded little additional information (in 2% of the patients). A rather poor correlation between skin test and RAST was found.

[The diagnosis of IgE-mediated allergy of the upper airways]. / Diagnostiek van IgE-gemedieerde allergie van de bovenste luchtwegen.

A statistical analysis was made of data concerning diagnosis of IgE-mediated allergy in the upper respiratory tract in 292 patients. It appears that screening for an IgE-mediated allergy can be performed with a limited number of skin tests (rye grass, timothy, birch, house dust mite and cat). In this study a larger number of skin tests (15 instead of 5) and investigation of specific IgE (RAST) yielded little additional information (in 2% and 3% of the patients, respectively). When indicated, an extended series of skin tests, immuno-assay (total IgE and specific IgE) and eosinophil counts in the serum and nasal smear should be performed.

Verification of the air-bone gap using cancellation and evoked responses.

Using the cancellation method as well as evoked responses, we investigated their application in the estimation of the air-bone gap in patients undergoing operation on middle ear structures. The cancellation method had a closer relationship with pure-tone audiometry than evoked responses in predicting the air-bone gap. This was caused by recruitment, by which the cancellation method was not affected. The results of brainstem-evoked responses to bone and air conduction signals showed therefore an underestimation in the prediction of the air-bone gap. Combining the cancellation method with evoked responses elicited to air conduction signals permitted the estimation of the cochlear reserve without the need of threshold measurement by bone conduction.

Phase relationship between bone and air conducted impulse signals in the human head.

Using the cancellation method we investigated the phase relationship as well as the amplitude ratio between impulse signals transmitted simultaneously via both bone and air conduction channels. The psychophysical findings indicated a phase difference corresponding to a time delay of about 0.9 ms by which the air input led the bone input in the case of frontal bone stimulation. Changing the polarity of the bone conduction signal a maximum in the loudness sensation was found just in the same phase difference. The psychophysical findings, predicted mathematically, were verified with brainstem evoked potentials elicited to similar signals. Preliminary results showed that the time lag observed relied on the location of the bone vibrator on the head and furthermore on the frequency used. This suggests different transmissions of vibratory energy reaching the inner ear along the skull.

Comparison of the latencies between bone and air conduction in the auditory brain stem evoked potential.

Brain stem auditory evoked potentials obtained by air and bone conduction were investigated in 22 subjects. The necessity for a carefully selected stimulus being presented to the headphone and the bone vibrator is discussed. Latency values of both responses are presented for wave V. Bone conduction showed latencies which were longer than those obtained by air conduction (about 0.9 msec). Possible mechanisms for this delay are discussed.

Effect of contralateral and ipsilateral masking of acoustic stimulation on the latencies of auditory evoked potentials from cochlea and brain stem.

Masking techniques were applied to a group of 10 subjects with normal hearing, to investigate the influence of masking on the latencies of brain stem auditory evoked potentials. The levels of sufficient masking and overmasking were determined. Central masking, as established in a psychophysiological manner, cannot be clearly demonstrated by auditory brain stem evoked potentials. When masking the untested ear, the alterations in the response are small and this can be ignored in clinical practice if masking of the untested ear is necessary.

The cancellation method for an accurate determination of an air-bone gap.

For a reliable detection of the bone conduction threshold blocking of the contralateral ear can be necessary but then the noise will affect the threshold in the test ear. To resolve this problem the cancellation method is used. The equalization of air and bone conduction sound level in the inner ear is very accurate, the relation between the intensities has been proved to be linear and independent of noise at the contralateral ear. An application for calibration of the bone conduction vibrator to the head phone is discussed. The benefit of the method for measuring an air-bone gap in an accurate way in patients suffering bilaterally from a severe conductive hearing loss is shown.