A genetically defined signature of responsiveness to erlotinib in early-stage pancreatic cancer patients: Results from the CONKO-005 trial.

BACKGROUND: high recurrence rates of up to 75% within 2 years in pancreatic ductal adenocarcinoma (PDAC) patients resected for cure indicate a high medical need for clinical prediction tools and patient specific treatment approaches. Addition of the EGFR inhibitor erlotinib to adjuvant chemotherapy failed to improve outcome but its efficacy in some patients warrants predictors of responsiveness. PATIENTS AND METHODS: we analysed tumour samples from 293 R0-resected patients from the randomized, multicentre phase III CONKO-005 trial (gemcitabine ± erlotinib) with targeted sequencing, copy number, and RNA expression analyses. FINDINGS: a total of 1086 mutations and 4157 copy-number aberrations (CNAs) with a mean of 17.9 /tumour were identified. Main pathways affected by genetic aberrations were the MAPK-pathway (99%), cell cycle control (92%), TGFß signalling (77%), chromatin remodelling (71%), and the PI3K/AKT pathway (65%). Based on genetic signatures extracted with non-negative matrix factorization we could define five patient clusters, which differed in mutation patterns, gene expression profiles, and survival. In multivariable Cox regression analysis, SMAD4 aberrations were identified as a negative prognostic marker in the gemcitabine arm, an effect that was counteracted when treated with erlotinib (DFS: HR=1.59, p = 0.016, and OS: HR = 1.67, p = 0.014). Integration of differential gene expression analysis established SMAD4 alterations with low MAPK9 expression (n = 91) as a predictive biomarker for longer DFS (HR=0.49; test for interaction, p = 0.02) and OS (HR = 0.32; test for interaction, p = 0.001). INTERPRETATION: this study identified five biologically distinct patient clusters with different actionable lesions and unravelled a previously unappreciated association of SMAD4 alteration status with erlotinib effectiveness. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that SMAD4 status might guide addition of erlotinib treatment in early-stage PDAC patients.

Increase in prostaglandin H synthase 2, but not prostaglandin F2alpha synthase mRNA in intrauterine tissues during betamethasone-induced premature labor and spontaneous term labor in sheep.

OBJECTIVE: Prostaglandin F synthase (PGFS) catalyzes reduction of prostaglandin H(2) to PGF2alpha. No information exists on PGFS expression and regulation during pregnancy, either mRNA or protein, in relation to labor in uterine tissues in any species. We characterized PGFS mRNA expression in ovine myometrium, endometrium, maternal and fetal placenta in betamethasone-induced premature labor and spontaneous term labor using our cloned ovine PGFS riboprobe. Prostaglandin H synthase (PGHS) 2 mRNA was evaluated simultaneously as a control whose pregnancy related changes are well known. METHODS: Poly-A or total RNA from fetal placenta, myometrium, and endometrium in control ewes at 143-147 days of gestational age (dGA, TCNL, n = 6), and ewes in spontaneous term labor at 145-147 dGA (STL, n = 6) and endometrium and maternal and fetal placenta in early control ewes not in labor (ECNL, n = 6) and betamethasone induced labor at 128-135 dGA (BL, n = 6) were analyzed for PGHS2 and PGFS mRNA. RESULTS: PGFS mRNA did not change at spontaneous term labor in myometrium, endometrium, and fetal placenta. PGFS mRNA decreased during betamethasone-induced premature labor in endometrium and maternal placenta (P < .05), but remained unchanged in fetal placenta and myometrium. PGHS2 mRNA increased in endometrium, placenta, and myometrium during betamethasone-induced premature labor and spontaneous term labor. CONCLUSION: Increased PGHS2, but not PGFS mRNA was tightly associated with the onset of betamethasone-induced premature labor as well as spontaneous term labor in the endometrium, placenta, and myometrium. Transcription of PGFS mRNA may not be the rate-limiting step in the pathway contributing to increased PGF(2alpha) at labor.

Differential expression of myometrial oxytocin receptor and prostaglandin H synthase 2, but not estrogen receptor alpha and heat shock protein 90 messenger ribonucleic acid in the gravid horn and nongravid horn in sheep during betamethasone-induced labor.

In the present study, we characterized four myometrial contraction-associated proteins (mCAPs): oxytocin receptor (OTR), prostaglandin H synthase 2 (PGHS2), estrogen receptor alpha (ERalpha), and heat shock protein 90 (Hsp90) messenger RNA (mRNA) expression in the nongravid horn of pregnant sheep and compared them with their expression in the gravid horn that is exposed to a greater degree of stretch. We also examined the regulatory effects of estrogen and progesterone on OTR mRNA expression in ovariectomized nonpregnant sheep. In addition, we determined the ontogeny of mCAP expression in the gravid horn throughout late pregnancy and during spontaneous term labor. Gravid horn and nongravid horn myometria were removed under general anesthesia from control ewes not in labor at 130-140 days gestational age (dGA; n = 3) and during betamethasone-induced labor (n = 6) at the same gestational age. Gravid horn myometrium was also collected from ewes not in labor at 95 dGA (n = 3), 101-110 dGA (n = 3), 111-120 dGA (n = 3), 121-130 dGA (n = 3), 131-140 dGA (n = 3), and 141-145 dGA (n = 4) and from ewes in spontaneous term labor (n = 4). All ewes were carrying single fetuses. Myometrium was also collected from ovariectomized nonpregnant ewes treated with saline (n = 5), estradiol (50 microg/day; n = 5), progesterone (0.3 g, intravaginally; n = 5), and estradiol plus progesterone (n = 5). Myometrial RNA was extracted and analyzed by Northern blot for OTR, PGHS2, ERalpha, and Hsp90 mRNA, normalized for 18S ribosomal RNA or beta-actin. ERalpha, Hsp90, OTR, and PGHS2 mRNA were all significantly up-regulated during betamethasone-induced labor (P < 0.01) in gravid and nongravid horn myometrium. The level of gravid horn OTR mRNA during labor was 3 times the level of nongravid horn OTR mRNA (P < 0.0001). Gravid horn PGHS2 mRNA was also higher than nongravid horn PGHS2 (P < 0.02). In contrast, in spontaneous term labor nongravid horn, ERalpha and Hsp90 mRNA were similar to gravid horn. Myometrial ERalpha and Hsp90 mRNA remained unchanged throughout late pregnancy and increased at spontaneous term labor (P < 0.05). In contrast, myometrial OTR increased around 130 dGA (P < 0.01) and further increased at spontaneous term labor (P < 0.02). Progesterone significantly inhibited myometrial OTR mRNA expression in nonpregnant sheep and estradiol antagonized progesterone's inhibitory effect. Mechanical stretch differentially regulated mCAP mRNA expression in the ovine gravid horn and nongravid horn. Mechanical stretch appears largely responsible for increased OTR mRNA and to a lesser degree PGHS2 mRNA. In addition, endocrine factors may be required for full activation of OTR and PGHS2 mRNA associated with labor. ERalpha and Hsp90 mRNA are not under the control of uterine stretch in keeping with our previous results, indicating that systemic hormones such as estradiol, are prime regulators for these two mCAP mRNA expression during labor.

Rhythmic changes in the stomach movement of the human fetus with congenital duodenal obstruction during the third trimester of pregnancy.

In order to reveal whether or not rhythmic changes exist in fetal stomach movement (FSM), in utero FSM was assessed in three fetuses between 27 and 33 weeks' gestation with congenital duodenal obstruction. A total of four observations, one each at 27, 29, 31 and 33 weeks' gestation, was obtained. The longitudinal transection of each fetal stomach was continuously observed for 60 min using real-time ultrasound. The configuration and the area of the stomach were analyzed for each 15-s epoch. The complexity of the stomach configuration was quantified and defined as stomach complexity. For each case, the chronological changes of the stomach complexity were analyzed using the least median square of regression. The correlation between changes of the stomach complexity and the area of the stomach was analyzed using the cross-correlation method. (1) For gestational ages of 27, 29, 31 and 33 weeks, the 240 sequential measurements of the stomach complexity were significantly stratified into outlying and non-outlying points. The outlying points were 13.3% (32/240), 30.8% (74/240), 32.9% (79/240) and 36.3% (87/240) of the total observation points, respectively. (2) The percentages in which outlying points lasted 3 min (12 points) or more were 0% (0/240), 5.0% (12/240), 28.3% (68/240) and 30.4% (73/240) of the total observation points, respectively. (3) For each gestational age, no significant time series correlation was found between the stomach complexity and the area of the stomach. These findings suggest that: (1) two different conditions emerge in the FSM, at the latest at 27 weeks' gestation, and begin manifesting from 29 weeks' gestation onwards. (2) These chronological changes cluster into 'active' and 'quiet' phases from 31 weeks' gestation onwards. (3) FSMs are not related to the changes in the stomach area throughout the observation periods. The underlying mechanism of this rhythmicity may represent the development of ultradian rhythm of the stomach movement, generated by the central nervous system.

To what extent does a given heart rate correlate with following heart rates in the developing human fetus?

To quantitatively determine the extent to which a given heart rate correlates with the following heart rate(s) at any gestational age, we studied 181 uncomplicated human fetuses between 23 and 41 weeks gestation. A continuous 90-120 min observation was made for each case using external Doppler-ultrasound cardiotocography. For every individual fetal heart rate dataset, 'probability distribution matrices' were calculated with fetal heart rates (termed FHRs) at 1-beat/min (bpm) intervals, and the beat-to-beat(s) difference (termed DFHRn: 1 < or = n < or = 1000), also at 1-bpm intervals arranged in rows and columns, respectively, with probability in each element. Using 'piecewise linear regression', (1) the difference between the DFHR1 (n = 1) probability distribution matrix as the 'control' and a given DFHRn (n > or = 2) probability matrix as the 'variable', was analyzed to obtain the statistically critical point(s) (termed 'beat-delay' in beats) for each fetus, and (2) a scattergram of 'beat-delay' vs. gestational age-group, made from all fetuses studied, was analyzed to reveal any critical age(s) in gestation. One statistically significant point was evident at 28-29 weeks gestation. During the period prior to the critical point, a linear decrement in 'beat-delay' with 21 beats (mean) at 23-25 weeks to 11 beats at 28-29 weeks gestation was noted, suggesting that the conduction system-oriented heart beat gradually comes under functional control of the autonomic nervous system as well as under possible regulation by the maturating medulla oblongata. From the critical point, through to term, there was no significant change in 'beat-delay' ranging from 7-11 beats, thereby implying that the fetal heart rate becomes stabilized as a result of the control rendered by the more developed autonomic nervous system function. Whether or not brain function cephalad to the medulla oblongata actually participates in this phenomenon remains to be elucidated.

Three-dimensional ultrasound image of the fetal stomach: congenital duodenal obstruction in utero.

To quantitatively characterize the stereographic stomach configuration in utero, 11 fetuses (subject-group) with congenital duodenal obstruction, diagnosed antenatally, between 29 and 37 weeks' gestation were studied. Also included were 879 uncomplicated fetuses between 20 and 40 weeks' gestation as a control-group. Applying the algorithm which we devised: "Modeling a three-dimensional shape from a silhouette by detecting symmetry", we reconstructed the three-dimensional stomach configuration from a two-dimensional ultrasound image for each case. The statistical differences in two parameters, stomach volume and sphericity, between subject- and control-group fetuses, were analyzed using the Grubbs-Smirnoff's test at corresponding gestational ages. From 29 to 37 weeks' gestation, the stomach volume in the subject-group fetuses was found to increase greatly with advancing gestation, having significantly higher values than the control-group fetuses, whereas the stomach sphericity remained unchanged with no significant differences from the control-group fetuses. These findings indicate that the fetal stomach with duodenal obstruction maintains almost the same three-dimensional portrayal in utero as that seen in uncomplicated fetuses, although the stomach becomes extremely enlarged.

Fetal urine production at different gestational ages: correlation to various compromised fetuses in utero.

To reveal which fetal life-threatening diseases significantly contribute to impairment of in-utero urine production and also to determine the gestational age at which time aberrant urine production becomes manifest, we observed 376 compromised fetuses (subject group) at 21-42 weeks' gestation using ultrasonography. A total of 358 uncomplicated fetuses, aged 21-40 weeks, were separately chosen as a control group. Statistical differences in the urine production rate between subject- and control-group fetuses were analysed using the Grubbs-Smirnoff test at corresponding gestational ages. Significant decreases were evident in: bilateral renal agenesis (100%) at 21-23 weeks; bilateral infantile polycystic kidney (100%) at 21-28 weeks; bilateral multicystic kidney disease (100%) at 21-31 weeks; donor fetuses with twin transfusion syndrome (TTS) (100%) at 21-28 weeks; post-term fetuses (100%) at 42 weeks; bilateral hydronephrosis (60%) at 21-38 weeks; non-immunologic hydrops fetalis (42%) at 21-35 weeks; intrauterine growth retardation (41%) at 29-40 weeks; and upper gastrointestinal tract obstruction (36%) at 30-38 weeks. Significant increases were noted in: recipient fetuses with TTS (100%) at 21-28 weeks, and unilateral hydronephrosis (36%) at 27-32 weeks. All indicate that urine production clearly delineates various fetal conditions in utero, in a closely disease-dependent relation to gestational age.

Quantification of fetal heart rate variation using a probability distribution matrix.

Analysis of FHR variability attempts to be able to distinguish which FHR changes are physiological and which are pathological. From the results of such studies, it is hoped that clinicians will be provided with the means to identify accurately which fetuses are healthy, which are at risk and which are actually in distress at any gestational age. The probability distribution matrix presented has the outstanding advantage of enabling one to condense any amount of FHR data into one uniform description for analysis en bloc. Not even one FHR is sacrificed; each one contributes to the FHR vs. DFHR distribution pattern. Where the probability distribution matrix is made from many FHR samples taken from healthy fetuses under natural day-to-day maternal circumferences, as we have done, this matrix can be considered as a physiologically unbiased reference. The subtraction matrix is characterized by the difference in FHR vs. DFHR distribution patterns between two probability distribution matrices compared, representing the equal residue of net absolute value. The larger the difference in FHR vs. DFHR distribution between two probability distribution matrices, the larger these kinds of residue values, and if two probability distribution matrices are identical, all the elements in the subtraction matrix indicate the residue values of zero. Our probability distribution matrix approach confirms the results of previously reported studies which utilized other analytical strategies. Therefore, we can evaluate quantitatively age-related FHR changes used as variables, such as the subtraction matrix and difference rate. By varying intervals between paired FHRs (e.g. selecting the original FHR--skip--select the following FHR--skip 2 FHR--select the next FHR, etc.), the probability distribution matrix could be adopted as a general tool for analysis of sequential changes in large numbers of FHR for further studies.

What is the minimum number of heart rates necessary to evaluate fetal conditions at different gestational ages?

Our objective was to determine the minimum number of fetal heart rates (FHRs) needed to assess various fetal conditions adequately, focusing on FHR changes in relation to gestational age. We used probability distribution matrices previously derived from 10,934,604 FHRs of 743 uncomplicated fetuses. These matrices were made at nine consecutive 2-week intervals between 23 and 40 weeks' gestation, from which samples were taken after assigning random numbers to FHRs in sequence. As a variable, the difference rate (%) between the sample probability distribution matrix and its corresponding age-group probability distribution matrix was calculated. Scattergrams of difference rates vs. a given number of FHR samplings were analyzed using piecewise linear regression. One critical point per age-group emerged, ranging between 9000 and 10,000 beats, for all age-groups. A linear decrease in the difference rate was noted with a step-by-step increase in random sampling size of FHRs until reaching the critical point, beyond which the difference rate remained constant between 25-33%. The critical points indicate that the minimum number of FHRs for assessment of the fetus at 23-40 weeks' gestation is almost the same, between 9000 and 10,000, with 67-75% baseline variability (so called beat-to-beat variability) and 25-33% long-term variability regardless of advance in gestation.

The relationship between age-related heart rate changes and developing brain function: a model of anencephalic human fetuses in utero.

We attempted to identify the brain segment which controls heart rate changes in human fetuses with advancing gestation. Twelve anencephalic and 165 normal fetuses (control-group fetuses) between 25-32 weeks' gestation were studied. The instantaneous fetal heart rate (FHR) data were obtained from each fetus for a continuous 90-120 min period, using an external cardiotocograph. Calculations included the 'individual probability distribution matrices' in which the FHRs at 1 beat/min intervals between 110 and 180 beats/min, the beat-to-beat differences (DFHRs) between +/- 5 beats/min and the probability values were arranged in rows, columns and the corresponding elements, respectively. Using 2-gestational-week intervals probability distribution matrices (age-group probability distribution matrices) obtained from 335 normal fetuses in our previous study as a reference, the difference between a given 'individual probability distribution matrix' and the corresponding age-group probability distribution matrix' was quantified as the 'difference rate' according to the formula in the text. From 25-26 to 27-28 weeks' gestation, the 'difference rates' in four anencephalic fetuses, with only the spinal cord preserved, were significantly higher in value than those of control-group fetuses, whereas the rates in four fetuses with both the spinal cord and medulla oblongata preserved, indicated no significant differences. From 29-30 to 31-32 weeks' gestation, the rates of the four fetuses with the spinal cord and medulla oblongata preserved, showed significant differences from the control-group fetuses. These findings suggest that there is a critical period between 27-28 and 29-30 weeks' gestation with regard to the developing brain function pertaining to FHR changes. In the early stage, the medulla oblongata plays a role in FHR changes, whereas, in the latter stage, the brain cephalad to the medulla also appears to take on the role of FHR regulator.

Fetal heart rate variation described using a probability distribution matrix.

We devised a new method using a probability distribution matrix to simultaneously describe variation in the characteristics of fetal heart rate (FHR) and beat-to-beat difference (DFHR) between the present and the immediately following FHR. The FHRs were plotted in columns, DFHRs in rows and probabilities in the corresponding elements of the matrix. The age-related changes of FHR data obtained from 743 fetuses at 23-40 weeks gestation were analysed using a pulsed Doppler cardiotocograph with an autocorrelation system. While keeping an almost symmetrical spread around 0 beats/min of DFHR, three particular probability distribution patterns of FHR versus DFHR emerged with advance in gestation: (i) oval with a monomodal peak from 23/24 to 29/30 weeks, (ii) ellipsoid with a monomodal peak and plateau from 31/32 to 33/34 weeks and (iii) elongated ellipsoid with bimodal peaks from 35/36 weeks of gestation onwards. The probability distribution matrix presented enables one to condense any amount of FHR data into one uniform description. This allows analysis of data, en bloc, achieving a quantitative inter-group comparison, on an equivalent scale.

How antenatal ultrasound diagnosis of congenital malformations has contributed to fetal outcome: a 22-year review.

We herein reviewed 630 malformed fetuses delivered from 24 weeks of gestation onwards in our institute over the past 22 years. These fetuses were divided into 2 groups: 210 from 1970 to 1982 (group 1) and 420 from 1983 to 1991 (group 2). Twenty-two varieties of congenital malformations were diagnosed antenatally in group 1, whereas 55 additional malformations became diagnosable in group 2. Thirty-four varieties of congenital malformations still remained undiagnosed throughout the 22-year study period. Cases receiving 'fetal therapy' and 'close obstetric care' increased, with statistical significance, from 1.8 to 15.7%, and from 22.9 to 55.7% in groups 1 and 2, respectively. A significant increase was also noted in the survival rate from group 1 (63.3%: 105/166) to group 2 (75.5%: 259/343). This study has revealed that the steady advancement in antenatal diagnosis of congenital malformations, along with great efforts in terms of intensive care, has contributed to improved fetal outcome.

Fetal eye movement assessed with real-time ultrasonography: are there rapid and slow eye movements?

The goal of this study was to determine whether rapid eye movement and slow eye movement exist during the eye-movement period in the human fetus in utero. We studied 21 fetuses with real-time ultrasonography, 10 from 33 to 36 weeks and 11 from 37 to 41 weeks' gestation. We used the duration of eye-movement unit as a parameter and calculated the cumulative duration from the shortest to a given duration of eye movement per individual case. A scattergram of cumulative duration versus given duration obtained from all cases in each age group was analyzed with piecewise linear regression to search for a critical point(s). Critical given duration points were noted and reached statistical significance at 0.62 second and at 0.76 second during 33 to 36 and 37 to 40 weeks of gestation, respectively. These findings reveal two different types of eye movement: one with a duration of less than 0.6 to 0.8 second and the other with a duration of greater than 0.6 to 0.8 second. These findings are compatible with previous criteria on rapid and slow eye movements, respectively, at 33 weeks of gestation onward. The mean value of cumulative duration at the critical point increased from 29.0% between 33 and 36 weeks to 47.1% between 37 and 41 weeks of gestation, indicating an increase in the proportionate amount of time maintaining rapid eye movement as gestation advances.

Relationship between heart rate and rhythm, and cardiac performance assessed in the human fetus in utero.

To evaluate the relationship between heart rate and rhythm, and cardiac performance, in the human fetus in utero, observed over a long-term period in gestation, we made a total of 138 studies in 114 fetuses from 18 to 41 weeks of gestation; 104 having heart rate changes without rhythm disturbances ("control group"), 6 with complete atrioventricular block and 4 with supraventricular tachycardia. Using M-mode echocardiogram, we measured end-diastolic dimension and fractional shortening in the right and left ventricles. The corresponding heart rate for each cardiac cycle was measured using the interval between two consecutive end-systolic points. In fetuses in the group of controls, the values for fractional shortening in both ventricles were almost constant with advancing gestational age, unrelated to an increase in end-diastolic dimensions of either ventricle. There was no correlation between changes in heart rate and changes in the value of ventricular fractional shortening at any period of gestation studied. In the fetuses with atrioventricular block, dimensions and fractional shortenings in both ventricles were significantly larger than those in the group of control fetuses at the same stage of gestation. This indicates that the fetal heart is capable of acclimatizing itself, beginning as early as 18-25 weeks of gestation, to long-lasting bradycardia in which an increased stroke volume would be required. In fetuses with supraventricular tachycardia, end-diastolic dimensions were larger and fractional shortening was significantly smaller in both ventricles than in fetuses from the control group from 26-30 weeks of gestation onwards. This suggests tachycardia-induced cardiomyopathy occurring during intrauterine life.

Safety and utility of endomyocardial biopsy in infants, children and adolescents: a review of 66 procedures in 53 patients.

The benefits and risks of endomyocardial biopsy in infants, children and adolescents were determined by reviewing the indications for and complications and results of 66 procedures in 53 patients aged 2 months to 20 years. One patient had a pneumothorax, and three had a right ventricular perforation. Ventricular tachycardia developed in four patients; it was treated with lidocaine in three and was self-limited in one. The procedure was unsuccessful in two patients. Among 25 patients with a prebiopsy diagnosis of idiopathic dilated cardiomyopathy, microscopic features were consistent with cardiomyopathy in 24 (96%) and were normal in 1. Of nine patients with clinically suspected myocarditis, only two (22%) had microscopic evidence of inflammation, and seven had chronic nonspecific features suggestive of dilated cardiomyopathy. Of eight patients with unexplained arrhythmias, six (75%) had microscopic findings compatible with dilated cardiomyopathy and two had myocarditis. Biopsy tissue samples from seven patients with nondilated forms of cardiomyopathy (four hypertrophic, three restrictive) were consistent with the clinical diagnosis in six and were inadequate in one. Cardiac biopsies were also performed in four patients with other disorders. Among the 51 patients with adequate biopsy specimens, microscopic features were considered diagnostic in 5, confirmatory in 44 and not helpful in 2 with normal tissue. The results indicate that endomyocardial biopsy is safe in infants, children and adolescents. It is useful for the evaluation of cardiomyopathy and specific secondary forms of myocardial disease. There seems to be little correlation, however, between clinical and tissue diagnoses of myocarditis.

Truncus arteriosus revisited: an angiographic demonstration.

The 1965 reclassification of truncus arteriosus by Van Praagh and Van Praagh greatly enhanced our understanding of this interesting anomaly. This brief review article attempts to illustrate the various types of truncus arteriosus identified in this classification by demonstrating their angiographic features. Reemphasis of the usefulness of this classification should help students of congenital heart disease recognize the advantages of a uniform diagnostic approach to this entity.

Pulmonary artery catheter placement with two-dimensional echocardiographic guidance: a technique applicable in critically ill neonates.

In nine critically ill neonates with persistent fetal circulation, femoral venous catheters were inserted at the bedside to initiate treatment and provide venous access. After femoral vein puncture or cutdown, a 5-F sheath was placed in the inferior vena cava through the femoral vein. With use of two-dimensional echocardiographic guidance, a 5-F balloon angiographic catheter was advanced through the inferior vena cava into the right atrium and subsequently manipulated through the tricuspid valve and into the main pulmonary artery. No major complications were attributable to the procedure. When performed by a pediatric cardiologist, this technique is as safe as umbilical catheter placement.