Utility of fluorescence in situ hybridization in pancreatic ductal adenocarcinoma.

Often the diagnosis of pancreas cancer needs to be established from limited cytology specimens or small biopsies. Most ductal adenocarcinomas are histologically well to moderately differentiated and mimicked closely by pancreatitis, and therefore the microscopic diagnosis can be difficult. In addition, there appears to be significant heterogeneity in the outcome of the patients with pancreatic cancer, which cannot be predicted accurately by current prognosticators such as the grade and stage of the tumor. Therefore, there is need for methods that can be used as adjuncts to routine diagnostic and prognostic parameters. This study was designed to test the utility of the fluorescent in situ hybridization (FISH) method in identifying the molecular alterations, particularly the ones that have been detected with relatively high frequency in pancreas cancer. Formalin-fixed and paraffin-embedded tissues of 10 cases were enumerated for chromosome 7, 8, 17, 18, and 20 copy numbers by using alpha-satellite probes, and for c-myc by using a gene-specific probe. The number of signals per nucleus (reflecting chromosomal copy number and status of c-myc amplification) were counted in more than two areas containing 50-500 cells. Because of tumor heterogeneity, monosomy (loss of one chromosome copy) was defined arbitrarily as one signal in >25% of nuclei. C-myc amplification was defined as more than two gene copies in >20% of the cells. The most frequent signal losses were found in chromosomes 8 (four of 10 cases) and chromosome 17 (four of 10), followed by 20 (three of 10) and 18 (two of 10). No loss of chromosome 7 was detected. In contrast, gains in chromosome copy number were identified in only one of 10 tumors, which showed gain of both chromosome 7 and 18. Amplification of c-myc gene was detected in two of 10 cases, but neither of the two had aneuploidy for chromosome 8, where the c-myc gene is located. In addition, loss in c-myc signal was observed in one case that also showed loss of chromosome 8 copy number. FISH can be used to detect chromosomal changes in pancreatic cancer; abundance of lytic enzymes in this organ is not an impediment for the applicability of this technique. Therefore it can potentially be used in the future as an adjunct to the conventional diagnostic and prognostic markers. This study confirms that loss of chromosomes, particularly chromosomes 17 and 18, which carry the p53 and DCC genes, are common in pancreas cancer. Chromosome 20 is also frequently lost. In addition, in this study, alterations of chromosome 8, which is seen commonly in prostatic adenocarcinoma but has not been previously documented in pancreatic cancer, also was detected in five of 10 tumors. Furthermore, amplification of the c-myc gene, which is located in chromosome 8, was found in the two of the remaining five cases. Further studies are needed to confirm this high incidence of chromosome 8 and c-myc alterations and their possible role in the pathogenesis of pancreatic adenocarcinoma.

Viable pregnancies after diagnosis of trisomy 16 by CVS: lethal aneuploidy compartmentalized to the trophoblast.

Increasing utilization of chorionic villus sampling (CVS) has lead to the discovery that the placenta can karyotypically be a very heterogeneous organ, and chromosomal mosaicism within the placental can confuse cytogenetic interpretation. Recently, confined placental mosaicism (confined regions of aneuploidy in the otherwise normal diploid placental and fetus) has been described involving a number of chromosomal abnormalities. Fetal trisomy 16 is considered uniformly lethal early in gestation. However, we present 3 cases of nonmosaic trisomy 16 confined regionally to the placenta. We discuss the possible etiology, impact on the developing fetus, and suggest an approach to the workup and evaluation of cases where the karyotype obtained on CVS is not compatible with the findings on ultrasound.

Postmortem chorionic villus sampling: correlation of cytogenetic and ultrasound findings.

We performed chorionic villus samplings (CVS) in 795 cases in the first trimester during a 13-month period. Of these 35 were found to have a blighted ovum or missed abortion prior to the procedure. Nineteen women consented to have CVS. Ultrasonographic and cytogenetic findings in these 19 pregnancies were correlated. Expected gestational age was determined by last menstrual period. Observed gestational age was determined by crown rump length (CRL) (12 pregnancies) or gestational sac (GS) (7 pregnancies without fetal pole). The differences in days between the estimated and observed gestational ages was determined for each pregnancy. In all 19 CVS samples cytogenetic diagnosis documented aneuploidy. Ten cases had chromosome abnormalities virtually always lethal in the embryonic period (group I). Nine pregnancies had defects with moderate potential for fetal viability (group II). Gestations with low viability potential (group I) had estimated minus observed gestational age discrepancies (23.4 +/- 8.3 days) significantly greater than gestations with moderate viability potential (group II) (8.9 +/- 4.3 days) (P less than .001). The absence of a fetal pole was more common in group I. CVS in pregnancies with missed abortion or blighted ovum is feasible and has a high likelihood of documenting aneuploidy. Furthermore, the more severe the anomaly the more likely there will be very early fetal demise or intrauterine growth retardation.

Postmortem chorionic villus sampling is a better method for cytogenetic evaluation of early fetal loss than culture of abortus material.

In utero chorionic villus sampling at the time of diagnosis of intrauterine fetal death is compared with more traditional use of cultured fetal skin, products of conception, or amniocentesis. A total of 102 specimens from early fetal losses were evaluated for success in karyotyping and chromosomal results. We found postmortem chorionic villus sampling is technically possible, offers the highest likelihood of getting a cytogenetic result, and is a rapid, reliable, and safe technique. The extraembryonic component of intrauterine fetal deaths appears to remain viable and continues to grow long after the embryo has died. Samples obtained at the time of diagnosis of fetal death offer the greatest changes of successfully obtaining a karyotype. The incidence of chromosome abnormalities associated with fetal loss, particularly trisomies, is higher than previous data suggested.

Prenatal genetic diagnosis following recurrent early pregnancy loss.

Recurrent pregnancy loss affects 1% of patients, an incidence higher than expected from the prevalence of spontaneous abortion in the general population. Some couples may show a tendency for aneuploid conceptions. Genetic counseling and amniocentesis or chorionic villus sampling were offered to 305 couples with a history of two or more pregnancy losses and normal parental karyotypes, with no additional known risk factors for aneuploidy. Prenatal diagnostic procedures were performed in 96 pregnancies. Two hundred nine couples declined active intervention, and these pregnancies were followed to delivery. Five chromosomal abnormalities (1.6%) were diagnosed in the study group. A group of 979 prenatal diagnostic procedures performed in "low-risk" pregnancies in Hutzel Hospital was used as controls, and three chromosomal anomalies (0.3%) were diagnosed. That the rate of aneuploid conceptions was statistically significantly (P = .02) higher in low-risk couples experiencing recurrent pregnancy loss than in controls points to a tendency for chromosomal aberrations in their offspring and suggests a place for prenatal diagnosis in subsequent pregnancies.

Prenatal diagnosis of and midtrimester pathology with karyotype 46,XY,del(4)(q22q26). A case report.

A prenatal diagnosis of an interstitial deletion with chromosome 4,46,XY,del(4)(q22q26) was obtained on amniotic fluid cells drawn at 19 weeks' gestation from a 35-year-old gravida. Counseling on the basis of unusual or tenuous data is always difficult, but comparisons with similar deletions in 4q suggested a substantial risk of anomalies. A comparison of the postabortal autopsy findings with those from other reported cases of interstitial deletions of chromosome 4q suggested different pathology with this area of deletion than previously reported for other areas of 4q.

Does the color of amniotic fluid still matter?

Second trimester amniotic fluid (AF) is generally clear or very light yellow. We examined the color of 2,141 AF samples. Fifty-six specimens were brown, 35 were green. There were 71 samples with abnormal karyotype (3.46%). In the group with brown AF, there were 7 abnormal karyotypes out of 56 (12.5%). There was 1 case of aneuploidy out of the 35 green samples (2.86%). We conclude that green AF in the second trimester, absent any other findings, carries no special significance, but brown AF carries a fourfold increased incidence of chromosomal aneuploidy in patients undergoing genetic amniocentesis.

Interpretation of chorionic villus sampling laboratory results is just as reliable as amniocentesis.

Karyotypes of chorionic villi have been said to be less accurate than karyotypes of amniocytes. Karyotypic differences between placental and fetal tissue and maternal-cell contamination could potentially complicate clinical management. We compared cytogenetic results obtained by chorionic villus sampling and amniotic fluid cells in our center during a 2-year period (1986-1987). Chorionic villus sampling material was processed for direct analysis and backed up when indicated (now routinely) with tissue cultures. The incidence of inconclusive results requiring additional studies was 1.2% for chorionic villus sampling and 0.75% for amniotic fluid cells. Mosaicism was the most common problem for both chorionic villus sampling and amniotic fluid cells. Failure of growth was more frequent in amniocentesis material (0.35 versus 0.09%), but polyploidy and atypical aneuploidies were greater with chorionic villus sampling. The accuracy of cytogenetic evaluation by chorionic villus sampling and amniotic fluid cells and the need for additional invasive procedures appear to be equal in our laboratory.

Genetic diagnosis in the first trimester: the norm for the 1990s.

Increasing technical capabilities and patient motivation for earlier and more private prenatal genetic diagnosis have allowed us to alter the concept of first-trimester genetic diagnosis from being rare to routine in our tertiary Reproductive Genetics Center. As public awareness of available services has increased, we have seen steadily increasing numbers and proportion of patients who are referred by their physicians earlier, who schedule tests earlier, opting to have earlier testing, and accept slightly higher risks in return for earlier results and privacy. Analysis of our clinical and laboratory results and complication rates suggests that first-trimester genetic diagnosis by either chorionic villus sampling or early amniocentesis may be offered to virtually all patients who would be candidates in the midtrimester. We believe that this trend will accelerate, making first-trimester diagnosis the norm, rather than the exception, for the 1990s.

Counseling for low maternal serum alpha-fetoprotein should emphasize all chromosome anomalies, not just Down syndrome.

In common usage, a low maternal serum alpha-fetoprotein (MSAFP) value is associated with an increased risk of Down syndrome. We have performed amniocenteses for the indication of age-adjusted low MSAFP in 1154 patients and found 13 chromosomally abnormal conceptions. Autosomal trisomies were detected in half the cases. Additional abnormalities included sex-chromosome aberrations, deletions, or triploidy in proportions consistent with those seen in an advanced-maternal-age population. Patients with low serum AFP should be counseled that not only Down syndrome, but other aneuploidies as well, may be diagnosed. The risk quoted should be that of all chromosomal abnormalities, which is about twice the risk calculated for Down syndrome.

Amniotic fluid alpha-fetoprotein levels in the differential diagnosis of cystic hygroma.

In 7 second trimester pregnancies ultrasound (US) demonstrated cystic hygroma colli (CHC); amniocentesis was performed in 6 patients. In the 7th patient, because of oligohydramnios, the fluid for karyotype was aspirated from the CHC. Five pregnancies had been referred secondary to abnormalities on US and 2 others because of low maternal serum alpha-fetoprotein (MSAFP). Four karyotypes were abnormal (45,X;47,XX+21; 47,XY+21; 46,XX/45,X), and 3 had normal karyotypes. Amniotic fluid alpha-fetoprotein (AFAFP) was normal in 4 pregnancies and low in 2 (0.09 MOM, 0.41 MOM). Of 2 pregnancies with trisomy 21 one had been referred for low MSAFP. In 2 pregnancies with normal karyotypes, US findings at early gestational age (14-17 weeks) of small, nonseptated, bilateral CHC disappeared during pregnancy; these women delivered normal, term babies. Most prenatally diagnosed CHC are not in fetuses with Turner syndrome. With a normal karyotype and CHC as the only finding on early US in utero, normal neonatal survival is possible. AFAFP is not elevated in pregnancies with CHC. If AFAFP is elevated with a positive acetylcholinesterase, such results may suggest that the CHC was inadvertently aspirated.

Effects of karyotype, gestational age and medium on the duration of amniotic fluid cell culturing.

Third-trimester genetic amniocentesis has often been underutilized because of the long period required to obtained results. While fetal cord sampling can be used to obtain rapid karyotypes, its availability is still very limited. With more defined media, the culture duration (CD) for amniotic fluid cells has decreased markedly. We investigated CD as a function of gestational age (GA) and karyotype and found that: (1) there is an inverse correlation of GA and CD; (2) aneuploid karyotypes, translocations and inversions all grow more slowly regardless of GA; (3) since late taps are performed most often on highly suspicious cases, the general impression of late-GA slow growth may be because of a higher likelihood of aneuploidy. Our data thus support increased utilization of third-trimester genetic amniocentesis in selected cases.

Amniotic fluid acetylcholinesterase: implications of an inconclusive result.

The implications of an "inconclusive" acetylcholinesterase test (a faint but true band) in amniotic fluid were studied over a 2 1/2-year period in our laboratory. One thousand one hundred fifty-four amniotic fluid samples were tested for acetylcholinesterase and alpha-fetoprotein; the rate of an inconclusive acetylcholinesterase result was 3.3% (38 of 1154). Fourteen such results were found in patients with a high amniotic fluid alpha-fetoprotein level (23.3%), and 24 results were associated with normal amniotic fluid alpha-fetoprotein levels (2.19%). The rates of congenital fetal malformation associated with an inconclusive acetylcholinesterase result in the two groups were 57.14% and 37.5%, respectively. In amniotic fluid samples obtained before 15 weeks' gestation, there was a higher rate of inconclusive acetylcholinesterase tests (9.29%), but a lower percentage of malformed fetuses were found compared with later in pregnancy (2.46% and 56%, respectively). Thus we suggest the terminology "equivocal" for early specimens and "suspicious" for later specimens. If obtained in early second trimester and the ultrasound scan is normal, such findings implicate the need for a careful search for fetal malformations. A positive pregnancy outcome may be expected in most cases.

Establishment of a collaborative university-commercial maternal serum alpha-fetoprotein screening program: a model for tertiary center outreach.

Expansion of the availability of tertiary level services beyond major medical centers has proved to be a major problem in health care delivery. Routine maternal serum alpha-fetoprotein screening for neural tube defects, and now also for aneuploidy, is a classic example in which there has been a schism between the clinical expertise to manage such a program within a tertiary level reproductive genetics center and the ability to reach patients in regions that are not routinely accessible to the tertiary center. To address this problem we have established a collaborative university-commercial laboratory statewide maternal serum alpha-fetoprotein program that we believe can serve as a model for others. In the first 4 months since its implementation, the program volume has increased tenfold. The detection frequency of neural tube defects has been consistent with that of other programs (1/1690). Three aneuploid karyotypes were found in amniotic fluid of 118 women less than 30 years old who underwent genetic amniocentesis because of a low maternal serum alpha-fetoprotein value. Thus we conclude that: the establishment of a joint university-commercial maternal serum alpha-fetoprotein program may provide a successful model for efficient tertiary center outreach, assessment of our data suggests that a population at high risk for abnormal fetuses can be identified among patients not generally considered at high risk, low maternal serum alpha-fetoprotein values may likely be a more important public health measure than high ones.

Lysosomal enzyme activities in fresh and frozen chorionic villi and in cultured trophoblasts.

Fifteen lysosomal enzyme activities were compared in 14 presumed normal chorionic villus specimens that were each divided, processed and analyzed as fresh tissue, tissue frozen for 1 week, and cultures established from minced whole villi. Most of the activities determined in the chorionic villus tissue were not affected significantly by freezing. However, activities for most enzymes were significantly different from those determined in the cultured cells. Our experience with first trimester prenatal evaluations for several lysosomal disorders showed that the limited amount of tissue obtained is not always sufficient for thorough analysis and thus, cultured trophoblasts derived from the tissue specimen should also be examined. The results of this study stress the importance of using appropriate tissue-type and cell-type controls to establish the normal range in the respective analyses.

Improving the yield of direct chorionic villus slide preparations.

Low cytogenetic yields in the processing of small chorionic villus sampling have in some instances, limited its applicability. We have modified "standard" techniques to increase the number of interpretable metaphases by (1) using a gravity method for cell suspension spreading and (2) using a special high-quality slide glass. Both modifications reduce cell damage and increase interpretable mitotic figures and may allow a cytogenetic diagnosis in some instances in which a diagnosis might not otherwise be possible with standard methods.

Sézary syndrome with a 14:14 (q12:q31) translocation.

The Sézary syndrome was diagnosed in a 71-year-old black woman with erythroderma, generalized lymphadenopathy and hepatosplenomegaly. The laboratory data revealed a white blood cell count of 65,000 mm3 with 81% lymphocytes, the majority having an indented or a cerebriform nucleus. The skin biopsy, the lymph node biopsy and immunologic surface marker studies confirmed the diagnosis of Sézary syndrome. The cytogenetic studies of the bone marrow and the peripheral blood revealed a (14:14) (q12:q31) translocation present, consistently in majority of the lymphocytes. This translocation is considered characteristic of ataxia-telangiectasia and, to our knowledge, has not been previously described in Sézary syndrome.