HER-2/neu testing in breast carcinoma: a combined immunohistochemical and fluorescence in situ hybridization approach.

We evaluated 750 consecutive invasive breast carcinomas for HER-2/neu utilizing a combination of immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) methodologies. IHC reactions of 3+ were considered HER-2/neu positive and 0 and 1+ IHC reactions were considered HER-2/neu negative. IHC reactions of 2+ were considered inconclusive and reflexed to FISH analysis. In addition, a 10% sampling and validation FISH analysis was performed on the positive and negative IHC tests. One hundred thirty-eight cases (18.4%) were HER-2/neu positive by IHC and/or FISH. One hundred twenty-three of the positive cases (89%) were 3+ IHC reactions and 14 positive cases were inconclusive by IHC and amplified by FISH. There was concordance with FISH in 77 of 78 (98.7%) of the positive or negative IHC cases that were tested (95% confidence interval [CI] = 93.1 to 100%). A single IHC-negative case showed HER-2/neu amplification by FISH. Thirty-nine cases were 2+ IHC (5.2%); 14 (36%) were amplified, 24 (62%) were not amplified, and one was not interpretable. HER-2/neu positivity was observed in 34% of grade 3 ductal carcinomas, 11.4% of grade 2 ductal carcinomas, 3.2% of grade 1 ductal carcinomas, and 3.2% of lobular carcinomas. Occasional cases with discordant IHC expression of HER-2/neu within the in situ and invasive carcinoma elements were also identified. IHC reliably characterized HER-2/neu in approximately 95% of the cases studied (95% CI = 93.0 to 96.2%) and was effective as a primary method for evaluating HER-2/neu status. In this study, 2+ IHC reactions were a heterogeneous group best regarded as indeterminate or inconclusive; in this series, only 36% were amplified by FISH analysis. Our findings suggest that a combination of IHC and FISH testing with FISH analysis performed reflexly on all 2+ IHC cases can optimize HER-2/neu testing.

Detection of mosaicism in amniotic fluid cultures: a CYTO2000 collaborative study.

PURPOSE: To evaluate the assumptions on which the American College of Medical Genetics (ACMG) Standards and Guidelines for detecting mosaicism in amniotic fluid cultures are based. METHODS: Data from 653 cases of amniotic fluid mosaicism were collected from 26 laboratories. A chi-square goodness-of-fit test was used to compare the observed number of mosaic cases with the expected number based on binomial distribution theory. RESULTS: Comparison of observed data from the in situ colony cases with the expected distribution of cases detected based on the binomial distribution did not reveal a significant difference (P = 0.525). CONCLUSIONS: The empirical data fit the binomial distribution. Therefore, binomial theory can be used as an initial discussion point for determining whether ACMG Standards and Guidelines are adequate for detecting mosaicism.

Identification of an unusual marker chromosome by spectral karyotyping.

We ascertained a newborn girl with multiple congenital anomalies including severe hypotonia, cardiovascular defects, hearing loss, central nervous system anomalies, and facial anomalies. The infant died at 12 days. Cytogenetic analysis showed a de novo supernumerary marker chromosome. Fluorescence in situ hybridization (FISH) with a combination of chromosome specific alpha-satellite probes and an all-human centromere probe failed to show hybridization to the marker, indicating that the marker chromosome lacked detectable alpha satellite sequences. Spectral karyotyping (SKY) was performed and showed that the marker was chromosome 15 in origin. This was confirmed by FISH with a 15q specific subtelomerie probe, which showed hybridization to both ends of the marker chromosome. Based on FISH information and G-banding pattern, the marker was determined to be an inverted duplication of 15q25-qter, leading to partial tetrasomy for chromosome 15. Although the marker chromosome lacked detectable centromeric alpha-satellite sequences, it seemed to have a functional centromere as it is mitotically stable. This observation is consistent with previous studies on acentric marker chromosomes, which suggested that the DNA sequence at the breakpoint could function similarly to alpha-satellite sequences once activated through marker formation.

Donor origin of a posttransplant liver allograft malignancy identified by fluorescence in situ hybridization for the Y chromosome and DNA genotyping.

Posttransplantation malignancy in the allograft is a rare complication of orthotopic liver transplantation. In the described case, an abnormal T-tube cholangiogram, performed 6 months after orthotopic liver transplantation between a male donor and a female recipient, prompted needle liver biopsy. A moderately differentiated adenocarcinoma was found. Fluorescence in situ hybridization for the Y chromosome indicated male origin of malignancy. Donor-related disease was confirmed by comparative DNA analysis of genomic sequences from the donor liver, associated tumor, and recipient peripheral blood. Results of these investigations qualified the recipient for a second liver transplant.

Miller-Dieker syndrome. Detection of a cryptic chromosome translocation using in situ hybridization in a family with multiple affected offspring.

OBJECTIVE: To describe a family in whom fluorescence in situ hybridization allowed for accurate diagnosis of Miller-Dieker syndrome in an at-risk pregnancy and determination of parental carrier status. DESIGN: Retrospective case analysis and application of a new molecular tool to evaluate the family. SETTING: Health maintenance organization. The family was followed up by the Departments of Medical Genetics, Pediatrics, and Obstetrics and Gynecology, Kaiser Permanente Medical Center, Panorama City, Calif. PARTICIPANTS: Members of a single family. INTERVENTIONS: Clinical evaluation and neuroimaging studies of the proband. Prenatal diagnosis via ultrasonography and amniocentesis. Chromosomal evaluation of the couple and their offspring. In situ hybridization studies in both parents and an affected fetus. MEASUREMENTS/MAIN RESULTS: We describe a family in whom fluorescence in situ hybridization detected a submicroscopic deletion of the Miller-Dieker syndrome critical region 17p13.3 arising from a cryptic translocation in one of the parents. The proband was determined at birth owing to the presence of multiple congenital anomalies, including low birth weight, microcephaly, agenesis of the corpus callosum, lissencephaly, cerebral atrophy, unilateral ptosis, polydactyly, and omphalocele. High-resolution chromosome-banding analysis findings were normal in the parents and proband, who died at age 4 years. There were four subsequent pregnancies: two ended in first-trimester spontaneous abortion, and in the other two, large omphaloceles were detected in fetuses at 15 and 13 weeks' gestation. Both pregnancies were terminated. Fluorescence in situ hybridization probes for 17p13.3 had become available before the most recent pregnancy and were used to study parental and fetal cells. As a result, a balanced cryptic translocation between chromosome 17 and chromosome 19 was identified in the father: 46,XY,t(17;19)(p13.3q13.33). An unbalanced form of the translocation, involving a deletion of 17p13.3, was detected with fluorescence in situ hybridization in the fetus. This finding was in accordance with a clinical diagnosis of Miller-Dieker syndrome. CONCLUSIONS: Molecular cytogenetic technology should be used in cases of suspected Miller-Dieker syndrome when high-resolution cytogenetic analysis fails to detect del(17) (p13.3). Positive findings should be followed up with parental studies. In addition, omphalocele should be included among the list of malformations that make up the Miller-Dieker syndrome.

Incomplete EEC syndrome in a patient with mosaic monosomy 21.

Chromosome anomalies in cases of ectrodactyly, ectodermal dysplasia and cleft of the lip and palate (EEC syndrome) have now been reported. We now report a child with the above syndrome and mosaic monosomy of chromosome 21.