Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms.

Familial cancer syndromes have helped to define the role of tumor suppressor genes in the development of cancer. The dominantly inherited Li-Fraumeni syndrome (LFS) is of particular interest because of the diversity of childhood and adult tumors that occur in affected individuals. The rarity and high mortality of LFS precluded formal linkage analysis. The alternative approach was to select the most plausible candidate gene. The tumor suppressor gene, p53, was studied because of previous indications that this gene is inactivated in the sporadic (nonfamilial) forms of most cancers that are associated with LFS. Germ line p53 mutations have been detected in all five LFS families analyzed. These mutations do not produce amounts of mutant p53 protein expected to exert a trans-dominant loss of function effect on wild-type p53 protein. The frequency of germ line p53 mutations can now be examined in additional families with LFS, and in other cancer patients and families with clinical features that might be attributed to the mutation.

Spontaneous abnormalities in normal fibroblasts from patients with Li-Fraumeni cancer syndrome: aneuploidy and immortalization.

Families of patients with the Li-Fraumeni cancer syndrome have an inherited pattern of sarcomas and various other types of cancers that follow a dominant mode of transmission, an early age of onset, and exhibit multiple primary tumors. As soft tissue sarcomas (including fibrosarcomas) are frequently observed with this syndrome, the in vitro growth characteristics of fibroblasts derived from skin biopsies of Li-Fraumeni syndrome patients were studied. Control fibroblasts maintained a normal morphology and eventually senesced in culture. Fibroblasts from seven of eight affected individuals developed changes in morphology, anchorage-independent growth, and chromosomal abnormalities. In a fashion similar to that of fibroblasts from normal donors they underwent a growth crisis during which their growth was slow, but they continued to grow past the point at which control samples had stopped dividing (35 population doublings). Fibroblasts from Li-Fraumeni cancer patients escape senescence, growing well beyond 35 population doublings with growth rates similar to early-passage cells. Patient fibroblasts maintain the morphology of a transformed cell but remain nontumorigenic in nude mice. These observations of the behavior of fibroblasts from patients with the Li-Fraumeni syndrome may have predictive value for the determination of gene carriers within these families who are at high risk of cancer.

Tumorigenic transformation of spontaneously immortalized fibroblasts from patients with a familial cancer syndrome.

Immortal cell lines arose spontaneously during in vitro culture of initially normal fibroblasts, MDAH041 and MDAH087, from patients with Li-Fraumeni familial cancer syndrome. Fibroblasts from a control donor, MDAH170, maintained a normal morphology and senesced at 31 population doublings. The immortal fibroblasts have several properties of transformed cells. In addition to having acquired an altered morphology and chromosomal anomalies, MDAH041 and MDAH087 have escaped from senescence, growing beyond 300 and 100 population doublings (pd), respectively. As early as 50 pd, these cells can be transformed by an activated H-ras oncogene to form tumors in nude mice. However, MDAH041 immortal cells were resistant to tumorigenic transformation by transfection with the v-abl oncogene.

Correlation of hormone receptor status between circulating tumor cells, primary tumor, and metastasis in breast cancer patients.

PURPOSE: Estrogen receptor (ER) and progesterone receptor (PR) status is prognostic and predictive in breast cancer. Because metastatic breast tumor biopsies are not routinely feasible, circulating tumor cells (CTCs) offer an alternative source of determining ER/PR tumor status. METHODS/PATIENTS: Peripheral blood was collected prospectively from 36 patients with metastatic breast cancer. CTCs were isolated using the microfluidic OncoCEE™ platform. Detection was accomplished with an expanded anti-cytokeratin (CK) cocktail mixture and anti-CD45. ER/PR protein expression was assessed by immunocytochemistry (ICC) on the CK+ cells and compared to the primary and/or metastatic tumor by immunohistochemistry (IHC). RESULTS: Among the 24 CK + CTC cases, a concordance of 68 % (15/22) in ER/PR status between primary breast tumor and CTCs and 83 % (10/12) between metastatic tumor and CTCs was observed. An overall concordance of 79 % (19/24) was achieved when assessing CTC and metastatic tumor (primary tumor substituted if metastatic breast biopsy not available). A test sensitivity of 72 % and specificity of 100 % was identified when comparing CTCs to tumor tissue. Of the 7 discordant cases between CTCs and primary tumor tissue, 2 were concordant with the metastatic biopsy. CONCLUSIONS: CTC ER/PR status using the OncoCEE™ platform is feasible, with high concordance in ER/PR status between tumor tissue (IHC) and CTCs (ICC). The prognostic and predictive significance of CTC ER/PR protein expression needs further evaluation in larger trials.

Optimized fixation and storage conditions for FISH analysis of single-cell suspensions.

In our protocol to isolate and identify fetal cells in maternal peripheral blood, antibody (Ab)-stained cells are preserved with paraformaldehyde (PF) before batch flow cytometric sorting. However, PF fixation compromises the quality of subsequent interphase fluorescence in situ hybridization (FISH). We therefore examined the effect of PF concentrations and storage time in phosphate-buffered saline (PBS) on the quality of FISH signals. Cells were analyzed for changes in light scatter, morphology, and accessibility of target cell DNA. Fixation in 3% PF for 1 hr was ideal for both flow cytometry and subsequent FISH detection. However, beyond 10 days of storage, FISH quality deteriorated. (J Histochem Cytochem 46:971-973, 1998)

Evidence of skewed X-chromosome inactivation in 47,XXY and 48,XXYY Klinefelter patients.

Klinefelter (47,XXY) syndrome occurs in approximately 1:800 male births and accounts for about 10-20% of males attending infertility clinics. Recent studies have shown no obvious phenotypic differences between subjects in which the extra X-chromosome is of paternal or maternal origin; however, a minority of Klinefelter patients are adversely affected clinically and intellectually to an exceptional level, and the underlying basis of this phenotypic variation is not known. We hypothesize that skewed X-inactivation and possibly parental origin of the X-chromosomes is involved. In this study, we determined parental origin and inactivation status of the X-chromosomes in 17 cytogenetically confirmed 47,XXY cases, two 48,XXYY cases and one mosaic 46,XY/47,XXY case. Eight highly polymorphic markers specific to the X-chromosome and the polymorphic human androgen-receptor (HUMARA) methylation assay were used to determine the parental origin and X-inactivation status of the X-chromosomes, respectively. Overall, 17 cases were fully informative, enabling parental origin to be assigned. In 59% of cases, both X-chromosomes were of maternal origin (Xm); in the remaining 41%, one X was of maternal (Xm) and one was of paternal origin (Xp). In 5 of 16 (31%) cases informative at the HUMARA locus, skewed X-inactivation was observed as defined by greater than 80% preferential inactivation involving one of the two X-chromosomes. The two 48,XmXpYY cases both showed preferential paternal X-chromosome (Xp) inactivation. Three 47,XmXmY cases also showed preferential inactivation in one of the two maternal X-chromosomes. These results suggest that skewed X-inactivation in Klinefelter (47,XXY and 48,XXYY) patients may be common and could explain the wide range of mental deficiency and phenotypic abnormalities observed in this disorder. Further studies are warranted to examine the role of X-inactivation and genetic imprinting in Klinefelter patients.

Comprehensive 4-year follow-up on a case of maternal heterodisomy for chromosome 16.

Uniparental disomy for chromosome 16 has been previously identified in fetal deaths and newborn infants with limited follow-up. Thus there is a lack of information about the long-term effects of maternal uniparental disomy 16 on growth and development. We present a case of maternal heterodisomy for chromosome 16 and a comprehensive 4-year physical and cognitive evaluation. Cytogenetic analysis of chorionic villus obtained at 10 weeks gestation for advanced maternal age showed trisomy 16. At 15 weeks, amniocentesis demonstrated low level mosaicism 47,XY,+16[1]/46,XY[25]. Decreased fetal growth was noted in the last 2 months of pregnancy and the infant was small for gestational age at birth. Molecular studies revealed only maternal alleles for chromosome 16 in a peripheral blood sample from the child, consistent with maternal uniparental heterodisomy 16. Although short stature remains a concern, there appears to be no major cognitive effects of maternal disomy 16. Clinical evaluation and follow-up on additional cases should further clarify the role of placental mosaicism and maternal disomy 16 in intrauterine growth retardation and its effects on long-term growth in childhood.

Effect of sexual abstinence on the proportion of X-bearing sperm as assessed by multicolor fluorescent in situ hybridization.

OBJECTIVE: To document the relation between sexual abstinence and the proportion of X-bearing sperm in the ejaculate. DESIGN: Prospective cohort study. SETTING: Medical college. PATIENT(S): Ten normospermic men, aged 30 to 40 years, provided two semen samples: the first sample was obtained 1.0 to 1.5 days after ejaculation; the second, 7 to 10 days after ejaculation. INTERVENTION(S): Abstinence. MAIN OUTCOME MEASURE(S): Proportion of X- and Y-bearing sperm in two ejaculates. RESULT(S): Multicolor fluorescent in situ hybridization using directly labeled alpha-satellite probes specific for chromosomes 18, X and Y were used to analyze 40,273 sperm. After 1.0 to 1.5 days of abstinence, there were 47.6% +/- 1.7% (mean +/- SD) X-bearing sperm, and after 7 to 10 days of abstinence, there were 49.6% +/- 2.1% X-bearing sperm. The X:Y ratio increased marginally from 0.905 to 0.981. CONCLUSION(S): Sexual abstinence marginally increases the proportion of X-bearing sperm in the ejaculate as assessed by multicolor fluorescent in situ hybridization. This change of borderline statistical significance probably has little impact on the secondary sex ratio.

Increased chromosome X, Y, and 18 nondisjunction in sperm from infertile patients that were identified as normal by strict morphology: implication for intracytoplasmic sperm injection.

OBJECTIVE: To determine the incidence of nondisjunction for chromosomes X, Y, and 18 using fluorescence in situ hybridization (FISH) on morphologically normal sperm from infertile men who are candidates for ICSI. DESIGN: After standard hematoxylin staining, sperm with normal morphology were identified using Kruger's strict morphology criteria. The location of each normal-appearing sperm was recorded using an electronic microstage locator. Slides were subsequently subjected to FISH for detection of chromosomes X, Y, and 18 (control probe). Nuclei were relocated and analyzed under the fluorescent microscope. SETTING: University-affiliated IVF and intracytoplasmic sperm injection program. PATIENT(S): Men classified as infertile on the basis of abnormal strict morphology (<4% by Kruger's criteria). For controls, normal fertile men (n=6) were also analyzed. INTERVENTION(S): Semen smears were obtained retrospectively from infertile (n=8) and fertile (n=6) men. MAIN OUTCOME MEASURE(S): Ploidy of each cell was determined according to the number of signals detected for each probe. RESULT(S): Approximately 100-150 morphologically normal sperm were identified and located in each case. Subsequent FISH analysis of these normal sperm showed aneuploidy to range from 1.8% to 5.5% in the infertile group as compared with 0 to 2.6% among the control fertile group. Statistically significant differences in the incidence of aneuploidy for the sex chromosomes as well as for all three (X, Y, and 18) chromosomes was observed. CONCLUSION(S): Although 95% to 98% of the sperm were found to be normal for X, Y, and 18, our findings show that infertile couples undergoing ICSI are likely to be at an increased risk for having a genetically abnormal conceptus as compared with the fertile controls. These results demonstrate that normal morphology is not an absolute indicator for the selection of genetically normal sperm. Hence, observed pregnancy failures among ICSI patients may in part be due to the selection of aneuploid sperm.

Noninvasive determination of fetal RhD status using fetal DNA in maternal serum and PCR.

OBJECTIVE: Because prenatal testing of fetal RhD status by amniocentesis carries small yet finite risks to the fetus and mother, this study sought to determine whether fetal DNA in maternal serum could be used to detect fetal RhD status by polymerase chain reaction (PCR). METHODS: A retrospective analysis was made of frozen serum specimens from 20 sensitized RhD-negative pregnant women (ranging from 15.0 to 36.0 weeks' gestation) who were confirmed by serology at birth to have been carrying RhD-positive fetuses. Eleven serum specimens from RhD-negative individuals served as controls. DNA was isolated from serum and used in two PCR-based methods to detect a 99 base pair (bp) DNA fragment specific for the RhD gene and a 113 bp fragment specific for the RhCE gene as control. RESULTS: Overall, in 14 (70%) of 20 RhD-positive fetuses the 99 base pair RhD-specific PCR product was detected. There was no false positive detection among the 11 control serum specimens. CONCLUSION: The results illustrate the ability to detect fetal RhD sequences in maternal serum of sensitized women. Moreover, the findings demonstrate that fetal single-gene disorders can be detected prenatally by using DNA isolated only from maternal serum.

The effect of the elapsed time between blood draw and processing on the recovery of fetal cells from maternal blood.

OBJECTIVE: To test the hypothesis that a delay in initial fetal cell enrichment processing of maternal blood samples (defined as the time between blood draw and the initial density gradient centrifugation step) compromises the ability to recover fetal cells, we performed a randomized comparison of immediate (within 4 hours of draw) versus delayed (between 18-24 hours of draw) processing. METHODS: Four centers participated: two centers utilized flow cytometry (FLOW), and two centers utilized magnetic-activated cell sorting (MACS) techniques. Each center collected 34 samples. The outcome was the percentage of gamma positive (gamma(+)) cells for FLOW or the number of nucleated red blood cells (NRBCs) for MACS, found in the final enriched cell population. Both outcomes reflect cell properties that are potentially fetal in origin, thus making them representative of the ability to recover fetal cells. RESULTS: Our results did not support our hypothesis that delay in processing compromises fetal cell recovery. Instead, in MACS processing, we observed an increase in recovered NRBCs when blood sample processing was delayed compared with immediate processing. There was no significant difference in gamma(+) cells with FLOW. CONCLUSION: Time-related changes in the density of target cells, perhaps associated with their progress towards apoptosis during the delay period, may result in increased intact fetal cells with the study methods utilized.

Placental release of distinct DNA-associated micro-particles into maternal circulation: reflective of gestation time and preeclampsia.

BACKGROUND: The aim of this study was to determine whether DNA-associated micro-particles (MPs) in maternal plasma express fetal-derived human leukocyte antigen-G (HLA-G) or placental alkaline phosphatase (PLAP) and whether the levels differ between women with normotensive pregnancies and preeclampsia. METHODS: DNA-associated MPs expressing HLA-G or PLAP were examined in the plasma of normal pregnant women and preeclamptic patients using flow cytometric analysis. RESULTS: DNA-associated HLA-G(+) MPs were significantly increased in maternal plasma compared to plasma from non-pregnant controls (p<0.005), with highest levels found in the first and second trimesters. DNA-associated PLAP(+) MPs were also increased in maternal plasma compared to plasma from non-pregnant controls (p<0.006), with highest levels in the second and third trimesters. Term preeclamptic women had higher levels of DNA-associated MPs than control pregnant women. HLA-G(+) MPs from the plasma of preeclamptic women had more DNA per MP than HLA-G(+) MPs from the plasma of normal pregnant women (p<0.03). CONCLUSIONS: HLA-G(+) and PLAP(+) MPs increase in maternal circulation at different times during gestation. DNA amounts per HLA-G(+) MP increase in preeclamptic women which might indicate dysfunctional extravillous cytotrophoblasts.

Heritability and molecular genetic studies of endometriosis.

Endometriosis is a common disease defined as the growth of endometrial tissue outside the uterine cavity that often results in a vast array of gynaecological problems including dyspareunia, dysmenorrhoea, pelvic pain and infertility. Despite the increasing evidence that supports a genetic component to this common gynaecological condition, the basic aetiology and pathogenesis of endometriosis remain unknown. It is likely that endometriosis is a common polygenic/multifactorial disease caused by an interaction between multiple genes as well as the environment. Such conditions do not have a clear Mendelian pattern of inheritance. Recent molecular cytogenetic studies on endometriotic tissue and an established endometriosis-derived cell line provide novel evidence that acquired chromosome-specific alterations may be involved in endometriosis, possibly reflecting clonal expansion of chromosomally abnormal cells. Molecular DNA studies examining the role of loss of heterozygosity in endometriotic lesions has identified candidate tumour suppressor gene loci, including 5q, 6q, 9p, 11q and 22q, that may play a role in the malignant transformation of endometriotic implants to endometrioid ovarian cancers. Evidence of mutations in the tumour suppressor PTEN gene in the endometrioid subtype of epithelial ovarian cancer further suggests that somatic genetic alterations represent early events in the transformation of benign endometriotic cells. Genetic factors are also likely to influence individual susceptibility to endometriosis. There is now evidence that heritable allelic differences in drug-metabolizing enzymes play an important role in the development of endometriosis. Further studies are warranted to identify major susceptibility gene(s) and the mechanism involved in endometriosis to assist in the development of better methods for early detection, diagnosis and prevention.

Genomic instability due to germline p53 mutations drives preneoplastic progression toward cancer in human cells.

Cells heterozygous for mutations in p53 demonstrate extreme genomic instability and develop mutations detectable at the chromosome level as well as the molecular level. This genomic instability causes initially nontumorigenic ras-expressing immortal LFS cells to progress to a tumorigenic state presumably due to additional mutational events. It is not surprising that LFS families with these p53 mutations develop the additional mutations necessary for cancer to occur at such high frequencies. This observation is consistent with increased cancer rates in these families being due to abrogation of a rate limiting step rather than a rate expected for one less step in a multistep carcinogenic process. Although p53 has been shown to be able to function as a transcription factor, mutations in p53 appear to affect genomic stability in LFS fibroblasts with double minutes and telomeric associations being prominent early events. One possibility is that p53 controls the expression of genes required for fidelity of replication or telomerase activity. Alternatively p53 may itself be a replication factor like the transcription factor CTF. In the future, we plan to investigate whether p53 plays a direct role in replication.

Isolating fetal nucleated red blood cells from maternal blood: the Baylor experience--1995.

In our previous work we have isolated fetal cells from maternal blood and used fluorescent in situ hybridization (FISH) for chromosome-specific probes to detect aneuploidy. Current efforts in the Baylor College of Medicine programme are focusing on obtaining consistency in flow-sorting methodology and on determining sensitivity and specificity. To this end, systematic evaluation of five glycophorin A (gly A) antibodies all produced agglutination, leading us to abandon the use of gly A antibodies for positive selection of fetal cells. Conversely, we have found LDS-751 to be useful for nuclear selection. CD45 negative selection can best be accomplished by the use of flasks coated with goat antibodies against mouse antibodies. Positive selection by flow sorting for either CD71+ cells or gamma-globin-positive cells seems to be successful. Using these two approaches, we have recently detected male (fetal) cells in pregnancies in which the fetus was 46,XY in 10 of 18 and in 12 of 14 cases, respectively.

Estimates of aneuploidy using multicolor fluorescence in situ hybridization on human sperm.

Single color fluorescence in situ hybridization (FISH) has been utilized on sperm to estimate nondisjunction rates for chromosomes 1, 12, 15, 16, X and Y. Using single-color FISH, one cannot distinguish nonhybridization from nullisomy nor disomy from diploidy. In order to provide an internal control, a multicolor FISH strategy was employed. Satellite probes specific for 13 human chromosomes were used on multiple semen samples from two normal donors. Two or three probes were hybridized simultaneously and scored by two independent observers. Over all experiments, 40,641 sperm were analyzed. The majority of autosomes had no significant difference in aneuploidy between chromosomes or between donors. However, a significant difference was observed for chromosome 18 between donors (chi 2(2) = 7.078, 0.025 < P < 0.05). Additionally, no significant difference was found between donors for sex chromosome aneuploidy. The frequency of sex chromosome aneuploidy was similar to that seen in paternally derived 47,XXY and 47,XYY conceptuses. Furthermore, 0.15% of sperm were found to be diploid. Based on the results of this study, as much as 19% of all sperm may be chromosomally abnormal. This method proved to be useful for determining aneuploidy of human chromosomes in sperm and valuable in exploring whether individual differences of nondisjunction exist.

Genetic concerns for the subfertile male in the era of ICSI.

The treatment of severe male factor infertility has seen remarkable advances in the last five years with the introduction and widespread use of intracytoplasmic sperm injection (ICSI). Although ICSI represents one of the most important advances in the treatment of the subfertile male, significant concerns exist regarding the potential for transmission of abnormal genes to the offspring because many of the natural barriers to conception have been bypassed. Because these couples were not able to conceive prior to ICSI, the long-term genetic consequences in these offspring are largely undefined at this time. Genetic abnormalities related to male infertility need to be considered in terms of being (1) causative for male infertility and (2) potentially transmissible to the offspring. Reasons for pursuing a genetic evaluation include (1) establishing a diagnosis, (2) establishing a possible genetic origin, (3) clarifying the pattern of inheritance, and (4) providing information on natural history, variation and expression. The three most common known genetic factors related to male infertility are cystic fibrosis gene mutations leading to congenital absence of the vas deferens, Y-chromosome microdeletions leading to spermatogenic impairment, and karyotype abnormalities. When congenital bilateral absence of the vas deferens with azoospermia is encountered, cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations are commonly the underlying cause. When testicular failure is manifest by azoospermia or severe oligoszoospermia, Y-chromosome microdeletions may be present in approximately 10-15 per cent of otherwise normal appearing men. Karyotyping can uncover potentially transmissible genetic abnormalities in the infertile male including structural chromosomal disorders such as Klinefelter's (classic 47,XXY), mixed gonadal dysgenesis, chromosomal translocations and XYY syndromes. Finally, potential male infertility genes in animal models are reviewed. Without question, advances in clinical and basic research raise scientific and social issues that must be addressed.

The current state of oncogenes and cancer: experimental approaches for analyzing oncogenetic events in human cancer.

The development of cancer is a multistage process. The activation of proto-oncogenes and the inactivation of tumor suppressor genes play a critical role in the induction of tumors. Using human cell model systems of carcinogenesis, we have studied how oncogenes, tumor suppressor genes, and recessive cancer susceptibility genes participate in this multistep process. Normal human cells are resistant to the transforming potential of oncogenes, such as ras oncogenes, which are activated by specific point mutations. Since as many as 40% of some tumor types contain activated ras oncogenes, a preneoplastic transition in multistage carcinogenesis must involve changing from an oncogene-resistant stage to an oncogene-susceptible stage. The analysis of such critical steps in carcinogenesis using rodent systems has usually not represented the human disease with fidelity. In order to study this carcinogenic process, we have developed human cell, in vitro systems that represent some of the genetic changes that occur in cellular genes during human carcinogenesis. Using these systems, we have learned some of the functions of dominant activated-transforming oncogenes, tumor suppressor genes, and cellular immortalization genes and how they influence the carcinogenic process in human cells. Using our understanding of these processes, we are attempting to clone critical genes involved in the etiology of familial cancers. These investigations may help us to develop procedures that allow us to predict, in these cancer families, which individuals are at high risk for developing cancer.

Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles.

Loss of cell cycle control and acquisition of chromosomal rearrangements such as gene amplification often occur during tumor progression, suggesting that they may be correlated. We show here that the wild-type p53 allele is lost when fibroblasts from patients with the Li-Fraumeni syndrome (LFS) are passaged in vitro. Normal and LFS cells containing wild-type p53 arrested in G1 when challenged with the uridine biosynthesis inhibitor PALA and did not undergo PALA-selected gene amplification. The converse occurred in cells lacking wild-type p53 expression. Expression of wild-type p53 in transformants of immortal and tumor cells containing mutant p53 alleles restored G1 control and reduced the frequency of gene amplification to undetectable levels. These studies reveal that p53 contributes to a metabolically regulated G1 check-point, and they provide a model for understanding how abnormal cell cycle progression leads to the genetic rearrangements involved in tumor progression.