Kurt J. Isselbacher

Isolation of circulating tumor cells using a microvortex-generating herringbone-chip

Rare circulating tumor cells (CTCs) present in the bloodstream of patients with cancer provide a potentially accessible source for detection, characterization, and monitoring of nonhematological cancers. We previously demonstrated the effectiveness of a microfluidic device, the CTC-Chip, in capturing these epithelial cell adhesion molecule (EpCAM)-expressing cells using antibody-coated microposts. Here, we describe a high-throughput microfluidic mixing device, the herringbone-chip, or “HB-Chip,” which provides an enhanced platform for CTC isolation. The HB-Chip design applies passive mixing of blood cells through the generation of microvortices to significantly increase the number of interactions between target CTCs and the antibody-coated chip surface. Efficient cell capture was validated using defined numbers of cancer cells spiked into control blood, and clinical utility was demonstrated in specimens from patients with prostate cancer. CTCs were detected in 14 of 15 (93%) patients with metastatic disease (median = 63 CTCs/mL, mean = 386 ± 238 CTCs/mL), and the tumor-specific TMPRSS2-ERG translocation was readily identified following RNA isolation and RT-PCR analysis. The use of transparent materials allowed for imaging of the captured CTCs using standard clinical histopathological stains, in addition to immunofluorescence-conjugated antibodies. In a subset of patient samples, the low shear design of the HB-Chip revealed microclusters of CTCs, previously unappreciated tumor cell aggregates that may contribute to the hematogenous dissemination of cancer.

Importance of dosage standardization for interpreting transcriptomal signature profiles: Evidence from studies of xenoestrogens

To obtain insights into similarities and differences in the biological actions of related drugs or toxic agents, their transcriptomal signature profiles (TSPs) have been examined in a large number of studies. However, many such reports did not provide proper justification for the dosage criteria of each agent. Using a well characterized cell culture model of estrogen-dependent proliferation of MCF7 human breast cancer cells, we demonstrate how different approaches to dosage standardization exert critical influences on TSPs, leading to different and even conflicting conclusions. Using quantitative cellular response (QCR)-based dosage criteria, TSPs were determined by Affymetrix microarray when cells were proliferating at comparable rates in the presence of various estrogens. We observed that TSPs of the xenoestrogens (e.g., genistein or bisphenol A) were clearly different from the TSP of 17β-estradiol; namely, the former strongly enhanced expression of genes involved in mitochondrial oxidative phosphorylation, whereas the latter showed minimal effects. In contrast, TSPs for genistein and 17β-estradiol were indistinguishable by using the marker gene expression-based dosage criteria, conditions in which there was comparable expression of the mRNA transcripts for the estrogen-inducible WISP2 gene. Our findings indicate that determination and interpretation of TSPs in pharmacogenomic and toxicogenomic studies that examine the transcriptomal actions of related agents by microarray require a clear rationale for the dosage standardization method to be used. We suggest that future studies involving TSP analyses use quantitative and objective dosage standardization methods, such as those with quantitative cellular response or marker gene expression-based dosage criteria.

Germ-Line BRCA1 Mutations in Jewish and Non-Jewish Women with Early-Onset Breast Cancer

BACKGROUND Mutations in a germ-line allele of the BRCA1 gene contribute to the familial breast cancer syndrome. However, the prevalence of these mutations is unknown in women with breast cancer who do not have the features of this familial syndrome. We sought BRCA1 mutations in women who were given a diagnosis of breast cancer at an early age, because early onset is characteristic of a genetic predisposition to cancer. METHODS Clinical information and peripheral-blood mononuclear cells were obtained from 418 women from the Boston metropolitan area in whom breast cancer was diagnosed at or before the age of 40. A comprehensive BRCA1 mutational analysis, involving automated nucleotide sequencing and a protein-truncation assay, was undertaken in 30 of these women, who had breast cancer before the age of 30. In addition, the BRCA1 mutation 185delAG, which is prevalent in the Ashkenazi Jewish population, was sought with an allele-specific polymerase-chain-reaction assay in 39 Jewish women among the 418 women who had breast cancer at or before the age of 40. RESULTS Among 30 women with breast cancer before the age of 30, 4 (13 percent) had definite, chain-terminating mutations and 1 had a missense mutation. Two of the four Jewish women in this cohort had the 185delAG mutation. Among the 39 Jewish women with breast cancer at or before the age of 40, 8 (21 percent) carried the 185delAG mutation (95 percent confidence interval, 9 to 36 percent). CONCLUSIONS Germ-line BRCA1 mutations can be present in young women with breast cancer who do not belong to families with multiple affected members. The specific BRCA1 mutation known as 185delAG is strongly associated with the onset of breast cancer in Jewish women before the age of 40.

Differential Contributions of BRCA1 and BRCA2 to Early-Onset Breast Cancer

BACKGROUND Germ-line mutations in the BRCA1 and BRCA2 genes predispose women to breast cancer. BRCA1 mutations are found in approximately 12 percent of women with breast cancer of early onset, and the specific mutation causing a deletion of adenine and guanine (185delAG), which is present in 1 percent of the Ashkenazi Jewish population, contributes to 21 percent of breast cancers among young Jewish women. The contribution of BRCA2 mutations to breast cancer of early onset is unknown. METHODS Lymphocyte specimens from 73 women with breast cancer diagnosed by the age of 32 were studied for heterozygous mutations of BRCA2 by a complementary-DNA-based protein-truncation assay, followed by automated nucleotide sequencing. In addition, specimens from 39 Jewish women with breast cancer diagnosed by the age of 40 were tested for specific mutations by an allele-specific polymerase chain reaction. RESULTS Definite BRCA2 mutations were found in 2 of the 73 women with early-onset breast cancer (2.7 percent; 95 percent confidence interval, 0.4 to 9.6 percent), suggesting that BRCA2 is associated with fewer cases than BRCA1 (P=0.03). The specific BRCA2 mutation causing a deletion of thymine (6174delT), which is found in 1.3 percent of the Ashkenazi Jewish population, was observed in 1 of the 39 young Jewish women with breast cancer (2.6 percent; 95 percent confidence interval, 0.09 to 13.5 percent), indicating that it has a small role as a risk factor for early-onset breast cancer. Among young women with breast cancer, there are BRCA2 mutations that cause truncation of the extreme C terminus of the protein and that may be functionally silent, along with definite truncating mutations. CONCLUSIONS Germ-line mutations in BRCA2 contribute to fewer cases of breast cancer among young women than do mutations in BRCA1. Carriers of BRCA2 mutations may have a smaller increase in the risk of early-onset breast cancer.

The pathogenesis of arthritis associated with viral hepatitis. Complement-component studies.

Abstract Eighteen patients with acute viral hepatitis had initial symptoms of arthralgia, arthritis or urticaria. Total serum hemolytic complement (CH50) and C4 tended to be severely depressed in the nine patients studied in the acute phase of their joint symptoms. The C3 levels were moderately depressed, and the C1q levels were widely variable. C1 inhibitor and C9 serum concentrations were normal. Patients with viral hepatitis in convalescence or remission from arthritis, or without a history of these symptoms, had a normal or elevated serum CH50 and C3 level, a widely variable C1q level and low normal C4 levels. Low serum levels of complement in viral hepatitis were associated with high titers of hepatitis-associated antigen. Five patients with acute nonviral hepatitis had normal or elevated serum complement levels. The correlation of joint and skin symptoms with hepatitis-associated antigen and depressed levels of complement activity in the acute state suggests that this serum-sickness-like syndrome ma...

STUDIES ON LIPID METABOLISM IN THE SMALL INTESTINE WITH OBSERVATIONS ON THE ROLE OF BILE SALTS

It is now well established that the esterification of long chain fatty acids is an important step in their transport across the small intestinal mucosa before they appear in the lymph in chylomicrons. Evidence for this process has been well reviewed recently (1, 2). The main ester formed by the mucosa is triglyceride but a small proportion of the fatty acids is incorporated into phospholipids and cholesterol esters. We have recently reported some in vitro studies on the incorporation of long chain fatty acids into glycerides using homogenates of rat and human small intestine mucosa and have delineated some of the cofactor requirements involved (3). This work has now been extended to the intact mucosa of the rat. Observations are presented on factors influencing the uptake and esterification of palmitate-1-C4 and the incorporation of label into lipid from C14glucose. In addition, evidence is presented for the concept that conjugated bile salts directly stimulate glyceride metabolism in the intestinal

Galactosemia, a congenital defect in a nucleotide transferase.

Abstract Galactosemia seems to furnish an example of a congenital human metabolic disease in which a specific enzyme is missing. The enzyme which catalyzes the exchange of α-galactose- I -phosphate with uridinediphospho-glucose, forming α-glucose- I -phosphate and uridinediphospho-galactose is absent in blood from galactosemic subjects. It is known that this enzymic exchange is an important reaction by which administered galactose is used in general carbohydrate metabolism. Several of the metabolic manifestations of the disease might readily be explained on the basis of this enzymatic defect.

Sugar and Amino Acid Transport by Cells in Culture — Differences between Normal and Malignant Cells

IN the last few years, studies of malignant cells have provided increasing evidence that there are important alterations in the structure and function of the malignant cell-surface membranes. Most ...

Very low density lipoproteins in intestinal lymph: origin, composition, and role in lipid transport in the fasting state.

The transport of endogenous lipids in the lipoproteins of mesenteric lymph was studied in fasting rats with mesenteric lymph fistulas. The lymph was found to contain, in addition to chylomicrons (S(f) >400), a significant amount of another, more dense, triglyceride-rich fraction, the very low density lipoproteins (VLDL), which showed a peak S(f) of 102. The VLDL differed from chylomicrons not only in flotation, but also in per cent lipid composition and electrophoretic mobility in agarose gel. The VLDL fraction was found to contain 47% of the triglyceride and 54% of the cholesterol of fasting lymph and, in the fasting state, was the major lipoprotein species present. When cholestyramine resin was administered intraduodenally, or bile flow was acutely diverted from the intestine, it was demonstrated that the lipids in lymph VLDL, like those in chylomicrons, were derived from the intestine and bile. These data indicate that the VLDL in intestinal lymph are not derived from the plasma but are of intestinal origin. Because certain properties of lymph VLDL were similar to those reported for plasma VLDL (per cent lipid composition, flotation coefficient, and continuing entry into plasma in the fasting state), additional comparisons between these fractions were made. Although lymph VLDL moved to the alpha(2) region in agarose gel, when they were mixed with VLDL-free serum immediately before electrophoresis they showed the alpha(2) mobility of rat serum VLDL. Furthermore, immunoelectrophoretic comparison of partially delipidated lymph and serum VLDL revealed that these fractions shared in common their major apoprotein, and possibly others as well. The fatty acid composition of lymph and serum triglycerides, as determined by gas-liquid chromatography, revealed that although they were generally similar, differences existed which most likely reflected the presence in serum of triglycerides of hepatic origin. These experiments demonstrate the importance of intestinal VLDL in the transport of endogenous lipids in mesenteric lymph in the fasting state. The similarities between intestinal lymph VLDL and plasma VLDL suggest that the latter may be derived in part from the former.

Intestinal Uptake of Macromolecules: Effect of Oral Immunization

Animals were orally immunized with horseradish peroxidase and bovine serum albumin, and absorption of these antigens was studied. In comparison with controls, a consistent and significant decrease in peroxidase uptake was noted in both germ-free and conventional rats immunized with peroxidase; a similar decrease in serum albumin uptake was also noted in animals immunized with serum albumin. There was no difference in the uptake of an unrelated macromolecule. These observations suggest that local immunization interferes specifically with the intestinal uptake of macromolecular antigens.