Barbara L. Weber

Metabolic monitoring of breast cancer chemohormonotherapy using positron emission tomography: initial evaluation.

PURPOSE We assessed the feasibility of noninvasive metabolic monitoring of cancer chemohormonotherapy using sequential quantitative positron emission tomographic (PET) scans of tumor glucose metabolism with the glucose analog 2-[18F]-fluoro-2-deoxy-D-glucose (FDG). PATIENTS AND METHODS Eleven women with newly diagnosed primary breast cancers larger than 3 cm in diameter beginning a chemohormonotherapy program underwent a baseline and four follow-up quantitative PET scans during the first three cycles of treatment (days 0 to 63). Tumor response was sequentially determined clinically, radiographically, and then pathologically after nine treatment cycles. RESULTS Eight patients had partial or complete pathologic responses. Their maximal tumor uptake of FDG assessed by PET decreased promptly with treatment to the following: day 8, 78 +/- 9.2% (P < .03); day 21, 68.1 +/- 7.5% (P < .025); day 42, 60 +/- 5.1% (P < .001); day 63, 52.4 +/- 4.4% (P < .0001) of the basal values. Tumor diameter did not decrease significantly during this period through 63 days. Prompt decreases in the FDG influx rate (K) from basal levels (from .019 to .014 mL/cm3/min) after 8 days of treatment (P < .02) and in the estimated rate of FDG phosphorylation to FDG-6-phosphate (k3) from .055 to .038 min-1 after 8 days of treatment (P < .02) to .029 +/- .004 min-1 at 21 days) (P < .02) were observed. Three nonresponding patients had no significant decrease in tumor uptake of FDG (81 +/- 18% of basal value), influx rate (.015 to .012 mL/cm3/min), or tumor size (81 +/- 12% of basal diameter) comparing basal versus 63-day posttreatment values. CONCLUSION Quantitative FDG PET scans of primary breast cancers showed a rapid and significant decrease in tumor glucose metabolism after effective treatment was initiated, with the reduction in metabolism antedating any decrement in tumor size. No significant decrease in FDG uptake (SUV) after three cycles of treatment was observed in the nonresponding patients. FDG PET scanning has substantial promise as an early noninvasive metabolic marker of the efficacy of cancer treatment.

Effect of Radiotherapy After Breast-Conserving Treatment in Women With Breast Cancer and Germline BRCA1/2 Mutations

PURPOSE Recent laboratory data suggest a role for BRCA1/2 in the cellular response to DNA damage. There is a paucity of clinical data, however, examining the effect of radiotherapy (RT), which causes double-strand breaks, on breast tissue from BRCA1/2 mutation carriers. Thus the goals of this study were to compare rates of radiation-associated complications, in-breast tumor recurrence, and distant relapse in women with BRCA1/2 mutations treated with breast-conserving therapy (BCT) using RT with rates observed in sporadic disease. PATIENTS AND METHODS Seventy-one women with a BRCA1/2 mutation and stage I or II breast cancer treated with BCT were matched 1:3 with 213 women with sporadic breast cancer. Conditional logistic regression models were used to compare matched cohorts for rates of complications and recurrence. RESULTS Tumors from women in the genetic cohort were associated with high histologic (P =.0004) and nuclear (P =.009) grade and negative estrogen (P=.0001) and progesterone (P=.002) receptors compared with tumors from the sporadic cohort. Using Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer toxicity scoring, there were no significant differences in acute or chronic morbidity in skin, subcutaneous tissue, lung, or bone. The 5-year actuarial overall survival, relapse-free survival, and rates of tumor control in the treated breast for the patients in the genetic cohort were 86%, 78%, and 98%, respectively, compared with 91%, 80%, and 96%, respectively, for the sporadic cohort (P = not significant). CONCLUSION There was no evidence of increased radiation sensitivity or sequelae in breast tissue heterozygous for a BRCA1/2 germline mutation compared with controls, and rates of tumor control in the breast and survival were comparable between BRCA1/2 carriers and controls at 5 years. Although additional follow-up is needed, these data may help in discussing treatment options in the management of early-stage hereditary breast cancer and should provide reassurance regarding the safety of administering RT to carriers of a germline BRCA1/2 mutation.

Breast conservation and prolonged chemotherapy for locally advanced breast cancer: the University of Michigan experience.

PURPOSE To determine whether breast conservation and prolonged neoadjuvant chemotherapy have efficacy in locally advanced breast cancer (LABC), as measured by survival and rate of breast conservation. MATERIALS AND METHODS Eighty-nine patients with stage III disease were enrolled at the University of Michigan (UM) onto a prospective nonrandomized trial. Patients received nine 21-day cycles of neoadjuvant chemohormonal therapy that consisted of doxorubicin 30 mg/m2 and cyclophosphamide 750 mg/m2 intravenously on day 1, conjugated estrogens 0.625 mg orally twice daily on days 6 to 8, methotrexate 40 mg/m2 and fluorouracil 500 mg/m2 intravenously on day 8, and tamoxifen 10 mg orally twice daily on days 9 to 14. Patients with a negative biopsy received radiation only, while those with residual disease underwent mastectomy and postoperative radiotherapy. Eight more cycles of chemohormonal therapy were administered after local-regional therapy. RESULTS The clinical response rate to neoadjuvant therapy was 97%, 28% of patients had a complete pathologic response evaluated at biopsy. Five-year overall and disease-free survival probabilities were 54% and 44%, respectively. The median disease-free survival time was 2.4 years. The 5-year actuarial rates of local-regional control with local failure as only first failure were 82% and 78% following radiotherapy, and mastectomy and radiotherapy, respectively (P = .99). CONCLUSION Prolonged neoadjuvant chemohormonal therapy and biopsy-driven local therapy have efficacy in LABC, with 28% of patients being candidates for breast conservation and a 5-year overall survival rate of 54%.

Progesterone receptor variant increases ovarian cancer risk in BRCA1 and BRCA2 mutation carriers who were never exposed to oral contraceptives.

Oral contraceptives have been shown to be protective against hereditary ovarian cancer. The variant progesterone receptor allele named PROGINS is characterized by an Alu insertion into intron G and two additional mutations in exons 4 and 5. The PROGINS allele codes for a progesterone receptor with increased stability and increased hormone-induced transcriptional activity. We studied the role of the PROGINS allele as a modifying gene in hereditary breast and ovarian cancer. The study included 195 BRCA1 and BRCA2 carriers with a prior diagnosis of ovarian cancer, 392 carriers with a diagnosis of breast cancer and 249 carriers with neither cancer. Fifty-eight women had both forms of cancer. Five hundred and ninety-five women had a BRCA1 mutation and 183 women had a BRCA2 mutation. Overall, there was no association between disease status and the presence of the PROGINS allele. Information on oral contraception use was available for 663 of the 778 carriers of BRCA1 or BRCA2 mutations. Among the 449 subjects with a history of oral contraceptive use (74 cases and 365 controls), no modifying effect of PROGINS was observed [odds ratio (OR) 0.8; 95% confidence interval (CI) 0.5-1.3]. Among the 214 carriers with no past exposure to oral contraceptives, the presence of one or more PROGINS alleles was associated with an OR of 2.4 for ovarian cancer, compared to women without ovarian cancer and with no PROGINS allele (P = 0.004; 95% CI 1.4-4.3). The association was present after adjustment for ethnic group and for year of birth.

Chromosomal mapping of the rat Slc4a family of anion exchanger genes, Ae1, Ae2, and Ae3.

Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, 02129, USA Department of Internal Medicine, University of Michigan, 1560 MSRB II, 1150 West Medical Center Drive, and Ann Arbor Veterans Administration Hospital, Ann Arbor, Michigan, 48109, USA Department of Molecular Biology, Universite Libre de Bruxelles, Rhode-St-Genese, Belgium Molecular Medicine and Renal Units, Beth Israel Hospital and Depts. of Cell Biology and Medicine, Harvard Medical School, Boston, Massachusetts, 02215, USA

A YAC-, P1-, and cosmid-based physical Map of the BRCA1 region on chromosome 17q21

A familial early-onset breast cancer gene (BRCA1) has been localized to chromosome 17q21. To characterize this region and to aid in the identification of the BRCA1 gene, a physical map of a region of 1.0-1.5 Mb between the EDH17B1 and the PPY loci on chromosome 17q21 was generated. The physical map is composed of a yeast artificial chromosome (YAC) and P1 phage contig with one gap. The majority of the interval has also been converted to a cosmid contig. Twenty-three PCR-based sequence-tagged sites (STSs) were mapped to these contigs, thereby confirming the order and overlap of individual clones. This complex physical map of the BRCA1 region was used to isolate genes by a number of gene identification techniques and to generate transcript maps of the region, as presented in the three accompanying manuscripts of Brody et al. (1995), Osborne-Lawrence et al. (1995), and Friedman et al. (1995).

Microdissection and microcloning of chromosomal alterations in human breast cancer

SummaryThe recognition of recurring sites of chromosome changes in malignancies has greatly facilitated the identification of genes implicated in the pathogenesis of human cancers. Based especially upon recent studies [1–4], it appears increasingly likely that a subset of recurring chromosome alterations will be recognized in human breast cancer. Currently recognized chromosome changes characterizing breast carcinoma include the recognition of cytologic features of gene amplification (e.g. double minutes [dmins] and homogeneously staining regions [HSRs]) [5–8]. As these and other chromosome regions are implicated in recurring abnormalities in breast cancer, it will become increasingly important to have band-or region-specific genomic libraries and probes in order to facilitate high resolution physical mapping and ultimately to clone breast cancer related genes [9]. Toward this end an important recent development in physical mapping has been the establishment of chromosome microdissection as a rapid and reproducible approach to rapidly isolate and characterize chromosome region-specific DNA, greatly facilitating the initial steps in positional cloning of disease-related genes [10–13]. In this brief report, we will highlight the application of chromosome microdissection to the generation of region-specific probes for both fluorescent in situ hybridization (FISH) and the generation of genomic microclone libraries. Additionally, efforts using this methodology to generate a microclone library encompassing the early onset breast/ovarian cancer (BRCA1) gene will be presented.

Transcript identification in the BRCA1 candidate region

SummaryChromosome 17q12-21 is known to contain a gene (or genes) which confers susceptibility to early-onset breast cancer and ovarian cancer (BRCA1). Identification and isolation of BRCA1 will likely provide the basis for increased understanding of the pathogenesis of breast and ovarian cancer, the development of targeted diagnostic and therapeutic approaches, and a means of screening women at risk of being BRCA1 mutation carriers. Genetic and physical maps of the BRCA1 candidate region have been largely completed and efforts are being directed at identification of candidate genes from within this region. We have begun the task of identifying transcripts from this region employing three complementary strategies. These include: 1) direct cDNA screening with cosmids derived from the BRCA1 region; 2) exon amplification; and 3) magnetic bead capture. Transcripts identified using these approaches are being characterized for: 1) tissue expression pattern; 2) the presence of genomic rearrangement in DNA derived from affected members of families believed to show linkage between breast cancer and genetic markers in the BRCA1 candidate interval; 3) altered size and/or expression pattern in RNA prepared from such individuals; and 4) homology to known genes or functional motifs. Germline mutations in affected individuals from these families will serve as presumptive evidence of BRCA1 identity.

Isolation of Expressed Sequences from the Chromosome 17q21 BRCA1 Region by Magnetic Bead Capture

Magnetic bead capture of cDNAs utilizes biotin-streptavidin magnetic bead technology to isolate expressed sequences from large genomic regions, resulting in several thousand-fold enrichment of selected cDNAs. The technique allows parallel analysis of several genomic segments of varying complexity. Expressed sequences from a variety of tissue sources can also be identified simultaneously. To evaluate this approach, we have applied it to pools of cosmid clones from the interval on chromosome 17q21 which contains the familial early onset breast cancer gene (BRCA1). We describe the characterization of 9 potentially unique cDNAs which were isolated from one pool of 7 minimally overlapping cosmids representing a subset of the BRCA1 candidate region. Overall, the method is shown to detect a large fraction of coding sequences in cosmid clones. Advantages and limitations of the approach are discussed.

Isolation of Gene Sequences from the BRCA1 Region of Chromosome 17q21 by Exon Amplification

A variety of techniques now exist for the identification and isolation of gene sequences from cloned genomic DNA. We report the use of exon amplification to isolate candidate exons of genes in the chromosome 17q21 region associated with familial breast and ovarian cancer. We have used the second generation splicing vector pSPL3, which provides greater flexibility for cloning genomic fragments and which reduces the frequency of the major classes of false positive clones. In two experiments, exon amplification was performed using DNAs of approximately 170 cosmids spanning 1–2 Mb of this region. Cosmid DNAs were pooled in groups of 6–10 each. More than 2000 candidate exon clones from these experiments have been arrayed in microtiter dishes. The average size determined for nearly 400 cloned inserts was approximately 200 base pairs. Ongoing efforts to identify and eliminate clone redundancy have thus far yielded more than 100 unique exon clones. Less than 10% of the clones were found to be repetitive or to be artifacts resulting from cryptic splicing involving sequences present in the splicing vector. Thus the great majority of clones were found to be single copy and to derive from the correct chromosomal location. These exons have been used as hybridization probes to isolate cDNA clones derived from normal breast tissue. The cloned exons and corresponding cDNAs are being localized within developing cosmid contigs in order to assemble a transcription map of the region, and to position transcribed sequences with respect to critical recombinants in breast/ovarian cancer families. While database searches suggest that many of the exon sequences are unique, these searches have also identified several genes which were either mapped previously to proximal 17q or which appear to be homologs of genes in other species. Exon amplification represents a rapid and efficient means for isolating candidate gene sequences from genomic clones, facilitating efforts to identify specific genes associated with disease using positional cloning strategies. Utilization of this technique to survey large genomic regions will also assist in efforts to construct transcription maps of chromosomes.