Ulf Lönn

Prognostic value of erb-B2 and myc amplification in breast cancer imprints

Background. Amplification of erb‐B2 and myc shows prognostic value in patients with operable breast cancer. Amplification is usually detected in tumor samples remaining after pathologic work‐up, preventing the examination of small tumors.

Gene Amplification Detected in Carcinoma Cells from Human Urinary Bladder Washings by the Polymerase Chain Reaction Method

Background. Urinary bladder carcinoma often is diagnosed from malignant cells in bladder washings obtained at cystoscopic examination. In some cases, there are difficulties distinguishing between cytologic Grades 1 and 2. Detection of genetic alterations in combination with morphologic analysis may facilitate the diagnosis.

Higher frequency of gene amplification in breast cancer patients who received adjuvant chemotherapy

Amplification of certain genes is involved in resistance to chemotherapy. The development of such amplification in patients by drug treatment has not yet been established. We have assessed the appearance of gene amplification in breast cancer patients with recurrent disease. One group of patients had previously received adjuvant chemotherapy (cyclophosphamide, methotrexate, 5‐fluorouracil [CMF]) after surgery. The second group had not. All of these patients had developed recurrent disease and were receiving first‐line endocrine treatment (tamoxifen).

Detection and temporal appearance of multiple copies of c-erb-B2 genes in advanced mammary carcinoma using fine needle biopsies and the polymerase chain reaction

Aspiration of tumor cells by the fine-needle biopsy method yields only a small number of cells, which hampers conventional molecular analysis for the presence of multiple copies of oncogenes. We have therefore adopted the polymerase chain reaction (PCR) method to study semi-quantitatively the level of the c-erb-B2 gene in human breast tumor samples. Of 39 patients with mammary carcinoma, 7 (19%) contained multiple copies of c-erb-B2 genes, whereas only two samples failed to give informative data. Next the temporal appearance of multiple gene copies was examined in 20 patients with clinical stage IV disease. Tumor samples were obtained every second to third month from the same tumor lesion of each patient. None of the initial samples from each patient contained multiple copies of c-erb-B2. Of 16 patients that showed progressive clinical disease, 5 developed multiple gene copies, showing that the event occurs in clinical stage IV disease.

Breast cancer: prognostic significance of c-erb-B2 and int-2 amplification compared with DNA ploidy, S-phase fraction, and conventional clinicopathological features

SummaryThe prognostic value of oncogene amplification and conventional clinicopathological features was determined in consecutive breast cancers detected during 5 months in 1975–1976 in 4 Swedish counties. Material was collected from 162 of the 179 patients and tumor size, nodal status, FSH, estrogen/progesterone receptor status, DNA ploidy and S-phase fraction determined. Tissues remaining from 80 patients were stored frozen until 1991, when amplification of the oncogenes c-erb-B2 and int-2 was determined. We show that c-erb-B2 amplification (but not int-2 amplification) and positive axillary nodal status show prognostic significance for both survival and relapse-free survival in univariate and multivariate analysis. The other examined factors showed no significance.

Detection of a 10 kb DNA replication intermediate in human melanoma cells.

DNA replication in human melanoma cells is investigated by lysing the cells in dilute alkali. This lysis condition results in the release from parental DNA of the single-stranded DNA fragments located in active replicating units. The size of the released DNA should theoretically range from that of Okazaki-fragments up to that of the entire replication unit. However, the results showed that the released DNA replication intermediates which are detected range in the size between Okazaki-fragments up to 10 kb DNA fragments. The 10 kb DNA fragments show a discrete appearance in agarose gel electrophoresis. Moreover the kinetic results indicate that the ligation of the 10 kb DNA fragments to form high molecular weight DNA is a late step. A prerequisite for the release of this DNA fragment as a discrete population is that there are gaps in the continuity of the newly synthesized DNA spaced roughly 10 kb away from each other.

C‐ERB‐B2/INT‐2 amplification appears faster in breast‐cancer patients receiving second‐line endocrine treatment

We have examined the appearance of c‐erb‐b2 and int‐2 amplification in 2 different groups of breast‐cancer patients. The groups differed with regard to their clinical status in that one group was receiving first‐line endocrine treatment (tamoxifen) whereas the second was receiving second‐line endocrine treatment (after failing on tamoxifen). The latter group of patients showed clinically a more advanced disease (higher frequency of stage‐IV as compared to stage‐III disease). Consecutive tumor samples were obtained using fine‐needle biopsies from individual tumor lesions of each patient every second or third month. Median time from diagnosis to the last biopsy for patients receiving tamoxifen was 25 months and, for patients receiving second‐line treatment, 55 months. The presence of amplification was determined using semi‐quantificative PCR. We found that both genes developed amplification during tumor progression. The appearance of amplification was more pronounced in the clinically more advanced patients receiving second‐line treatment (p = 0.018).

Progressive formation of DNA lesions during treatment with anti-metabolites without incorporation of the drugs into DNA.

Anti-metabolites, such as methotrexate, 5-fluoropyrimidines or hydroxyurea, induce progressive formation of DNA lesions. 5-Fluoropyrimidines induce DNA lesions either by incorporation of the drug into DNA or by a mechanism not involving incorporation. The second mechanism, not involving incorporation, is also seen with methotrexate and hydroxyurea. The three anti-metabolites have in common their ability to reduce intracellular levels of nucleotides, resulting in reduced efficiency of repair of DNA lesions. The lesions probably appear spontaneously, independently of the drug treatment.

Protein synthesis inhibitors and export of ribosomal subunits

Puromycin and cycloheximide inhibit nuclear export of ribosomal RNA in Chironomus salivary gland cells like in mammalian cells. The drugs do not prevent other types of RNA like heterodisperse messengerlike RNA, 75-S RNA of Balbiani ring origin and 4-S RNA from appearing in the cytoplasm. Newly exported ribosomal subunits have previously been found to enter polysomes close to the nuclear envelope in these cells. The basis for the specific export-blocking action of the drugs may be that immediate engagement in polysomes is a prerequisite for export of ribosomal subunits.

Prognostic significance of c-erb-B2 amplification in fine-needle biopsies of breast cancer patients not operated at diagnosis

Prognostication of breast cancer patients, not operated at diagnosis, poses a clinically difficult problem. To use gene amplification we examined cytological samples and determined c-erb-B2 gene copy number with semiquantitative PCR. Control experiments showed the same gene-copy number in aliquots that were either air-dried (and MGG-stained), fixed in methanol (and air-dried), or snap-frozen in liquid nitrogen. Therefore we examined the prognostic value of c-erb-B2 amplification in 95 breast cancer patients that had not been operated at diagnosis (up to 12 years previously). Tumor cells were obtained from routine archival cytological smears. 15 patients (16%) had developed amplification. Univariate and multivariate analysis showed that c-erb-B2 amplification is a significant prognostic factor (p < 0.0001). Hence routine cytological MGG smears can be used for prognostic determination.