Palbociclib dose reductions and the effect on clinical outcomes in patients with advanced breast cancer.

BACKGROUND: This study aimed to provide insights into the real-world use of palbociclib, dose reductions, and drug effectiveness in (older) patients with advanced breast cancer (BC). PATIENTS AND METHODS: Patients with advanced BC treated with palbociclib from 2017 to 2020 were included. The Kaplan-Meier method was used to calculate time to next treatment (TTNT) and overall survival (OS) for patients with or without dose reductions. These clinical outcomes were also compared in subgroup analyses for older patients (≥70 years) and younger patients (<70 years) and for patients discontinuing palbociclib early (<4 administrations). RESULTS: A total of 598 patients with advanced BC were included, with a median age of 64 years. Palbociclib dose reductions occurred in 33% of all patients. Early discontinuation of palbociclib without dose reductions occurred in 23% of the patients. Patients who required a palbociclib dose reduction were older (median age 67 years vs. 63 years). Patients with dose reductions had a significantly higher TTNT of 16.9 vs. 11.4 months (p < 0.001) and median OS of 29.7 vs. 21.9 months (p = 0.003) compared to patients without dose reductions. The TTNT in older patients was significantly longer (16.9 vs. 11.6 months, p = 0.013) than younger patients, but OS was similar (20.7 vs. 26.7 months, p = 0.051). CONCLUSION: Palbociclib dose reductions occurred in real-world practice similarly to the PALOMA-3 trial. Patients with dose reductions had no poorer outcomes compared to patients not requiring a dose reduction. Older patients treated with palbociclib had more frequent dose reductions, but this did not appear to affect OS.

Tamoxifen resistance in breast cancer: elucidating mechanisms.

Tamoxifen has been used for the systemic treatment of patients with breast cancer for nearly three decades. Treatment success is primarily dependent on the presence of the estrogen receptor (ER) in the breast carcinoma. While about half of patients with advanced ER-positive disease immediately fail to respond to tamoxifen, in the responding patients the disease ultimately progresses to a resistant phenotype. The possible causes for intrinsic and acquired resistance have been attributed to the pharmacology of tamoxifen, alterations in the structure and function of the ER, the interactions with the tumour environment and genetic alterations in the tumour cells. So far no prominent mechanism leading to resistance has been identified. The recent results of a functional screen for breast cancer antiestrogen resis- tance (BCAR) genes responsible for development of tamoxifen resistance in human breast cancer cells are reviewed. Individual BCAR genes can transform estrogen-dependent breast cancer cells into estrogen-independent and tamoxifen-resistant cells in vitro. Furthermore, high levels of BCAR1/pl30Cas protein in ER-positive primary breast tumours are associated with intrinsic resistance to tamoxifen treatment. These results indicate a prominent role for alternative growth control pathways independent of ER signalling in intrinsic tamoxifen resistance of ER-positive breast carcinomas. Deciphering the differentiation characteristics of normal and malignant breast epithelial cells with respect to proliferation control and regulation of cell death (apoptosis) is essential for understanding therapy response and development of resistance of breast carcinoma.

Immunohistochemical study of the BCAR1/p130Cas protein in non-malignant and malignant human breast tissue.

BCAR1/p130Cas is a docking protein involved in intracellular signaling pathways and in vitro resistance of estrogen-dependent breast cancer cells to antiestrogens. The BCAR1/p130Cas protein level in primary breast cancer cytosols was found to correlate with rapid recurrence of disease. A high BCAR1/p130Cas level was also associated with a higher likelihood of resistance to first-line tamoxifen treatment in patients with advanced breast cancer. Using antibodies raised against the rat p130Cas protein, we determined by immunohistochemical methods the BCAR1/p130Cas localization in primary breast carcinomas, in tumors of stromal origin, and in non-neoplastic breast tissues. The BCAR1/p130Cas protein was detected in the cytoplasm of non-malignant and neoplastic epithelial cells and in the vascular compartment of all tissue sections analyzed. Immunohistochemistry demonstrated variable intensity of BCAR1/p130Cas staining and variation in the proportion of BCAR1/p130Cas-positive epithelial tumor cells for the different breast carcinomas. Double immunohistochemical staining for BCAR1/p130Cas and estrogen receptor confirmed coexpression in non-malignant luminal epithelial cells and malignant breast tumor cells. The stromal cells in non-malignant tissues and tumor tissues as well as breast tumors of mesodermal origin did not stain for BCAR1/p130Cas. This immunohistochemical study demonstrates a variable expression of BCAR1/p130Cas in malignant and non-malignant breast epithelial cells, which may be of benefit for diagnostic purposes.

BCAR1/p130Cas expression in untreated and acquired tamoxifen-resistant human breast carcinomas.

High BCAR1/p130Cas expression in primary breast tumour cytosol predicts a poor chance of response recurrent disease to tamoxifen treatment in patients with oestrogen receptor (ER)-positive breast carcinomas. In this study, we assessed whether BCAR1/p130Cas expression is altered during acquisition of anti-oestrogen resistance. BCAR1/p130Cas protein was quantitatively measured by chemiluminescent Western blot analysis in the cytosol of 34 predominantly ER(+) carcinomas that initially responded to primary tamoxifen treatment and subsequently progressed (n = 22 ) or developed during adjuvant tamoxifen treatment (n = 12) and compared to 54 untreated ER(+) human breast carcinomas. We did not detect significant differences in the level of BCAR1/p130Cas protein in untreated and acquired tamoxifen-resistant carcinomas. Our results indicate that in tumour progression towards tamoxifen resistance, increase of BCAR1/p130Cas may be only one of the molecular mechanisms. Thus, high BCAR1/p130Cas protein levels appear to be a hallmark for intrinsic resistance to tamoxifen in breast carcinomas.

BCAR1, a human homologue of the adapter protein p130Cas, and antiestrogen resistance in breast cancer cells.

BACKGROUND: Treatment of breast cancer with the antiestrogen tamoxifen is effective in approximately one half of the patients with estrogen receptor-positive disease, but tumors recur frequently because of the development of metastases that are resistant to tamoxifen. We have previously shown that mutagenesis of human estrogen-dependent ZR-75-1 breast cancer cells by insertion of a defective retrovirus genome caused the cells to become antiestrogen resistant. In this study, we isolated and characterized the crucial gene at the breast cancer antiestrogen resistance 1 (BCAR1) locus. METHODS/RESULTS: Transfer of the BCAR1 locus from retrovirus-mutated, antiestrogen-resistant cells to estrogen-dependent ZR-75-1 cells by cell fusion conferred an antiestrogen-resistant phenotype on the recipient cells. The complete coding sequence of BCAR1 was isolated by use of exon-trapping and complementary DNA (cDNA) library screening. Sequence analysis of human BCAR1 cDNA predicted a protein of 870 amino acids that was strongly homologous to rat p130Cas-adapter protein. Genomic analysis revealed that BCAR1 consists of seven exons and is located at chromosome 16q23.1. BCAR1 transcripts were detected in multiple human tissues and were similar in size to transcripts produced by retrovirus-mutated ZR-75-1 cells. Transfection of BCAR1 cDNA into ZR-75-1 cells again resulted in sustained cell proliferation in the presence of antiestrogens, confirming that BCAR1 was the responsible gene in the locus. CONCLUSIONS: Overexpression of the BCAR1 gene confers antiestrogen resistance on human ZR-75-1 breast cancer cells. Overexpression of BCAR1 in retrovirus-mutated cells appears to result from activation of the gene's promoter. The isolation and characterization of this gene open new avenues to elucidating mechanisms by which the growth of human breast cancer becomes independent of estrogen.

Bcar1/p130Cas protein and primary breast cancer: prognosis and response to tamoxifen treatment.

BACKGROUND: The product of the Bcar1/p130Cas (breast cancer resistance/p130Crk-associated substrate) gene causes resistance to antiestrogen drugs in human breast cancer cells in vitro. To investigate its role in clinical breast cancer, we determined the levels of Bcar1/p130Cas protein in a large series of primary breast carcinomas. METHODS: We measured Bcar1/p130Cas protein in cytosol extracts from 937 primary breast carcinomas by western blot analysis. The levels of Bcar1/p130Cas protein were tested for associations and trends against clinicopathologic and patient characteristics, the lengths of relapse-free survival and overall survival (n = 775), and the efficacy of first-line treatment with tamoxifen for recurrent or metastatic disease (n = 268). RESULTS: Bcar1/p130Cas levels in primary tumors were associated with age/menopausal status and the levels of estrogen receptor and progesterone receptor. In univariate survival analysis, higher Bcar1/p130Cas levels were associated with poor relapse-free survival and overall survival (both two-sided P =.04; log-rank test for trend). In multivariate analysis, a high level of Bcar1/p130Cas was independently associated with poor relapse-free survival and overall survival. The response to tamoxifen therapy in patients with recurrent disease was reduced in patients with primary tumors that expressed high levels of Bcar1/p130Cas. In multivariate analysis for response, Bcar1/p130Cas was independent of classical predictive factors, such as estrogen receptor status, age/menopausal status, disease-free interval, and dominant site of relapse. CONCLUSION: Patients with primary breast tumors expressing a high level of Bcar1/p130Cas protein appear to experience more rapid disease recurrence and have a greater risk of (intrinsic) resistance to tamoxifen therapy. Thus, measurement of Bcar1/p130Cas may provide useful prognostic information for patients with primary or metastatic breast cancer.

Relevance of ultraviolet-induced N-ras oncogene point mutations in development of primary human cutaneous melanoma.

Intermittent or recreational exposure to sunlight is thought to contribute to development of human cutaneous melanoma. We investigated the incidence of ras oncogene mutation in human cutaneous melanoma in connection to sun-exposed body sites in the patient, using a large series of DNA samples derived from paraffin-embedded material as well as from fresh tumor samples and cell lines. We first show that, of the ras family, predominantly N-ras is activated (15%), whereas rarely H-ras or K-ras are mutated. The occurrence of N-ras mutations correlates with continuous exposure to sunlight of the primary tumor site. Of all tumors initiated on chronically sun-exposed body sites, 26% contained mutated N-ras, in contrast to 0% of sun-protected melanomas. Melanoma lesions obtained from patients from North or Central Europe contained fewer N-ras mutations (12%) as compared with patients from Australia (24%). Mutations were specifically associated with nodular melanoma and to a lesser extent with lentigo malignant melanoma. N-ras mutations did not correlate with metastasis or survival parameters. This study identifies a subset of cutaneous melanomas that contain in the primary lesion ultraviolet-induced N-ras mutations, which are maintained through further progression.

Analysis of N-ras mutations in human cutaneous melanoma: tumor heterogeneity detected by polymerase chain reaction/single-stranded conformation polymorphism analysis.

Determination of the activation state of oncogenes as well as tumor suppressor genes is a main subject of interest in the analysis of the mechanism of tumor initiation. In human melanoma, the c-myc and N-ras oncogenes have been found to be activated in approximately 50% and 15% of the analyzed material, respectively. These studies have mostly been done on fresh tumor material or cell lines. Only in a few cases has an attempt been made to look at tumor heterogeneity or clonality with respect to the activation of oncogenes. We have adjusted the polymerase chain reaction (PCR)/single-stranded conformation polymorphism analysis (SSCP) technique to screen paraffin-embedded melanoma material for the presence of N-ras mutations and found genetic defects at particular progression stages. In one melanoma of the skin, we were able to sublocalize an N-ras mutation in the intraepidermal tumor part, that was absent in the part deeply invading the dermal layer. We conclude that a thorough investigation of N-ras activation in human melanoma should include analysis of histologically different parts of the tumor.

Quantification of inter- and intra-nuclear variation of fluorescence in situ hybridization signals.

This study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi-wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH. The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein-labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation. The intra-nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter-nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence ratio measurements of double-labeled probes for multiple in situ hybridization by digital imaging microscopy.

To expand the multiplicity of the in situ hybridization (ISH) procedure, which is presently limited by the number of fluorochromes spectrally separable in the microscope, a digital fluorescence ratio method is proposed. For this purpose, chromosome-specific repetitive probes were double-labeled with two haptens and hybridized to interphase nuclei of human peripheral blood lymphocytes. The haptens were immunocytochemically detected with specific antibodies conjugated with the fluorochromes FITC or TRITC. The FITC and TRITC fluorescence intensities of spots obtained with different double-haptenized probes were measured, and the fluorescence ratio was calculated for each ISH spot. Combinations of different haptens, such as biotin, digoxigenin, fluorescein, sulfonate, acetyl amino fluorene (AAF), and mercury (Hg) were used. The fluorescence intensity ratio (FITC/TRITC) of the ISH spots was fairly constant for all combinations used, with coefficients of variation between 10 and 30%. To study the feasibility of a probe identification procedure on the basis of probe hapten ratios, one probe was double-labeled with different ratios, by varying the relative concentrations of the modified nucleotides (biotin-11-dUTP and digoxigenin-11-dUTP) in the nick-translation reaction. Measurement of the FITC and TRITC intensities of the ISH spots showed that the concentration of modified nucleotides used in the labeling procedures was reflected in the mean fluorescence intensity of the ISH spots. Furthermore, the ratio distributions showed little overlap due to the relatively small coefficients of variation. The results indicate that a multiple ISH procedure based on fluorescence ratio imaging of double-labeled probes is feasible.

Quantification of fluorescence in situ hybridization signals by image cytometry.

In this study we aimed at the development of a cytometric system for quantification of specific DNA sequences using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. The instrumentation requirements are evaluated. Quantification of the fluorescence ISH signals was performed using an epi-fluorescence microscope with a multi-wavelength illuminator, equipped with a cooled charge couple device (CCD) camera. The performance of the system was evaluated using fluorescing beads and a homogeneously fluorescing specimen. Specific image analysis programs were developed for the automated segmentation and analysis of the images provided by ISH. Non-uniform background fluorescence of the nuclei introduces problems in the image analysis segmentation procedures. Different procedures were tested. Up to 95% of the hybridization signals could be correctly segmented using digital filtering techniques (min-max filter) to estimate local background intensities. The choice of the objective lens used for the collection of images was found to be extremely important. High magnification objectives with high numerical aperture, which are frequently used for visualization of fluorescence, are not optimal, since they do not have a sufficient depth of field. The system described was used for quantification of ISH signals and allowed accurate measurement of fluorescence spot intensities, as well as of fluorescence ratios obtained with double-labeled probes.

Multiple fluorescence in situ hybridization.

A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.

Human gametes and zygotes studied by nonradioactive in situ hybridization.

A nonradioactive in situ hybridization technique was applied to human gametes and abnormally fertilized or developed zygotes. Using haptenized chromosome-specific probes, visualization was obtained using immunocytochemistry to achieve a fluorescent stain on specific hybrids. Using a chromosome 1-specific DNA probe, almost all spermatozoa gave a positive result, i.e., one hybridization signal per cell could be observed. Furthermore, it was possible to identify sperm cells with two spots, suggesting nondisjunction. Two cleavage arrested embryos from different patients showed both: two brightly fluorescent spots and two weaker spots with the same DNA probe. Using a Y-specific DNA probe the percentages of positive spermatozoa from the normal males ranged between 48.1% and 49.1%. In an embryo with four grossly haploid chromosome sets, three fluorescent spots were obtained with the Y-specific DNA probe, indicating the penetration of three spermatozoa.

Detection of chromosome aberrations in interphase tumor nuclei by nonradioactive in situ hybridization.

In a blind study, chromosome aberrations in tumor cells were analyzed by conventional cytogenetic techniques (G banding) and nonradioactive in situ hybridization with chromosome-specific probes. The material was obtained directly from patients with hematologic diseases and from colon tumor derived cell lines. The cytogenetic data obtained with G banding were in accord with those obtained by in situ hybridization to metaphase chromosomes. Most importantly, in situ hybridization to interphase nuclei gave reliable results and even allowed detection of cell subpopulations that were not detected by analyzing metaphase chromosomes. Furthermore, in retrospect, even structural aberrations could be detected in interphase nuclei; abnormal cells with either an i(1q) or a translocation der(1)t(1;7) could be identified. Our results show that the application of in situ hybridization in combination with routine cytogenetic techniques offers significant advantages for cytogenetic analysis of solid tumors and hematologic malignancies.