Imatinib disposition and ABCB1 (MDR1, P-glycoprotein) genotype.

The aim of this study was to explore the impact of individual variation in drug elimination on imatinib disposition. Twenty-two patients with gastrointestinal stromal tumor or chronic myeloid leukemia initially received imatinib 600 mg daily with dosage subsequently toxicity adjusted. Pharmacokinetic parameters on day 1 and at steady-state were compared with elimination phenotype and single-nucleotide polymorphisms of CYP3A5 and ABCB1. A fivefold variation in estimated imatinib clearance (CL/F) was present on day 1 and mean CL/F had fallen by 26% at steady state. This reduction in imatinib CL/F was associated with ABCB1 genotype, being least apparent in thymidine homozygotes at the 1236T>C, 2677G>T/A and 3435C>T loci. Toxicity-related dose reduction also tended to be less common in these individuals. ABCB1 genotype was associated with steady-state CL/F due to an apparent genotype-specific influence of imatinib on elimination. Further evaluation of ABCB1 genotype and imatinib dosage is warranted.

Histopathological features of breast cancer in carriers of ATM gene variants.

AIMS: Germline variants in the ataxia telangiectasia mutated (ATM) gene have been implicated in increased breast cancer risk. The aim of this study was to determine whether the histopathology of breast cancers occurring in ATM variant carriers is distinctive or resembles the described BRCA1 mutation-associated phenotype. METHODS: The histopathological features of breast cancers occurring in ATM variant carriers from multiple-case breast cancer families were compared with matched controls. The test group included 21 cases of in situ and/or invasive cancer from carriers of either the IVS10-6T-->G, 2424V-->G or 1420L-->F ATM variants in the absence of BRCA1 or BRCA2 mutations. An additional four invasive cancers from carriers of a pathogenic BRCA1 mutation in the context of a familial ATM variant were also examined. RESULTS: The histopathology of breast cancers in ATM variant-only carriers was not significantly different from controls and known features of BRCA1 mutation-associated cancer were rarely seen. In contrast, these features were prominent in the small group of cases with a pathogenic BRCA1 mutation. CONCLUSIONS: Breast cancer occurring in carriers of ATM variants is not associated with distinctive histopathological features and does not resemble the tumour phenotype commonly observed in BRCA1 mutation carriers.

Histopathologic indicators of breast cancer biology: insights from population mammographic screening.

Histopathologic features of breast cancer such as tumour size, grade and axillary lymph node (LN) status variably reflect tumour biology and time. Recent evidence suggests that the biological character of breast cancer is established at an early stage and has a major impact on clinical course. The aim of this study was to distinguish the impact of biology on breast cancer histopathology by comparing features of breast cancers diagnosed following population mammographic screening with prevalent vs incident detection and screening interval. Central histopathology review data from 1147 cases of ductal in situ and/or invasive breast cancer were examined. Size, grade and LN status of invasive cancers were positively correlated (P < 0.001). Prevalent invasive cancers were larger (P < 0.001) and more likely to be LN positive (P = 0.02) than incident cases, but grade was not associated with screening episode (P = 0.7). Screening interval for incident cancers was positively associated with invasive cancer size (P = 0.05) and LN status (P = 0.002) but not grade (P = 0.1). Together, these data indicate that biology and time both impact on size and LN status of invasive breast cancer, but grade reflects biology alone. In view of the clinical importance of breast cancer biology, grade as its most direct indicator assumes particular significance.

Heterogeneity of progesterone receptors A and B expression in human endometrial glands and stroma.

The human progesterone receptor (PR) is expressed as two isoforms, PRA and PRB, which function as ligand-activated transcription factors. In-vitro studies suggest that the isoforms differ functionally and that their relative expression in a target cell may determine the nature and magnitude of response to progesterone. We have shown recently that PRA and PRB are co-expressed in target cells of the human endometrium. The purpose of this study was to investigate the homogeneity of expression of PRA and PRB in target cells of the human uterus throughout the menstrual cycle. In the functionalis, PRA and PRB were expressed in comparable levels in glandular epithelium during the proliferative phase of the cycle, whereas there was persistence of PRB but not PRA in the glands during mid-secretory phase. In the stroma, there was predominance of the PRA isoform throughout the cycle. There was remarkable homogeneity in the relative expression of PRA and PRB in adjacent cells within the same tissue compartment, suggesting that the mechanisms regulating relative PR isoform expression are similarly active in these cells. By contrast, heterogeneity between glands was observed under some circumstances in the functionalis of the endometrium, suggesting PR isoform down-regulation by progesterone to be asynchronous. Heterogeneity was also seen between the glands of the basalis and functionalis of the endometrium implying region-specific responses to hormonal stimuli. This study demonstrates adjacent cell homogeneity in the relative expression of PRA and PRB in normal human endometrial tissue and a differential response to ovarian steroid hormones between cell types and between different regions within the same tissue.

The hD52 (TPD52) gene is a candidate target gene for events resulting in increased 8q21 copy number in human breast carcinoma.

Chromosome band 8q21 is frequently overrepresented in human cancer, but to date no 8q21 target gene has been proposed. The hD52 (TPD52) gene is of potential significance in breast and other cancers due to its location and expression pattern. Fine mapping of hD52 placed this locus within the peak of the 8q21 amplicon delineated in the SK-BR-3 breast carcinoma cell line, and a positive association between hD52 gene dosage and transcript levels was subsequently demonstrated in four breast carcinoma cell lines, including SK-BR-3. Increased copy number (ICN) was measured using Southern blot analyses in 3/32 human breast carcinomas at hD52, and the related hD54 gene in 20q13.2-q13.3. Subsequent immunohistochemical analysis of hD52 expression in 19 breast carcinomas with varying hD52 gene dosages demonstrated a significant positive association between hD52 dosage and hD52 expression using a Spearman rank correlation coefficient (r(s) = 0.573, alpha = 0.01) and a Wilcoxon rank-sum test (alpha = 0.05). On the basis of its map location and expression pattern in breast carcinoma, we therefore propose hD52 as a candidate target gene at chromosome band 8q21.

Colocalization of progesterone receptors A and B by dual immunofluorescent histochemistry in human endometrium during the menstrual cycle.

The human progesterone receptor (PR) is expressed as two isoforms, PRA and PRB, that function as ligand-activated transcription factors. In vitro studies suggest that the isoforms differ functionally and that the relative levels in a target cell may determine the nature and magnitude of response to progesterone. However, it is not known whether the two isoforms are normally coexpressed in vivo. To understand the functional significance of relative PR isoform expression in normal physiology, it is essential to determine whether PRA and PRB are coexpressed in the same cell. This study reports the development of a dual immunofluorescent staining technique to demonstrate PRA and PRB proteins by single cell analysis in the same tissue section of human endometrium during the menstrual cycle. PRA and PRB are coexpressed in target cells of the human uterus. In the glands, PRA and PRB were expressed before subnuclear vacuole formation and glycogenolysis, implicating both isoforms in this process, whereas persistence of PRB during the midsecretory phase suggested its significance in glandular secretion. In the stroma, the predominance of PRA throughout the cycle implicates this isoform in post-ovulatory progesterone-mediated events. These results support the view that PRA and PRB mediate distinct pathways of progesterone action in the glandular epithelium and stroma of the human uterus throughout the menstrual cycle.

Expression of the oestrogen responsive protein pS2 in human breast cancer.

The trefoil peptide pS2 was discovered in a breast cancer cell line as a result of its oestrogen responsive character. The expression of pS2 in breast tumours in vivo is also likely to be an oestrogenic effect and as such, the presence of pS2 in oestrogen receptor positive breast cancer is evidence of an intact oestrogen response pathway and an indicator of putative hormone responsiveness. Consistent with this, clinical studies of breast cancer have revealed a correlation between pS2 expression and favourable tumour characteristics as well as response to endocrine therapy.

Coexpression of alternatively spliced estrogen and progesterone receptor transcripts in human breast cancer.

Primary transcripts of the human estrogen receptor (ER) and progesterone receptor (PR) are subject to a number of alternative splicing events resulting in a range of variant messenger ribonucleic acid species in receptor-positive tissues. Despite in vitro demonstrations of a possible role for some of these variants in hormonal sensitivity, the clinical significance of this process is uncertain. In this study the coexpression of variant ER and PR transcripts has been documented by RT-PCR and Southern blot analysis in a series of receptor-positive breast tumors. In 35 ER-positive tumors, a common profile of variant ER transcripts was present, with all tumors containing the delta2ER and delta7ER, 94% containing the delta4ER, and 83% containing the delta5ER. In 25 of these cases, which were also PR positive, the most highly expressed PR variants, the delta4PR, delta6PR, and delta(4/2)PR, a transcript from which a 126-bp portion of PR exon 4 was deleted, were detected in over 90% of the cases. The alternatively spliced ER variants were expressed at higher relative levels than the PR species, which had mean levels of expression less than 10% that of wild-type PR. The most abundant species was the delta7ER, which was present at levels ranging from 29-83% of the wild type. There was no relationship between the level of delta7ER in individual tumors and the pattern of expression of the estrogen-responsive proteins PR and pS2. The common profile of alternatively spliced ER and PR transcripts in breast tumors means that this feature cannot be used as a discriminator of hormone responsiveness or other clinical end points. Further, the low level of expression of the majority of variant species calls into question their potential for impacting significantly on receptor function.

Absence of progesterone receptor associated with secondary breast cancer in postmenopausal women.

The relationship between expression of receptors for oestrogen and progesterone (ER and PR) and disease progression in breast cancer was investigated by comparing immunocytochemical determinations of ER and PR in fine needle aspirates from primary and secondary breast tumours. Rates of receptor expression were significantly higher in primary than in secondary lesions: for ER 63.3% (n = 689) compared with 45.3% (n = 223), and for PR 53.7% (n = 443) compared with 33.1% (n = 121). The effect of menopausal status was examined by subdividing the patient cohort into those over or under the age of 50 years. In both instances, ER expression in secondary tumours was relatively low; however, only postmenopausal patients had significantly lower rates of PR expression in secondary tumours. Consistent with this, an increase in the ER+PR- profile in secondary tumours compared with primary cases from postmenopausal patients was seen, and in a multivariate analysis, a specific absence of PR expression in secondary tumours was revealed. Comparison of ER and PR expression in simultaneously sampled primary tumours and lymph node metastases from the same patient showed that receptor expression was stable with progression to a metastatic site as results were concordant for ER in 92% (n = 88) and PR in 93.8% of cases (n = 65). These results suggest that absence of PR expression in primary breast cancer is associated with disease progression and may be a marker of an aggressive tumour phenotype.

Characterization of a truncated progesterone receptor protein in breast tumors.

The progesterone receptor (PR) mediates the actions of progesterone in the normal and malignant breast. PR is expressed as two proteins, PR B and PR A, which are expressed in normal progesterone target tissues and in breast cancers. A significant proportion of breast cancers contain, in addition, a smaller PR protein of molecular mass 78 kDa (PR78 kDa). The significance of PR78 kDa expression is unknown, and in particular, there are no data on whether PR78 kDa is able to bind ligand and therefore potentially exhibit transcriptional activity. If this smaller PR species exhibits similar differences in function as have been evidenced in vitro for PR A relative to PR B, it is possible that this PR species may be an important component in determination of progesterone response in breast cancer. The purpose of this study was to determine whether the PR78 kDa protein in breast tumors is able to bind ligand and to determine whether posttranscriptional mechanisms contribute to its formation in breast cancers. There was no evidence that PR78 kDa was derived from proteolytic activity of either PR B or PR A. Similarly, although exon-deleted PR transcripts were detected (which could, if translated, give rise to a PR protein similar in size to PR78 kDa), neither the abundance of such transcripts nor their relationship to levels of expressed PR78 kDa protein supported a role for exon deletion in formation of this truncated PR protein. PR78 kDa was not recognized by an antibody specific for PR B, indicating that, like PR A, it lacks the N-terminal portion of PR. PR78 kDa was able to bind the progestin ligand, indicating that it may have transcriptional activity. In summary, this study has shown that a truncated PR protein, found in breast cancers, is ligand-binding and seems to be derived from PR A, indicating that it may have a role in progesterone signaling, although a deeper understanding of its role, if any, in breast cancer remains to be established.

Expression of osteonectin mRNA in human breast tumours is inversely correlated with oestrogen receptor content.

Osteonectin is a secreted glycoprotein which is detected in a number of normal and neoplastic human tissues in vivo. It is an extracellular matrix (ECM)-associated protein which is postulated to regulate cell migration, adhesion, proliferation and matrix mineralisation and previous reports suggest that it may be modulated by steroid hormones in target tissues. The aim of this study was to measure osteonectin mRNA and protein expression in breast tumour biopsies and compare these with oestrogen (ER) and progesterone receptor (PR) levels in the same tumours. An inverse correlation was seen between osteonectin mRNA expression and ER level. Samples with low ER protein expression had a mean osteonectin mRNA level which was almost 4-fold greater than the mean level of expression observed in tumours containing high concentrations of ER protein. This inverse correlation was statistically significant. Despite the strong inverse relationship between osteonectin mRNA levels and tumour ER content, no correlation was seen when osteonectin protein concentration was measured in tumour cytosols on immunoblots and compared to ER and PR levels in the same tumours. However, since it is a secreted protein, osteonectin protein expression may not reflect cellular osteonectin levels in breast tumours. In summary, these data suggest that ER-mediated suppression of osteonectin gene expression may contribute to the less aggressive characteristics associated with receptor-positive tumours and that loss of ER expression may lead to over-expression of osteonectin and contribute to a poorer differentiated, more invasive phenotype.

Progesterone receptor A and B protein expression in human breast cancer.

The human progesterone receptor (PR) is a ligand-activated nuclear transcription factor which mediates progesterone action in target tissues. Two PR proteins, PR A (81-83 kDa) and PR B (116-120 kDa), have been described and different physiological activities ascribed to each on the basis of in vitro studies, suggesting that their ratio of expression may control progesterone responsiveness in target cells. Presence of PR in breast tumors is an important indicator of likely responsiveness to endocrine agents. However, the relative expression of PR A and B in breast cancer has not been described and its clinical significance has not been addressed. We have examined the expression of PR A and B in PR-positive breast tumors and found that while in most tumors PR A and B were expressed in similar amounts there was a broad overall distribution of PR A:B ratio which deviated significantly from a normal log distribution with tumors containing a PR A:B ration greater than 4 being over-represented in the group. Linear regression analysis revealed that high PR A:B ratios, in general, derived from a low concentration of PR B rather than high expression of PR A. PR A:B protein ratios were not correlated with the age of the patient or with total PR concentration. A third PR protein band (PR 78 kDa) was detected which comprised greater than 20% of total PR protein in a quarter of the tumor samples examined. The characteristics of tumors containing PR 78 kDa were not different from the overall group. In summary, in PR-positive breast tumors the ratio of expression of PR A and B proteins is close to unity as is seen in a number of other progestin target tissues. However, a significant proportion of tumors expressed very low levels of PR B and a consequently high PR A:B ration. Although the clinical consequence of this observation is not known, the in vitro findings that PR A may act as a repressor for PR B suggests that tumors containing primarily PR A may identify a subset of patients with low or aberrant response to endocrine agents.

Characterization of progesterone receptor A and B expression in human breast cancer.

The human progesterone receptor (PR) is a ligand-activated nuclear transcription factor that mediates progesterone action in target tissues. Two PR proteins, PR-A (M(r) 81,000-83,000) and PR-B (M(r) 116,000-120,000), have been described and different physiological activities ascribed to each on the basis of in vitro studies, suggesting that their ratio of expression may control progesterone responsiveness in target cells. Presence of PR in breast tumors is an important indicator of likely responsiveness to endocrine agents. However, the relative expression of PR-A and B in breast cancer has not been described, and its clinical significance has not been addressed. Expression of PR-A and B was measured by immunoblot analysis of 202 PR-positive human breast tumor cytosols. The ratio of expression of the two PR proteins (PR-A/B) ranged from 0.04 to 179.3. The median PR-A/B ratio was 1.26, and 61.4% of samples had PR-A/B ratios between 0 and 2. PR-A/B ratios deviated significantly from a normal log distribution; tumors containing a PR-A/B ratio greater than 4 were overrepresented in the group. Linear regression analysis revealed that high PR-A/B ratios, in general, derived from a low concentration of PR-B rather than high expression of PR-A. PR-A/B protein ratios were not correlated with the age of the patient or with total PR concentration. A third PR protein band (PR78kDa) was detected in a number of samples and comprised greater than 20% of total PR protein in 52 (25.7%) of the 202 tumor samples examined. The range or frequency distribution of PR-A/B ratios in samples containing PR78kDa was not different to the overall group. In summary, in PR-positive breast tumors, the ratio of expression of PR-A and B proteins is close to unity, as is seen in a number of other progestin target tissues. However, a significant proportion of tumors expressed very low levels of PR-B and a consequently high PR-A/B ratio. Although the clinical consequence of this observation is not known, the in vitro findings that PR-A may act as a repressor of PR-B suggest that tumors containing primarily PR-A may identify a subset of patients with low or aberrant response to endocrine agents.