Relationship of ZNF423 and CTSO with breast cancer risk in two randomised tamoxifen prevention trials.

A case-control study from two randomised breast cancer prevention trials of tamoxifen and raloxifene (P-1 and P-2) identified single-nucleotide polymorphisms (SNPs) in or near genes ZNF423 and CTSO as factors which predict which women will derive most anti-cancer benefit from selective oestrogen receptor modulator (SERM) therapy. In this article, we further examine this question using blood samples from two randomised tamoxifen prevention trials: the International Breast Cancer Intervention Study I (IBIS-I) and the Royal Marsden trial (Marsden). A nested case-control study was designed with 2:1 matching in IBIS-I and 1:1 matching in Marsden. The OncoArray was used for genotyping and included two SNPs previously identified (rs8060157 in ZNF423 and rs10030044 near CTSO), and 102 further SNPs within the same regions. Overall, there were 369 cases and 662 controls, with 148 cases and 268 controls from the tamoxifen arms. Odds ratios were estimated by conditional logistic regression, with Wald 95 % confidence intervals. In the tamoxifen arms, the per-allele odds ratio for rs8060157 was 0.99 (95 %CI 0.73-1.34) and 1.00 (95 %CI 0.76-1.33) for rs10030044. In the placebo arm, the odds ratio was 1.10 (95 %CI 0.87-1.40) for rs8060157 and 1.01 (95 %CI 0.79-1.29) for rs10030044. There was no evidence to suggest that other SNPs in the surrounding regions of these SNPs might predict response to tamoxifen. Results from these two prevention trials do not support the earlier findings. rs8060157 in ZNF423 and rs10030044 near CTSO do not appear to predict response to tamoxifen.

Selective oestrogen receptor modulators in prevention of breast cancer: an updated meta-analysis of individual participant data.

BACKGROUND: Tamoxifen and raloxifene reduce the risk of breast cancer in women at elevated risk of disease, but the duration of the effect is unknown. We assessed the effectiveness of selective oestrogen receptor modulators (SERMs) on breast cancer incidence. METHODS: We did a meta-analysis with individual participant data from nine prevention trials comparing four selective oestrogen receptor modulators (SERMs; tamoxifen, raloxifene, arzoxifene, and lasofoxifene) with placebo, or in one study with tamoxifen. Our primary endpoint was incidence of all breast cancer (including ductal carcinoma in situ) during a 10 year follow-up period. Analysis was by intention to treat. RESULTS: We analysed data for 83,399 women with 306,617 women-years of follow-up. Median follow-up was 65 months (IQR 54-93). Overall, we noted a 38% reduction (hazard ratio [HR] 0·62, 95% CI 0·56-0·69) in breast cancer incidence, and 42 women would need to be treated to prevent one breast cancer event in the first 10 years of follow-up. The reduction was larger in the first 5 years of follow-up than in years 5-10 (42%, HR 0·58, 0·51-0·66; p<0·0001 vs 25%, 0·75, 0·61-0·93; p=0·007), but we noted no heterogeneity between time periods. Thromboembolic events were significantly increased with all SERMs (odds ratio 1·73, 95% CI 1·47-2·05; p<0·0001). We recorded a significant reduction of 34% in vertebral fractures (0·66, 0·59-0·73), but only a small effect for non-vertebral fractures (0·93, 0·87-0·99). INTERPRETATION: For all SERMs, incidence of invasive oestrogen (ER)-positive breast cancer was reduced both during treatment and for at least 5 years after completion. Similar to other preventive interventions, careful consideration of risks and benefits is needed to identify women who are most likely to benefit from these drugs. FUNDING: Cancer Research UK.

Lasofoxifene in postmenopausal women with osteoporosis.

BACKGROUND: The effects of lasofoxifene on the risk of fractures, breast cancer, and cardiovascular disease are uncertain. METHODS: In this randomized trial, we assigned 8556 women who were between the ages of 59 and 80 years and had a bone mineral density T score of -2.5 or less at the femoral neck or spine to receive once-daily lasofoxifene (at a dose of either 0.25 mg or 0.5 mg) or placebo for 5 years. Primary end points were vertebral fractures, estrogen receptor (ER)-positive breast cancer, and nonvertebral fractures; secondary end points included major coronary heart disease events and stroke. RESULTS: Lasofoxifene at a dose of 0.5 mg per day, as compared with placebo, was associated with reduced risks of vertebral fracture (13.1 cases vs. 22.4 cases per 1000 person-years; hazard ratio, 0.58; 95% confidence interval [CI], 0.47 to 0.70), nonvertebral fracture (18.7 vs. 24.5 cases per 1000 person-years; hazard ratio, 0.76; 95% CI, 0.64 to 0.91), ER-positive breast cancer (0.3 vs. 1.7 cases per 1000 person-years; hazard ratio, 0.19; 95% CI, 0.07 to 0.56), coronary heart disease events (5.1 vs. 7.5 cases per 1000 person-years; hazard ratio, 0.68; 95% CI, 0.50 to 0.93), and stroke (2.5 vs. 3.9 cases per 1000 person-years; hazard ratio, 0.64; 95% CI, 0.41 to 0.99). Lasofoxifene at a dose of 0.25 mg per day, as compared with placebo, was associated with reduced risks of vertebral fracture (16.0 vs. 22.4 cases per 1000 person-years; hazard ratio, 0.69; 95% CI, 0.57 to 0.83) and stroke (2.4 vs. 3.9 cases per 1000 person-years; hazard ratio, 0.61; 95% CI, 0.39 to 0.96) Both the lower and higher doses, as compared with placebo, were associated with an increase in venous thromboembolic events (3.8 and 2.9 cases vs. 1.4 cases per 1000 person-years; hazard ratios, 2.67 [95% CI, 1.55 to 4.58] and 2.06 [95% CI, 1.17 to 3.60], respectively). Endometrial cancer occurred in three women in the placebo group, two women in the lower-dose lasofoxifene group, and two women in the higher-dose lasofoxifene group. Rates of death per 1000 person-years were 5.1 in the placebo group, 7.0 in the lower-dose lasofoxifene group, and 5.7 in the higher-dose lasofoxifene group. CONCLUSIONS: In postmenopausal women with osteoporosis, lasofoxifene at a dose of 0.5 mg per day was associated with reduced risks of nonvertebral and vertebral fractures, ER-positive breast cancer, coronary heart disease, and stroke but an increased risk of venous thromboembolic events. (ClinicalTrials.gov number, NCT00141323.)

Anti-oestrogenic prevention of breast cancer--the make or break point.

Clinical trials have shown that giving anti-oestrogens to healthy women can reduce the early incidence of breast cancer by approximately 40%. However, the large numbers of women treated, compared with the few who get breast cancer, together with the not insignificant toxicity and the unknown long-term clinical benefits and risks, makes this strategy of prevention versus treatment precarious. So how can we improve the odds for the successful use of endocrine chemoprevention?

Breast cancer incidence in postmenopausal women with osteoporosis or low bone mass using arzoxifene.

The Generations trial, a multicenter, placebo-controlled, double-blind trial, compared arzoxifene 20 mg/day and placebo in 9,354 postmenopausal women with osteoporosis (N=5,252) or low bone mass (N=4,102). Primary outcomes were vertebral fracture in the osteoporotic population and invasive breast cancer in all study participants. Here, we report the detailed breast cancer findings from the trial. Breast cancers were detected by annual mammograms and clinical examination. After 48 months follow-up, breast cancer incidence was compared between treatment groups by estrogen receptor (ER) and progesterone receptor (PR) status and baseline risk factors. Baseline breast cancer risk factors, including age, estimated Gail risk, and bone mineral density, were well balanced between treatment groups. A total of 75 breast cancers occurred 53 in the placebo group and 22 in the arzoxifene group (HR 0.41, 95% CI 0.25-0.68, P<0.001). There were 62 invasive breast cancers, 39 identified as invasive ER-positive (placebo 30, arzoxifene 9; HR 0.30, 95% CI 0.14-0.63, P=0.001) and 30 identified as invasive PR-positive (placebo 23, arzoxifene 7; HR 0.30, 95% CI 0.13-0.71, P=0.003). Breast cancer risk reduction with arzoxifene was similar between Gail risk groups (P interaction=0.31) and between low bone mass and osteoporosis groups (P interaction=0.35). Although generally well tolerated, there was a significant increase in venous thromboembolism, vasomotor symptoms, muscle cramps, and some gynecological events with arzoxifene. These findings demonstrate that in this study arzoxifene reduced the risk of ER-positive breast cancer in this population of postmenopausal women with low bone mass or osteoporosis, an effect similar to that seen with other SERMs.

Prevention of breast cancer by newer SERMs in the future.

The selective oestrogen receptor modulators (SERMs) tamoxifen has been shown to reduce the incidence of oestrogen receptor positive breast cancer by about 60 to 70% in healthy high risk women. The oestrogenic effects of tamoxifen caused a beneficial effect of reduced bone loss and fracture risk in postmenopausal women. However there was also significant gynaecological toxicity including an increased risk of endometrial cancer. Further clinical trials have evaluated the newer SERMs raloxifene, arzoxifene and lasofoxifene. The latter has been shown to significantly reduce the incidence of breast cancer, vertebral and non vertebral fractures, major coronary events and stroke with no significant gynaecological toxicity.

Clodronate.

Two early observations about the first generation bisphosphonate, clodronate, suggested that it would likely have clinical utility; specifically, it was a more potent anti-resorptive but a less potent inhibitor of mineralisation than its predecessor etidronate. The known mechanism of action differs from that of the later nitrogen-containing bisphosphonates, as clodronate is metabolised intracellularly to a toxic analog of adenosine triphosphate, AppCCl2p, which causes mitochondrial dysfunction, impaired cellular energy metabolism and osteoclast apoptosis. For pre-clinical studies in a variety of disease models, liposomal clodronate has become the agent of choice for macrophage depletion, for example in a recent study to enhance haematopoietic chimerism and donor-specific skin allograft tolerance in a mouse model. For clinical use, clodronate was developed in oral and injectable formulations; while poorly absorbed from the gastro-intestinal tract, its absorption at 1-3% of the administered dose is approximately three-fold higher than for nitrogen-containing bisphosphonates. Following an early setback due to an erroneous association with toxic adverse events, a number of successful clinical studies have established clodronate, predominantly in its oral formulations, as a highly successful treatment in Paget's disease, hypercalcaemia (benign and malignant), multiple myeloma, and early or metastatic breast cancer. Novel uses in other disease areas, including veterinary use, continue to be explored.

Guidance for the management of breast cancer treatment-induced bone loss: a consensus position statement from a UK Expert Group.

In postmenopausal women, the use of aromatase inhibitors increases bone turnover and induces bone loss at sites rich in trabecular bone at an average rate of 1-3% per year leading to an increase in fracture incidence compared to that seen during tamoxifen use. The bone loss is much more marked in young women with treatment-induced ovarian suppression followed by aromatase inhibitor therapy (average 7-8% per annum). Pre-treatment with tamoxifen for 2-5 years may reduce the clinical significance of the adverse bone effects associated with aromatase inhibitors, particularly if this leads to a shortening in the duration of exposure to an aromatase inhibitor. However, skeletal status should still be assessed at the commencement of aromatase inhibitor therapy. The rate of bone loss in women who experience a premature menopause before the age of 45 or are receiving ovarian suppression therapy is accelerated by the concomitant use of aromatase inhibitors. These patients are considered to be at high risk of clinically important bone loss and should have a baseline dual energy X-ray absorptiometry (DXA) assessment of bone mineral density (BMD). Randomised clinical trials in postmenopausal women indicate that bisphosphonates prevent the bone loss and accelerated bone turnover associated with aromatase inhibitor therapy and are a promising strategy for the prevention and treatment of osteoporosis in this setting. Treatment initiation recommendations are based on a combination of risk factors for osteoporotic fracture and BMD levels. Bisphosphonates, along with a healthy lifestyle and adequate intake of calcium and vitamin D are the treatments of choice to prevent bone loss. Due to the rate of bone loss associated with breast cancer treatments, and uncertainties about the interaction between aromatase inhibitor use and BMD for fracture risk, the threshold for intervention has been set at a higher level than that generally recommended for postmenopausal osteoporosis. Management recommendations have been summarised in two algorithms, one for women experiencing a premature menopause and the other for postmenopausal women requiring adjuvant aromatase inhibitor therapy.

Prevention of breast cancer using SERMs.

The development of breast cancer is dependant in part on oestrogen. Suppression of ovarian function or use of anti-oestrogens will reduce the incidence of breast cancer. Many trials have now been done involving tens of thousands of healthy women evaluating the use of selective oestrogen receptor modulators to reduce the risk of breast cancer in healthy women. Tamoxifen will reduce the early incidence of breast cancer in pre and postmenopausal women by about 40% but causes vasomotor symptoms, thromboembolism and gynaecological toxicity including polyps, endometrial atypia and rarely cancer. In long follow up trials the risk reduction for breast cancer extends beyond the treatment period out to at least 15 years appearing to get larger with time indicating a true long term prevention effect. The toxicity of tamoxifen is for the most part confined to the treatment period. Raloxifene also has similar breast cancer risk reduction activity to tamoxifen but has less toxicity with no evidence of an increased risk of endometrial atypia or cancer. Tamoxifen is licensed for breast cancer risk reduction in the USA and raloxifene has also recently been approved by the FDA for such use.

Tamoxifen-induced anorexia is associated with fatty acid synthase inhibition in the ventromedial nucleus of the hypothalamus and accumulation of malonyl-CoA.

Fatty acid metabolism in the hypothalamus has recently been shown to regulate feeding. The selective estrogen receptor modulator tamoxifen (TMX) exerts a potent anorectic effect. Here, we show that the anorectic effect of TMX is associated with the accumulation of malonyl-CoA in the hypothalamus and inhibition of fatty acid synthase (FAS) expression specifically in the ventromedial nucleus of the hypothalamus (VMN). Furthermore, we demonstrate that FAS mRNA expression is physiologically regulated by fasting and refeeding in the VMN but not in other hypothalamic nuclei. Thus, the VMN appears to be the hypothalamic site where regulation of FAS and feeding converge. Supporting the potential clinical relevance of these observations, reanalysis of a primary breast cancer prevention study showed that obese women treated with TMX gained significantly less body weight over a 6-year period than obese women given placebo. The finding that TMX can modulate appetite through alterations in FAS expression and malonyl-CoA levels suggests a link between hypothalamic sex steroid receptors, fatty acid metabolism, and feeding behavior.

BRCA1/BRCA2 mutation status and analysis of cancer family history in participants of the Royal Marsden Hospital tamoxifen chemoprevention trial.

We have analysed the pedigrees of all 70 women who developed cancer in the Royal Marsden Hospital (RMH) tamoxifen chemoprevention trial, using the Claus model, to assess breast cancer susceptibility heterozygote risk (HR) and screened the entire coding regions of BRCA1 and 2 genes in 62 of these cases. We found a reduced incidence of breast cancers developing on tamoxifen in women who have a lower HR, but not in women with higher HR. There were too few BRCA1/2 mutations (4 cases) to be able to determine the efficacy of tamoxifen by BRCA status. Immunohistochemical analysis showed a significantly lower frequency of median ER (p=0.03) in the cancers developing in tamoxifen-treated patients. These results suggest that tamoxifen is less likely to be effective at reducing breast cancers which are ER negative and also in some individuals at higher HR.

Oral bisphosphonates as adjuvant therapy for operable breast cancer.

Bone is the most common site of metastatic spread from primary operable breast cancer, causing pain, fractures, and hypercalcemia. This spread depends on the release of osteolytic substances by the cancer cells, which activate osteoclasts to cause bone resorption. The osteoclasts also release growth factors that can act back on the cancer cells to activate growth. This vicious circle thereby facilitates the growth of metastases in bone, thus making this a preferential site for relapse. Agents, such as the bisphosphonates, which block osteoclast function, have been shown to reduce the progression of established bone metastases. The oral bisphosphonate clodronate (1,600 mg/d) is effective for treatment of patients with bone metastases. When used as adjuvant therapy, given to patients with operable breast cancer for 2 years, clodronate has been reported to significantly reduce the risk of bone metastases during the 2-year study period [19 clodronate patients versus 35 placebo patients; hazard ratio (HR), 0.546; P=0.03] and 5-year study period (51 clodronate patients versus 73 placebo patients; HR, 0.692; P=0.04) with a significant reduction in mortality (HR, 0.768; P=0.048). This benefit, together with the low toxicity and safety of clodronate, supports its use as additional adjuvant therapy for patients with primary breast cancer. Further, similarly designed trials are under way to establish the optimal duration of therapy, the efficacy in stage I disease, and the relative potential of other bisphosphonates, particularly the more powerful aminobisphosphonates, such as ibandronate and zoledronate.

Advances in treating metastatic bone cancer: summary statement for the First Cambridge Conference.

The First Cambridge Conference on Advances in Treating Metastatic Bone Cancer, a symposium held in Cambridge, Massachusetts, October 28 to 29, 2005, was convened to discuss recent advances and research related to the natural history of bone metastases and skeletal complications, bone cancer biology, treatment of myeloma and other solid tumors, and treatment-induced bone loss. The conference format combined brief presentations with extended periods of discussion. The conclusions reached during the 2-day meeting are summarized in this article and presented in more detail in the individual articles and accompanying discussion sessions that comprise the conference proceedings.

Immunohistochemical Phenotype of Breast Cancer during 25-Year Follow-up of the Royal Marsden Tamoxifen Prevention Trial.

The randomized, double-blinded Royal Marsden Tamoxifen Breast Cancer Prevention Trial in healthy high-risk women started in 1986 and is still blinded. Eligible participants (n = 2,471) were randomly assigned to tamoxifen (20 mg/d) or placebo for 8 years. Analysis in 2006 showed a 30% risk reduction of estrogen receptor (ER)-positive invasive breast cancer mostly in the posttreatment period. Biomarker analysis in this population may identify any subgroup-specific preventive effects tamoxifen. After a median follow-up of 18.4 years, 242 patients had developed invasive cancer, 134 on placebo and 108 on tamoxifen. From these, 180 tissue blocks were available and ER, progesterone receptor (PgR), Ki67, HER2, and EGFR were immunohistochemically analyzed. A 32% reduction in ER+ and PgR+ invasive cancers resulted after 8 years of treatment. Quantitative levels of ER and PgR were lower in the tamoxifen-treated group, significantly so for ER (P = 0.001). These lower ER levels were restricted to the posttreatment period (P = 0.018). Among the ER+ group, there was a similar proportional decrease in PgR+ and PgR- tumors by tamoxifen. The median levels of Ki67 were similar in both arms. The numbers of HER2-positive and EGFR-positive cancers were higher in the tamoxifen arm but not significantly so. In conclusion, the preventive effects of tamoxifen result in reduced ER-positive but not ER-negative tumors and reduced ER expression in the ER-positive cases largely confined to the posttreatment period. Overall reductions in PgR expression are explained by lower frequency of ER-positive cases. Impact on Ki67, HER2, and EGFR was modest. Cancer Prev Res; 10(3); 171-6. ©2017 AACR.

Impact of a Panel of 88 Single Nucleotide Polymorphisms on the Risk of Breast Cancer in High-Risk Women: Results From Two Randomized Tamoxifen Prevention Trials.

Purpose At least 94 common single nucleotide polymorphisms (SNPs) are associated with breast cancer. The extent to which an SNP panel can refine risk in women who receive preventive therapy has not been directly assessed previously. Materials and Methods A risk score on the basis of 88 SNPs (SNP88) was investigated in a nested case-control study of women enrolled in the International Breast Intervention Study (IBIS-I) or the Royal Marsden study. A total of 359 women who developed cancer were matched to 636 controls by age, trial, follow-up time, and treatment arm. Genotyping was done using the OncoArray. Conditional logistic regression and matched concordance indices (mC) were used to measure the performance of SNP88 alone and with other breast cancer risk factors assessed using the Tyrer-Cuzick (TC) model. Results SNP88 was predictive of breast cancer risk overall (interquartile range odds ratio [IQ-OR], 1.37; 95% CI, 1.14 to 1.66; mC, 0.55), but mainly for estrogen receptor-positive disease (IQ-OR, 1.44; 95% CI, 1.16 to 1.79; P for heterogeneity = .10) versus estrogen receptor-negative disease. However, the observed risk of SNP88 was only 46% (95% CI, 19% to 74%) of expected. No significant interaction was observed with treatment arm (placebo IQ-OR, 1.46; 95% CI, 1.13 to 1.87; tamoxifen IQ-OR, 1.25; 95% CI, 0.96 to 1.64; P for heterogeneity = .5). The predictive power was similar to the TC model (IQ-OR, 1.45; 95% CI, 1.21 to 1.73; mC, 0.55), but SNP88 was independent of TC (Spearman rank-order correlation, 0.012; P = .7), and when combined multiplicatively, a substantial improvement was seen (IQ-OR, 1.64; 95% CI, 1.36 to 1.97; mC, 0.60). Conclusion A polygenic risk score may be used to refine risk from the TC or similar models in women who are at an elevated risk of breast cancer and considering preventive therapy. Recalibration may be necessary for accurate risk assessment.

American Society of Clinical Oncology technology assessment on the use of aromatase inhibitors as adjuvant therapy for postmenopausal women with hormone receptor-positive breast cancer: status report 2004.

PURPOSE: To update the 2003 American Society of Clinical Oncology technology assessment on adjuvant use of aromatase inhibitors. RECOMMENDATIONS: Based on results from multiple large randomized trials, adjuvant therapy for postmenopausal women with hormone receptor-positive breast cancer should include an aromatase inhibitor in order to lower the risk of tumor recurrence. Neither the optimal timing nor duration of aromatase inhibitor therapy is established. Aromatase inhibitors are appropriate as initial treatment for women with contraindications to tamoxifen. For all other postmenopausal women, treatment options include 5 years of aromatase inhibitors treatment or sequential therapy consisting of tamoxifen (for either 2 to 3 years or 5 years) followed by aromatase inhibitors for 2 to 3, or 5 years. Patients intolerant of aromatase inhibitors should receive tamoxifen. There are no data on the use of tamoxifen after an aromatase inhibitor in the adjuvant setting. Women with hormone receptor-negative tumors should not receive adjuvant endocrine therapy. The role of other biomarkers such as progesterone receptor and HER2 status in selecting optimal endocrine therapy remains controversial. Aromatase inhibitors are contraindicated in premenopausal women; there are limited data concerning their role in women with treatment-related amenorrhea. The side effect profiles of tamoxifen and aromatase inhibitors differ. The late consequences of aromatase inhibitor therapy, including osteoporosis, are not well characterized. CONCLUSION: The Panel believes that optimal adjuvant hormonal therapy for a postmenopausal woman with receptor-positive breast cancer includes an aromatase inhibitor as initial therapy or after treatment with tamoxifen. Women with breast cancer and their physicians must weigh the risks and benefits of all therapeutic options.

Prevention of breast cancer using selective oestrogen receptor modulators (SERMs).

Placebo controlled trials in over 25,000 women showed that tamoxifen reduced breast cancer risk by about 40% and osteoporotic fracture risk by about 32%. Similarly placebo controlled trials in nearly 18,000 women showed that raloxifene reduced breast cancer risk by 44-72% and osteoporotic fractures risk by 30-50%. A direct comparison of tamoxifen with raloxifene showed similar risk reduction for breast cancer and osteoporotic fractures with less toxicity for raloxifene.

The effect of the stromal component of breast tumours on prediction of clinical outcome using gene expression microarray analysis.

INTRODUCTION: The aim of this study was to examine the effect of the cellular composition of biopsies on the error rates of multigene predictors of response of breast tumours to neoadjuvant adriamycin and cyclophosphamide (AC) chemotherapy. MATERIALS AND METHODS: Core biopsies were taken from primary breast tumours of 43 patients prior to AC, and subsequent clinical response was recorded. Post-chemotherapy (day 21) samples were available for 16 of these samples. Frozen sections of each core were used to estimate the proportion of invasive cancer and other tissue components at three levels. Transcriptional profiling was performed using a cDNA array containing 4,600 elements. RESULTS: Twenty-three (53%) patients demonstrated a 'good' and 20 (47%) a 'poor' clinical response. The percentage invasive tumour in core biopsies collected from these patients varied markedly. Despite this, agglomerative clustering of sample expression profiles showed that almost all biopsies from the same tumour aggregated as nearest neighbours. SAM (significance analysis of microarrays) regression analysis identified 144 genes which distinguished high- and low-percentage invasive tumour biopsies at a false discovery rate of not more than 5%. The misclassification error of prediction of clinical response using microarray data from pre-treatment biopsies (on leave-one-out cross-validation) was 28%. When prediction was performed on subsets of samples which were more homogeneous in their proportions of malignant and stromal cells, the misclassification error was considerably lower (8%-13%, p < 0.05 on permutation). CONCLUSION: The non-tumour content of breast cancer samples has a significant effect on gene expression profiles. Consideration of this factor improves accuracy of response prediction by expression array profiling. Future gene expression array prediction studies should be planned taking this into account.