Aju Mathew

Sensitive Detection of Mono- and Polyclonal ESR1 Mutations in Primary Tumors, Metastatic Lesions, and Cell-Free DNA of Breast Cancer Patients

Purpose: Given the clinical relevance of ESR1 mutations as potential drivers of resistance to endocrine therapy, this study used sensitive detection methods to determine the frequency of ESR1 mutations in primary and metastatic breast cancer, and in cell-free DNA (cfDNA). Experimental Design: Six ESR1 mutations (K303R, S463P, Y537C, Y537N, Y537S, D538G) were assessed by digital droplet PCR (ddPCR), with lower limits of detection of 0.05% to 0.16%, in primary tumors (n = 43), bone (n = 12) and brain metastases (n = 38), and cfDNA (n = 29). Correlations between ESR1 mutations in metastatic lesions and single (1 patient) or serial blood draws (4 patients) were assessed. Results: ESR1 mutations were detected for D538G (n = 13), Y537S (n = 3), and Y537C (n = 1), and not for K303R, S463P, or Y537N. Mutation rates were 7.0% (3/43 primary tumors), 9.1% (1/11 bone metastases), 12.5% (3/24 brain metastases), and 24.1% (7/29 cfDNA). Two patients showed polyclonal disease with more than one ESR1 mutation. Mutation allele frequencies were 0.07% to 0.2% in primary tumors, 1.4% in bone metastases, 34.3% to 44.9% in brain metastases, and 0.2% to 13.7% in cfDNA. In cases with both cfDNA and metastatic samples (n = 5), mutations were detected in both (n = 3) or in cfDNA only (n = 2). Treatment was associated with changes in ESR1 mutation detection and allele frequency. Conclusions: ESR1 mutations were detected at very low allele frequencies in some primary breast cancers, and at high allele frequency in metastases, suggesting that in some tumors rare ESR1-mutant clones are enriched by endocrine therapy. Further studies should address whether sensitive detection of ESR1 mutations in primary breast cancer and in serial blood draws may be predictive for development of resistant disease. Clin Cancer Res; 22(5); 1130–7. ©2015 AACR. See related commentary by Gu and Fuqua, p. 1034

Bisphosphonates in breast cancer

Bisphosphonates are osteoclast inhibitors, currently being used in oncology to prevent or delay bone morbidity in cancer. Oral and intravenous formulations of bisphosphonates have been found to be efficacious in preventing skeletal‐related events such as bone pain, pathologic fractures, spinal cord compression and hypercalcemia of malignancy, in patients with bone metastatic breast cancer. Bisphosphonates are also used to prevent bone loss associated with anti‐estrogen therapy using aromatase inhibitors. In addition to its role in preventing bone resorption, several pre‐clinical studies have noted an anti‐tumor role as well. Recent research effort has particularly focused on investigating an adjuvant role for bisphosphonates in early breast cancer. Recently, few randomized trials have found a beneficial effect for adjuvant use of the aminobisphosphonate, zoledronate, in older patients who are post‐menopausal. This review article will summarize the various clinical studies investigating the role of bisphosphonates in breast cancer.

Recurrent hyperactive ESR1 fusion proteins in endocrine therapy-resistant breast cancer

Abstract Background Estrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer. Patients and methods To identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287–395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots. Results We identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3′ partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations. Conclusions Collectively, these data indicate that N-terminal ESR1 fusions involving exons 6–7 are a recurrent driver of endocrine therapy resistance and are impervious to ER-targeted therapies.

Adjuvant endocrine therapy for premenopausal women with hormone-responsive breast cancer.

Multiple strategies for endocrine treatment of premenopausal women with hormone-responsive breast cancer have been assessed and results have been presented over the last two years. These include tamoxifen for 5-10 years (ATLAS and aTTom), tamoxifen for 5 years followed by aromatase inhibitor (AI) for 5 years for women who have become postmenopausal (MA-17); ovarian ablation (OA) by surgery (EBCTCG overview); ovarian function suppression (OFS) by LHRH agonist (LHRH agonist meta-analysis); or combinations of approaches including OFS plus tamoxifen or AI (SOFT, TEXT, ABCSG 12 and E3193). Many of these trials have taken place in the backdrop of (neo)adjuvant chemotherapy which can confound interpretation because such therapy can suppress ovarian function either transiently or permanently. Nonetheless these trials suggest in aggregate that 10 years of tamoxifen are better than 5 years and that a program of extended adjuvant therapy of tamoxifen for 5 years followed by aromatase inhibitor for 5 years is effective for suitable candidates. The SOFT and E3193 trials do not show a major advantage for use of OFS + tamoxifen compared to tamoxifen alone. The joint SOFT/TEXT analysis and ABCGS12 trials both suggest that outcomes can be excellent with the use of combined endocrine therapy alone in properly selected patients but give conflicting results with regard to potential benefits for OFS + AI compared with OFS + tamoxifen. Further work will be needed to ascertain long-term outcomes, identify factors that predict who will benefit from extended adjuvant endocrine therapy, and assess role of OFS by medical or surgical means. It is clear, however, that endocrine therapy is a critical part of the adjuvant regimen for most premenopausal women with hormone-responsive breast cancer, and a subset of these women with luminal A-type tumors can be safely treated with endocrine therapy alone.

Breast cancer: Zoledronic acid--more than just a bone drug.

Several clinical trials have investigated the antitumour effect of bisphosphonates when used as adjuvant treatment for early stage breast cancer. Among these, the results of the AZURE trial, although negative,highlight the potential benefit of treatment with zoledronic acid in postmenopausal women with high-risk early stage breast cancer.

Bisphosphonates, bone, and breast cancer recurrence

Bisphosphonates reduce bone turnover by inhibiting osteoclast maturation and function, and are important in the prevention of age-related osteoporosis and bone fracture, in the prevention of complications of bone metastases, and in the prevention of osteopenia and osteoporosis resulting from adjuvant aromatase inhibitor therapy of breast cancer. Over 125 years ago, in The Lancet, Paget hypothesised that the “soil” in which a tumour resides would be important for tumour propagation and growth. Interruption of the interaction of tumour micrometastases with their microenvironmental “soil” is now a subject of intense investigation. Bone is an active microenvironment, and, in bone with high turnover, excess osteoclastic activity could potentially lead to excess production of growth factors, which could aff ect survival of micrometastases. Bisphosphonates, by reducing osteoclast activity, could in theory reduce expression of these factors, thereby preventing establishment of micrometastatic disease. Clinical trials of bisphosphonates as adjuvant therapy for breast cancer have had mixed results. Clodronate, an oral fi rst-generation bisphosphonate, showed a disease-free survival benefi t versus placebo in one large randomised trial, but not in another. An early trial of zoledronic acid, a more powerful third-generation aminobisphosphonate, added to adjuvant aromatase inhibitor therapy for postmenopausal women to prevent bone loss, showed a non-signifi cant improve ment in disease-free survival, a secondary endpoint. Larger trials comparing zoledronic acid to no therapy in postmenopausal women, or in premenopausal women made menopausal with gonadotropin-releasing hormone agonists, showed signifi cant disease-free survival benefi ts, but no benefi t was seen in a large randomised trial of both premenopausal and postmenopausal women. In The Lancet, the Early Breast Cancer Trialists’ Collaborative Group presents a meta-analysis of randomised trials of bisphosphonates as adjuvant systemic therapy for breast cancer. This meta-analysis is comprised of individual patient data derived from randomised adjuvant bisphosphonate trials in breast cancer done over the past 20 years. The analysis received data on 18 766 women (18 206 in randomised trials of 2–5 years of adjuvant bisphosphonate vs control), with a median follow-up of 5·6 years, 3453 fi rst recurrences, and 2106 deaths. For all patients, there were borderline signifi cant reductions with the addition of bisphosphonates at 10 years for distant recurrence (20·4% vs 21·8%, rate ratio [RR]=0·92, 95% CI 0·85–0·99; 2p=0·03), bone recurrence (7·8% vs 9·0%, RR=0·83, 0·73–0·94; 2p=0·004), breast cancer mortality (16·6% vs 18·4%, RR=0·91, 0·83–0·99; 2p=0·04), and all-cause mortality (20·8% vs 22·3%, RR=0·92, 0·85–1·00; 2p=0·06). 8 Davies C, Pan H, Godwin J, et al. Long-term eff ects of continuing adjuvant tamoxifen to 10 years versus stopping at 5 years after diagnosis of oestrogen receptor-positive breast cancer: ATLAS, a randomised trial. Lancet 2013; 381: 805–16. 9 Goss PE, Ingle JN, Martino S, et al. Randomized trial of letrozole following tamoxifen as extended adjuvant therapy in receptor-positive breast cancer: updated fi ndings from NCIC CTG MA.17. J Natl Cancer Inst 2005; 16: 707–15. 10 Sestak I, Dowsett M, Zabaglo L, et al. Factors predicting late recurrence for estrogen receptor-positive breast cancer. J Natl Cancer Inst 2013; 105: 1504–11. 11 Viale G, Regan MM, Dell’Orto P, et al. Which patients benefi t most from adjuvant aromatase inhibitors? Results using a composite measure of prognostic risk in the BIG 1-98 randomized trial. Ann Oncol 2011; 22: 2201–07. 12 Gnant M, Mlineritsch B, Schippinger W, et al. Endocrine therapy plus zoledronic acid in premenopausal breast cancer. N Engl J Med 2009; 360: 679–91. 13 Francis PA, Regan MM, Fleming GF, et al. Adjuvant ovarian suppression in premenopausal breast cancer. N Engl J Med 2015; 372: 436–46. 14 Aiello Bowles EJ, Boudreau DM, Chubak J, et al. Patient-reported discontinuation of endocrine therapy and related adverse eff ects among women with early-stage breast cancer. J Oncol Pract 2012; 8: 149–57. 15 Hadji P, Jackisch C, Bolten W, et al. COMPliance and Arthralgia in Clinical Therapy: the COMPACT trial, assessing the incidence of arthralgia, and compliance within the fi rst year of adjuvant anastrozole therapy. Ann Oncol 2014; 25: 372–77.

Can Circulating Tumor Cells Predict Resistance in Metastatic Breast Cancer

Circulating tumor cell (CTC) enumeration provides prognostic but not predictive information for chemotherapy in metastatic breast cancer. Because CTC measurement is reproducible and allows molecular profiling, an assay to assess endocrine resistance was developed. Whether this CTC-based assay can be used to reliably select endocrine therapy must be tested. Clin Cancer Res; 21(11); 2421–3. ©2015 AACR. See related article by Paoletti et al., p. 2487

Decreased risk of breast cancer associated with oral bisphosphonate therapy

Preclinical studies and adjuvant trials using bisphosphonates have found them to have an antitumor effect. Although major advances have been made in chemoprevention strategies with selective estrogen receptor modulators and aromatase inhibitors, their use has been fraught with significant adverse effects such as venous thromboembolic events and an increased risk for endometrial cancer. In this context, several recent observational studies have investigated a chemoprevention role for oral bisphosphonates in decreasing risk for breast cancer. This review will aim to summarize these studies and present a critical evaluation of the association between oral bisphosphonate use and breast cancer risk reduction.

HIV-1 prevalence in young adults in south India

The article by Rajesh Kumar and colleagues (April 8, p 1164) begins with misrepresentations. Although the title speaks of trends in HIV-1 in young adults of south India, the southern state of Kerala was omitted from the analysis. Paradoxically, Maharashtra, always represented as a western Indian state, was included. We do not understand the logic behind redefi ning the regional geography of India. The omission of Kerala is signifi cant because Kerala stands out as a lowprevalence state. Its inclusion would have slightly reduced the initial prevalence and attenuated the slope of the decline. The deliberate inclusion of Maharashtra serves the purpose of increasing the credibility of the conclusion in the paper. We also note that the three states in north India—Mizoram, Nagaland, and Manipur—excluded by the authors are high-prevalence states that do not fi t into the pattern in north India. The title would have done more justice to the article if “south India” had been replaced by “selected states of India”. An examination of the crude prevalence of HIV-1 in antenatal clinics indicates no consistent trend in the south Indian states (fi gure). It is quite clear that the steady decline in the prevalence of HIV-1 among young pregnant women is mainly driven by prevalence in Tamil Nadu, followed by Maharashtra (see Kumar and colleagues’ reference 1 and webfi gure 1). The signifi cant decline of HIV-1 prevalence in Tamil Nadu seems to counteract the apparent lack of trend in other south Indian states. If Kerala was included and Maharashtra excluded (fi gure), one would conclude that the decline in HIV-1 prevalence in south India is a Tamil Nadu phenomenon. The aggregate analysis suggesting that the rate of infection in south India is declining among young women is likely to generate an air of optimism leading to complacency. The need of the hour is to establish more antenatal clinics in every Indian state and generate reliable state-level estimates of HIV-1 prevalence. Until that time, broad generalisations based on deliberate selection of states seem premature.

Bisphosphonates in Early Breast Cancer

Abstract Bisphosphonates are osteoclast inhibitors used to treat osteoporosis. In oncology, bisphosphonates are used to prevent or treat skeletal-related events such as pathologic fractures and hypercalcemia of malignancy. In early breast cancer, these drugs have been used to prevent or treat bone loss associated with the use of aromatase inhibitors. It is thought that bisphosphonates also exert an anticancer effect by altering the tumor microenvironment in the bone. Individual clinical trials of adjuvant bisphosphonates were inconclusive, although few of these trials generated the hypothesis that such an anticancer effect is limited to postmenopausal women. An individual patient-level data meta-analysis of 26 clinical trials including 18,766 women with early breast cancer, treated with bisphosphonates or control, found borderline significant reductions with the use of bisphosphonates for distant recurrence, bone recurrence, breast cancer mortality, and all-cause mortality. A subgroup analysis noted lack of benefit in premenopausal women. In postmenopausal women, there were significant reductions in bone recurrences and breast cancer mortality. Therefore all postmenopausal women (natural or induced), especially those on antiestrogen therapy, should be considered for adjuvant bisphosphonate treatment, and decision to treat should be individualized.