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CDK4/6 inhibition partly overcomes NF1 loss in T47D cells

Supplementary Table 1. Targets of The Breast NGS v1.0 capture panel. Supplementary Table 2. Gene list of NanoString nCounter{trade mark, serif} custom codeset. Supplementary Table 3. Gene list of Human Estrogen Receptor RT2 Profiler PCR Array (330231, PAHS-005ZA-24, Qiagen) Supplementary Table 4. Lines of treatment of patients with ESR1 and NF1 mutations. Supplementary Table 5. Disease free survival for mutated genes with incidence {greater than or equal to}5% presented by HR+/HER2-, HER2+ and TNBC subtype. Supplementary Table 6. Advanced overall survival for mutated genes with incidence {greater than or equal to}5% presented by HR+/HER2-, HER2+ and TNBC subtype

Supplementary Figure Legend

Genetic profile of advanced breast cancer subtypes

NCOR expression following NF1 loss

Purpose:Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC.Experimental Design:Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients.Results:We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant.Conclusions:Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.

NCOR expression in NF1 mutated cancers

Genetic profile of ER positive primary breast cancers that 'switch' to triple negative recurrent advanced breast cancer

Background: HER2 mutations occur in approximately 3% of HER2 non-amplified cancers and include missense substitutions and indels within the tyrosine kinase domain resulting in oncogenic HER2 activation. Neratinib, an irreversible EGFR, HER2 and ERBB4 tyrosine kinase inhibitor, has demonstrated activity in pre-clinical HER2 mutant models, and in a prior phase I trial with HER2 mutations identified through tumour testing. The plasmaMATCH trial Cohort B assessed the efficacy of neratinib in BC patients with a HER2 mutation identified in ctDNA testing. Methods: The plasmaMATCH trial was an open-label, multi-centre, multi-cohort platform trial, consisting of ctDNA testing in ~1000 patients with advanced BC. Patients with a HER2 mutation identified via ctDNA testing were registered to Cohort B. Patients were treated with 240mg neratinib once daily. Patients with ER positive BC were also treated with fulvestrant 500mg intramuscularly on Cycle 1 Days 1 and 15, and Cycle 2 onwards every 28 days. The primary endpoint for Cohort B was confirmed objective response rate as defined by RECIST v1.1. Using a single stage A’Hern design with a target response rate of 25%, unacceptable response rate of 5%, alpha=5% and power=80%, at least 3 responses out of 16 evaluable patients were required to infer efficacy. Results: Following ctDNA testing, 21 patients enrolled in Cohort B (58% of patients with HER2 mutations identified in ctDNA testing). Eighteen (86%) were ER positive, 2 (10%) were HER2 amplified, and 18 (86%) had visceral metastases. The most common HER2 mutations detected in baseline plasma were L755S (47.6%), V777L (19%), and S310F (14.3%). In the 20 evaluable patients, confirmed response rate was 25.0% (95%CI 8.7-49.1%, 5/20) (first 16 evaluable patients: 4/16, 25.0% (95%CI 7.3-52.4)). One patient had a complete response, ongoing at 29 months duration, and three additional patients had unconfirmed partial responses. Median progression free survival was 5.4 months (IQR 3.4-9.1) and median duration of response was 5.7 months (IQR 3.7-9.7 months) with 3 patients continuing on treatment. The most common clinically significant grade 3 or 4 adverse events were diarrhoea (20%) and hypertension (15%). Conclusions: Neratinib, with or without fulvestrant, was active in advanced BC patients with HER2 mutations identified in ctDNA testing, meeting the pre-specified threshold for efficacy. Citation Format: Andrew M Wardley, Lucy Kilburn, Sarah Kernaghan, Iain Macpherson, Richard D Baird, Rebecca Roylance, Peter Stephens, Olga Oikonomidou, Jeremy P Braybrooke, Mark Tuthill, Jacinta Abraham, Matthew C Winter, Belinda Kingston, Katie Wilkinson, Judith M Bliss, Alistair Ring, Nicholas Turner, on behalf of the plasmaMATCH Trial Management Group. Results from plasmaMATCH trial treatment cohort B: A phase II trial of neratinib plus fulvestrant in ER positive breast cancer or neratinib alone in ER negative breast cancer in patients with a HER2 mutation identified via ctDNA screening (CRUK/15/010) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-07.

Background: Circulating tumour DNA (ctDNA) testing may provide a more current assessment of the genetic profile of advanced breast cancer (BC) compared with analysis of the primary tumour, with repeat advanced disease biopsy conducted infrequently in routine clinical practice. The plasmaMATCH trial was designed to assess the clinical utility of using ctDNA testing to select patients for targeted therapies. Methods: The plasmaMATCH trial was an open-label, multi-centre, multi-cohort platform trial, consisting of ctDNA testing in ~1000 patients with advanced BC, with patients recruited into four parallel treatment cohorts with therapies matched to mutations identified in ctDNA (A: ESR1 mutation - extended-dose fulvestrant 500mg every 2 weeks, B: HER2 mutation - neratinib +/- fulvestrant (standard dosing), C: AKT1 in ER positive BC -capivasertib + fulvestrant (standard dosing), D: AKT1 in ER negative BC or PTEN inactivating mutation - capivasertib). A fifth cohort (E) recruited patients with triple negative BC with no actionable mutation to receive olaparib + AZD6738, and will be reported separately. Each cohort had a specific phase II single arm design. ctDNA testing was conducted with two technologies: digital droplet PCR (ddPCR) at a central laboratory prospectively in all patients, and error corrected sequencing with Guardant360 prospectively from part-way through recruitment and retrospectively for the remaining patients. Tumour sequencing from an advanced disease biopsy was conducted retrospectively, not influencing cohort entry. The primary endpoint for Cohorts A-D is confirmed objective response rate by RECIST v1.1. Secondary endpoints include clinical benefit rate, progression-free survival, safety and frequency of mutations identified in ctDNA screening. Results: Entry into ctDNA testing for Cohorts A-D was closed on 26/Apr/2019 with 1044 patients registered. ctDNA screening results were received for 1033 patients (99%), with 142 patients entered into Cohorts A-D (A 84, B 21, C 18, D 19). Agreement between ctDNA digital PCR and sequencing results was high (individual gene level agreement 95.5%-99.4%, kappa 0.89-0.93). Predefined efficacy criteria were met in Cohorts B (neratinib for HER2 mutations) and C (capivasertib for AKT mutations), with exploratory analysis of Cohort D identifying activity of capivasertib in AKT1 mutations (Table 1). Efficacy criteria were not met in Cohort A (extended-dose fulvestrant for ESR1 mutations). Adverse events were consistent with prior reports, with extended-dose fulvestrant well tolerated. Table 1: Efficacy results from plasmaMATCHMutationCohortConfirmed response rate, % (95%CI; n/N)Median PFS (IQR), monthsAll patientsFirst 16 evaluable patients*ESR1A8.1% (3.0-16.8; 6/74)-2.2 (1.7-5.3)HER2B25.0% (8.7-49.1; 5/20)25.0% (7.3-52.4; 4/16)5.4 (3.4-9.1)AKT1C22.2% (6.4-47.6; 4/18)18.8% (4.0-45.6; 3/16)10.2 (3.2-18.2)AKT basketD10.5% (1.3-33.1; 2/19)12.5% (1.6-38.3; 2/16)3.4 (1.8-5.5)AKT133.3% (4.3-77.7; 2/6)--PTEN0 % (0/13)--*Predefined cohort efficacy thresholds for response were set: 13/78 (A); 3/16 (B, C, D) Conclusion: Circulating tumour DNA testing offers accurate tumour genotyping, sufficient for routine clinical practice. This approach can be used to identify patients with rare HER2 and AKT1 mutations, who have clinically relevant response rates with matched targeted therapies. Citation Format: Nicholas Turner, Belinda Kingston, Lucy Kilburn, Sarah Kernaghan, Andrew M Wardley, Iain Macpherson, Richard D Baird, Rebecca Roylance, Peter Stephens, Olga Oikonomidou, Jeremy P Braybrooke, Mark Tuthill, Jacinta Abraham, Matthew C Winter, Hannah Bye, Michael Hubank, Claire Snowdon, Daniel Rea, David Cameron, Abeer Shaaban, Katrina Randle, Katie Wilkinson, Laura Moretti, Judith M Bliss, Alistair Ring, on behalf of the plasmaMATCH Trial Management Group. Results from the plasmaMATCH trial: A multiple parallel cohort, multi-centre clinical trial of circulating tumour DNA testing to direct targeted therapies in patients with advanced breast cancer (CRUK/15/010) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS3-06.

Background: Circulating tumor DNA (ctDNA) is found in the plasma of over 90% of patients with advanced breast cancer (BC). ctDNA analysis can establish the current genomic profile of an individual’s cancer and identify potentially targetable mutations. The genomic landscape of advanced BC and clinical associations has yet to be fully defined. This analysis describes the genomic landscape of ctDNA in patients screened for the UK plasmaMATCH study. Methods: The plasmaMATCH trial was an open-label, multi-centre, multi-cohort platform trial, consisting of ctDNA testing in ~1000 patients with advanced BC. The trial consists of parallel treatment cohorts (Cohorts A-E) with therapies matched to mutations identified in ctDNA testing. The genomic landscape of ctDNA in advanced BC was prospectively analysed for hotspot PIK3CA,AKT1, HER2 and ESR1 mutations using digital droplet PCR (ddPCR, Bio-Rad), and using an error-corrected 73-gene targeted panel (Guardant360, Guardant Health) prospectively from part-way through the trial, and retrospectively for the remaining patients. Results: Entry into ctDNA testing for Cohorts A-D was closed on 26/Apr/2019, at which time 1044 patients had been registered and 1025 tested with ddPCR. 800 patients had targeted panel ctDNA sequencing (364 prospective, 436 retrospective) (Table 1). Targeted sequencing identified a somatic alteration in 92.9% of patients (743/800). The most frequent mutations were TP53 (44.1%), PIK3CA (34.9%), ESR1 (33.1%), GATA3 (11.0%) and ARID1A (7.8%). ESR1 (33.1%) and KRAS (4.1%) alterations occurred at higher frequency in ctDNA compared to a metastatic tissue sequencing series (q Genomic AlterationddPCR*Targeted Panel**All % n = 1025HR+ HER2-% n = 658HR+ HER2+ % n = 62HR- HER2+ % n = 36TNBC % n = 172All % n = 800HR+ HER2- % n = 515HR+ HER2+ % n = 48HR- HER2+ % n = 26TNBC % n = 136PIK3CA mutation25.829.933.919.49.334.939.041.734.614.7ESR1 mutation27.737.819.40033.143.522.900.7AKT1 mutation4.24.01.604.75.05.22.104.4ERBB2 mutation2.73.208.31.26.46.612.515.42.2PTEN mutationNANANANANA6.98.54.23.84.4ERBB2 amplificationNANANANANA5.81.931.357.71.5MSI highNANANANANA1.10.42.101.5* Phenotype data based on 928 patients with known phenotype tested by ddPCR for targetable hotspot mutations.** Phenotype data based on 725 patients with known phenotype sequenced by targeted panel. All pathogenic mutations are shown, including mutations not tested by ddPCR. Citation Format: Belinda Kingston, Hannah Bye, Michael Hubank, Giselle Walsh, Claire Swift, Matthew Beaney, Lucy Kilburn, Sarah Kernaghan, Andrew M Wardley, Iain Macpherson, Richard D Baird, Rebecca Roylance, Katie Wilkinson, Isaac Garcia-Murillas, Judith M Bliss, Nicholas Turner, Alistair Ring, on behalf of the plasmaMATCH Trial Management Group. The genomic landscape of breast cancer based on ctDNA analysis: Data from the plasmaMATCH trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS3-07.

Background: Activation of the AKT pathway can result from diverse rare genetic events, including mutation of AKT1, AKT2/3 E17K, and through inactivating mutation or homozygous deletion of PTEN. AKT1 mutations and genetic loss of PTEN are associated with activation of AKT signalling and selective sensitivity to AKT inhibition in pre-clinical models. Capivasertib is a potent catalytic inhibitor of AKT1, AKT2 and AKT3 with activity both in vivo and in the clinic. The plasmaMATCH trial assessed the efficacy of capivasertib in BC patients with rare AKT activating mutations. Methods: The plasmaMATCH trial is an open-label, multi-centre, multi-cohort platform trial, consisting of ctDNA testing in ~1000 patients with advanced BC. Patients with AKT1 mutations in ER negative BC; or AKT2/3 E17K mutations, PIK3R1 or PTEN inactivating mutations orhomozygous deletion of PTEN in both ER positive and ER negative BC were recruited. Mutations were identified in ctDNA testing in plasmaMATCH or in prior tumour sequencing. Patients were treated with capivasertib 480mg BID 4 days on - 3 days off. The primary endpoint for Cohort D is confirmed objective response rate as defined by RECIST v1.1. Using a single stage A’Hern design with a target response rate of 25%, unacceptable response rate of 5%, alpha=5% and power=80%, at least 3 responses out of 16 evaluable patients were required to infer efficacy for capivasertib. Results: In total 19 patients were recruited in Cohort D, 12 following ctDNA testing and 7 on tumour testing. Fifteen (79%) were ER positive, all were HER2 non-amplified, and 14 (74%) had visceral metastases. Mutations were AKT1 E17K (5 patients), AKT1 L52R (1), PTEN inactivating mutation (12), and PTEN homozygous deletion (1). All patients were evaluable with overall confirmed response rate of 10.5% (95%CI 1.3-33.1%, 2/19) (first 16 evaluable patients: 2/16, 12.5% (95%CI 1.6-38.3)). Two further patients had unconfirmed responses. Median progression free survival was 3.4 months (IQR 1.8-5.5 months) and median duration of response 3.9 months (IQR 3.7-4.2 months) with 1 patient continuing treatment. In patients with AKT1 mutations there was a 33% response rate (2/6) with 2 further unconfirmed responses. There were no responses in patients with PTEN mutations (0/13). The most common clinically significant grade 3 or 4 adverse events were rash (26%), hypertension (11%), transaminase increase (11%) and vomiting (11%). Conclusions: Capivasertib monotherapy did not meet pre-specified criteria for efficacy in this group of patients with a range of AKT pathway activating mutations. In exploratory analysis, capivasertib was active in patients with AKT1 mutations. Citation Format: Richard D Baird, Lucy Kilburn, Sarah Kernaghan, Andrew M Wardley, Iain Macpherson, Rebecca Roylance, Peter Stephens, Olga Oikonomidou, Jeremy P Braybrooke, Mark Tuthill, Jacinta Abraham, Matthew C Winter, Belinda Kingston, Katie Wilkinson, Nicholas Turner, Alistair Ring, Judith M Bliss, on behalf of the plasmaMATCH Trial Management Group. Results from plasmaMATCH trial treatment cohort D: A phase II trial of capivasertib in patients with an AKT activation basket mutation identified via ctDNA testing or tumour sequencing (CRUK/15/010) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-14.

Background: AKT1 mutation occurs in approximately 3% of breast cancer (BC), enriched in advanced BC. The AKT1 E17K mutation results in constitutive activation of AKT1, associated with sensitivity to AKT inhibitor capivasertib in pre-clinical models, and in a prior phase I trial with AKT1 mutations identified through tumour testing. The plasmaMATCH trial Cohort C assessed the efficacy of capivasertib and fulvestrant in ER positive BC patients with an AKT1 mutation in ctDNA testing. Methods: The plasmaMATCH trial was an open-label, multi-centre, multi-cohort platform trial, consisting of ctDNA testing in ~1000 patients with advanced BC. Patients with an AKT1 mutation identified via ctDNA testing were registered to Cohort C. Patients were treated with capivasertib 400mg BID 4 days on - 3 days off, plus fulvestrant 500mg intramuscularly on Cycle 1 Days 1 and 15, and Cycle 2 onwards every 28 days. The primary endpoint for Cohort C was confirmed objective response rate as defined by RECIST v1.1. Using a single stage A’Hern design with a target response rate of 25%, unacceptable response rate of 5%, alpha=5% and power=80%, at least 3 responses out of 16 evaluable patients were required to infer efficacy. Results: Following ctDNA testing, 18 patients enrolled in Cohort C (42% of patients with AKT1 mutations identified in ctDNA testing). All were ER positive, 1 (5.6%) was HER2 amplified, and 17 (94%) had visceral metastases. Mutation was AKT1 E17K in 17 patients and AKT1 L52R in 1 patient. All patients were evaluable with a confirmed response rate of 22.2% (95%CI 6.4-47.6%, 4/18) (first 16 evaluable patients: 3/16, 18.8% (95%CI 4.0-45.6)). A further 4 patients had an unconfirmed partial response. Median progression free survival was 10.2 months (IQR 3.2-18.2 months) and the median duration of response was 7.5 months (IQR 4.1-9.8 months) with 4 patients continuing on treatment. The most common clinically significant grade 3 or 4 adverse events were fatigue (22%), rash (17%), diarrhoea (11%) and hyperglycaemia (11%). Conclusions: Capivasertib plus fulvestrant was active in patients with ER positive breast cancer and AKT1 mutations identified in ctDNA testing, meeting the pre-specified threshold for efficacy Citation Format: Rebecca Roylance, Lucy Kilburn, Sarah Kernaghan, Andrew M Wardley, Iain Macpherson, Richard D Baird, Peter Stephens, Olga Oikonomidou, Jeremy P Braybrooke, Mark Tuthill, Jacinta Abraham, Matthew C Winter, Belinda Kingston, Katie Wilkinson, Alistair Ring, Judith M Bliss, Nicholas Turner, on behalf of the plasmaMATCH Trial Management Group. Results from plasmaMATCH trial treatment cohort C: A phase II trial of capivasertib plus fulvestrant in ER positive breast cancer patients with an AKT1 mutation identified via ctDNA screening (CRUK/15/010) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-11.

Purpose: Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC. Experimental Design: Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients. Results: We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant. Conclusions: Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.

Background Palbociclib is approved in 1st line for hormone receptor (HR)-positive HER2-negative advanced breast cancer (ABC). A Compassionate Access Program (CAP) previously allowed patients to receive it in 4th line. However, Palbociclib has not been specifically tested in this population. We aimed to determine the safety and efficacy profile of Palbociclib within the CAP across ten institutions in the United Kingdom. Methods We retrospectively identified HR-positive HER2-negative ABC patients on the Palbociclib CAP between December 2015 and September 2017. Demographics, disease characteristics, prior treatments, blood tests, toxicities, treatment delays and responses were recorded. Simple statistics, Fisher's exact test, chi-squared method and Cox regression were used as appropriate. Results 118 patients identified had median age of 59 (32-82). 97 (82.20%) were postmenopausal and 109 (92.37%) had ECOG Performance Status 0-1. 96 (81.36%) had visceral involvement, 19 (16.10%) bony only and 15 (12.71%) visceral only disease. Patients received a median 5 (range 3-11) prior lines of treatment and 3 (range 0-8) prior chemotherapy lines. 105 patients (89.74%) developed neutropenia (grade ≥3 in 59 [56.19%]). 6 experienced febrile neutropenia (5.13%). 57 (48.72%) had a dose reduction, down to 100mg in 48 (41.03%) and 75mg in 9 (7.69%) due to hematologic toxicity in 46 (80.70%). Dose delays were in median 7 days long (range: 0-56). Palbociclib was discontinued due to disease progression in 97 (82.91%) and to toxicity in 5 (4.27%). Grade 3 neutropenia occurred in 45 patients (67.16%) who received ≥3 and in 22 (32.84%) who received Palbociclib produced clinical benefit rate (CBR) of 47.52% and overall response rate of 15.84% in 101 patients assessed. CBR was 45.45% with previous endocrine treatment (ET) progression-free survival (PFS) Overall, median PFS was 4.5 months (95% CI 3.7-5.9). The PFS seen in different subgroups showed no impact in relation to the number of lines of prior chemotherapy ( Median OS was 15.8 months (95% CI 13.3-18.7) for the whole cohort and it was greater in patients who derived longer PFS from prior ET (≥6 months: 18.1 months [95% CI 13.0-NR]; Conclusions To date, this is the most extensive analysis of Palbociclib outcomes in ≥4th-line setting. In this heavily pretreated population clinical benefit was confirmed particularly for endocrine-sensitive disease and predominantly involving the bones and in earlier lines of treatment. Grade ≥3 neutropenia rates were similar to PALOMA trials, but the higher incidence of febrile neutropenia need to be carefully considered. Citation Format: Battisti NML, Kingston B, King J, Denton A, Waters S, Sita-Lumsden A, Rehman F, Stavraka C, Kristeleit H, Sawyer E, Houghton D, Davidson N, Howell S, Choy J, Harper P, Roylance R, Fharat R, Mohammed K, Ring A, Johnston S. Palbociclib and endocrine therapy in fourth line and beyond for hormone receptor-positive HER2-negative advanced breast cancer: The UK compassionate access program experience [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-18-23.

The genomics of advanced breast cancer (ABC) has been described through tumour tissue biopsy sequencing, although these approaches are limited by geographical and temporal heterogeneity. Here we use plasma circulating tumour DNA sequencing to interrogate the genomic profile of ABC in 800 patients in the plasmaMATCH trial. We demonstrate diverse subclonal resistance mutations, including enrichment of HER2 mutations in HER2 positive disease, co-occurring ESR1 and MAP kinase pathway mutations in HR + HER2− disease that associate with poor overall survival (p = 0.0092), and multiple PIK3CA mutations in HR + disease that associate with short progression free survival on fulvestrant (p = 0.0036). The fraction of cancer with a mutation, the clonal dominance of a mutation, varied between genes, and within hotspot mutations of ESR1 and PIK3CA. In ER-positive breast cancer subclonal mutations were enriched in an APOBEC mutational signature, with second hit PIK3CA mutations acquired subclonally and at sites characteristic of APOBEC mutagenesis. This study utilises circulating tumour DNA analysis in a large clinical trial to demonstrate the subclonal diversification of pre-treated advanced breast cancer, identifying distinct mutational processes in advanced ER-positive breast cancer, and novel therapeutic opportunities., Circulating tumour DNA can provide useful information on disease burden. Here, the authors analysed circulating tumour DNA from 800 patients from a breast cancer clinical trial and investigate the subclonal nature of the disease, and identify DNA mutations associated with resistance and poor survival.

Background: Early changes in circulating tumour DNA (ctDNA) levels may identify which patients respond to therapy earlier than imaging, with ctDNA levels falling rapidly in patients who respond to therapy. The plasmaMATCH trial assessed the utility of ctDNA testing with an error-corrected 73-gene targeted panel (Guardant360, Guardant Health) to allocate patients to four mutation matched therapy cohorts. ESR1-extended fulvestrant (A), HER2-neratinib +/- fulvestrant (B), AKT1-capivasertib + fulvestrant (C), AKT basket-capivasertib (D). Here, we report paired baseline and early on treatment ctDNA analysis from plasmaMATCH, to establish the optimal criteria for predicting progression free survival (PFS). Methods: In plasmaMATCH treatment cohorts, plasma samples were collected for ctDNA analysis pre-treatment at cycle 1-day 1 (C1D1) and cycle 2-day 1 (C2D1) timepoints, and sequenced with the Guardant 360 assay. Patients were included if they had a minimum of 14 days of treatment in the first cycle. Multiple different methods were investigated to integrate variant allele fractions (VAF) of mutations identified at each timepoint to estimate the level of ctDNA, including maximum VAF, mean VAF and weighted mean VAF, and weighted mean VAF of clonal mutations at C1D1. Variants with a VAF Table 1ctDNA dynamics categoryMedian PFS months (95%CI)6-month PFSORRUndetectable (N=11) CDR=018.2 (10.2-NA)91%9/11 (82%)Suppressed (N=14) CDR 05.4 (4.6-NA)48%6/14 (43%)High (N=52) CDR >=0.1322.4 (2.0-3.7)8%4/52 (8%) Citation Format: Javier Pascual, Rosalind J Cutts, Belinda Kingston, Sarah Hrebien, Lucy S Kilburn, Sarah Kernaghan, Laura Moretti, Katie Wilkinson, Andrew M Wardley, Iain R Macpherson, Richard D Baird, Rebecca Roylance, Michael Hubank, Giselle Walsh, Iris Faull, Kimberly C Banks, Richard B Lanman, Isaac Garcia-Murillas, Judith M Bliss, Alistair Ring, Nicholas C Turner. Assessment of early ctDNA dynamics to predict efficacy of targeted therapies in metastatic breast cancer: Results from plasmaMATCH trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-02.

1009 Background: The selective estrogen receptor modulator (SERD) fulvestrant is commonly used to treat patients with hormone receptor positive advanced breast cancer, although potential mechanisms of acquired resistance are poorly understood. plasmaMATCH cohort A (NCT03182634) investigated the activity of fulvestrant in patients with activating ESR1 mutations in circulating tumor DNA (ctDNA). Here we present analysis of baseline and end-of-treatment (EOT) ctDNA to identify potential resistance mutations to fulvestrant. Methods: Paired baseline and EOT plasma samples from patients enrolled into plasmaMATCH underwent ctDNA sequencing (Guardant360, Guardant Health) to identify acquired mutations. For F404 analysis, MCF-7 cells were transiently transfected with estrogen receptor expression constructs containing either wildtype ESR1 (WT), single ESR1 mutations (D538G, E380Q, F404L), or compound mutations (D538G_F404L, E380Q_F404L), alongside an estrogen response element (ERE)-luciferase reporter construct. Transfected cells were treated with or without fulvestrant and ERE-luciferase activity compared. Results: Of 84 patients enrolled in cohort A, 69 had paired baseline and EOT sequencing. Patients with baseline ESR1 Y537S had shorter progression free survival (PFS), and Y537C longer PFS, than those wild-type for each respective mutation (p = 0.03 and p = 0.04). Patients frequently acquired mutations at EOT (n = 35, 51%), including potentially targetable mutations in 25% (including 3 PTEN, 3 BRCA1/2, 2 PIK3CA, 2 HER2, 1 BRAF). Three (4%) patients acquired ESR1 p.F404 mutations (F404L, F404I, F404V), with 7 mutations in total. Of 26 patients with PFS of ≥16 weeks, 3 patients (12%) acquired ESR1 F404. In 800 patients screened for entry to plasmaMATCH, one harbored a F404 mutation (0.13%), with a prior history of fulvestrant. F404 mutations resided in cis with E380Q in 6/7 assessable mutations. In vitro structural modelling revealed that ESR1 p.F404 resides within the ESR1 ligand binding domain, and contributes to estrogen and fulvestrant binding through a pi-stacking bond between the aromatic ring of phenylalanine and estrogen/fulvestrant, with all F404 mutations disrupting this bond. Transient transfection demonstrated that single mutations D538G, E380Q, F404L and wild-type ESR1 were sensitive to fulvestrant (p < 0.0001, p = 0.0006, p = 0.04 and p = 0.0001), whereas compound mutations D538G_F404L and E380Q_F404L were resistant. Further investigation of relative sensitivity of the F404 mutant ESR1 to other anti-estrogens will be presented. Conclusions: We have identified a novel resistance mechanism to fulvestrant, with F404 mutations acquired in patients with pre-existing activating ESR1 mutations. F404 confers fulvestrant resistance through the loss of a pi-stacking bond and likely reduced fulvestrant binding affinity, identifying a new potential target to overcome endocrine therapy resistance.

Palbociclib is approved in 1st line for hormone receptor (HR)-positive HER2-negative advanced breast cancer (ABC). A Compassionate Access Programme previously allowed patients to receive it in 4th line. However, Palbociclib has not been specifically tested in this population. We aimed to determine the safety and efficacy profile of Palbociclib within the Programme across ten institutions in the United Kingdom. We retrospectively identified HR-positive HER2-negative ABC patients on the Programme between December 2015 and September 2017. Demographics, disease characteristics, prior treatments, blood tests, toxicities, treatment delays and responses were recorded. Simple statistics, Fisher’s exact test, χ2 method and Cox regression were used. 118 patients identified had a median age of 59. 82.2% were postmenopausal and 92.4% performance status 0–1. 81.4% had visceral involvement and 6.8% bone-only disease after a median of 5 prior treatments and 3 prior chemotherapies. Clinical benefit rate was 47.5%, overall response rate 15.8%, median PFS 4.5 months and median OS 15.8 months. Longer progression-free survival on prior endocrine therapy was a predictor of longer PFS and OS. 89.7% developed neutropenia (grade ≥ 3 in 56.8%). 5.1% experienced febrile neutropenia. 48.3% had dose reductions and 3.4% discontinued Palbociclib following toxicity. No statistically significant difference in grade ≥ 3 neutropenia was observed according to metastatic sites nor previous treatments. This is the most extensive analysis of palbociclib in ≥ 4th-line setting. Clinical benefit was confirmed particularly for endocrine-sensitive, predominantly bony disease and in earlier lines of treatment. Safety was similar to PALOMA trials with higher febrile neutropenia rate.

Purpose Leptomeningeal disease (LMD) is an uncommon complication of advanced breast cancer. The prognosis is poor, and although radiotherapy (RT), systemic and intra-thecal (IT) chemotherapy are accepted treatment modalities, efficacy data are limited. This study was designed to evaluate potential predictors of survival in this patient group. Methods Breast cancer patients with LMD diagnosed by MRI in a 10-year period (2004–2014) were identified from electronic patient records. PFS and OS estimates were calculated using Kaplan-Meier method, with planned sub-group analysis by treatment modality. Cox regression was employed to identify significant prognostic variables. Results We identified 182 eligible patients; all female, median age at LMD diagnosis 52.5 years (range 23–80). Ninety patients (49.5%) were ER positive/HER2 negative; 48 (26.4%) were HER2 positive, and 27 (14.8%) were triple negative. HER2 status was unknown in 17 (9.3%). Initial management of LMD was most commonly whole or partial brain RT in 62 (34.1%), systemic therapy in 45 (24.7%) or supportive care alone in 37 (20.3%). Fourteen patients (7.7%) underwent IT chemotherapy, of whom two also received IT trastuzumab. From diagnosis of LMD, the median PFS was 3.9 months (95%CI 3.2–5.0) and median OS was 5.4 months (95%CI 4.2–6.6). Patients treated with systemic therapy had the longest OS (median 8.8 months, 95%CI 5.5–11.1), compared to RT; 6.1 months (95%CI 4.2–7.9 months), IT therapy; 2.9 months (95%CI 1.2–5.8) and supportive care; 1.7 months (95%CI 0.9–3.0). On multivariable analysis, triple negative histology, concomitant brain metastases, and LMD involving both the brain and spinal cord were associated with poor OS. Conclusions Breast cancer patients with triple negative LMD, concomitant brain metastases or LMD affecting both the spine and brain have the poorest prognosis. Clinical trials to identify more effective treatments for these patients are urgently needed.

Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC.Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients.We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations inOur research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of

e13039Background: Palbociclib is approved in the UK in first line for hormone receptor (HR)-positive HER2-negative advanced breast cancer (ABC). A Compassionate Access Program (CAP) allowed patient...

Fatigue is a common symptom amongst cancer survivors which can have a significant impact on quality of life even in those who have completed treatment many years previously. It appears that a combination of genetic predisposition, environmental exposure, tumor and treatment factors combine to put women at increased risk of fatigue, but the precise mechanisms are not well understood. The fatigue experienced by cancer survivors is best regarded as a multidimensional symptom involving the subjective experience of tiredness, weakness and lack of energy. However there is a lack of consensus as to the optimal means by which to measure fatigue, and partly as a consequence of this, interventions to ameliorate this distressing symptom are not well established. This chapter examines what is known of the prevalence and natural history of fatigue amongst breast cancer survivors, with guidance as to the differential diagnosis and potential management strategies.

New therapies are changing the outlook for breast cancer worldwide. Breast cancer is traditionally divided into hormone receptor-positive, HER2-positive, and triple-negative disease. We have seen the development of new drugs targeting each of these breast cancer groups with promising results. In this article, the novel treatments are discussed with reference to recent clinical trial data. In each group, treatments are emerging that have had positive results in clinical trials. We discuss the new therapies aimed at evading hormone resistance in hormone receptor-positive disease, which have substantially increased the disease-free survival of patients with metastatic breast cancer in clinical trials. We have also seen multiple new therapies targeting HER2 receptors, working synergistically with Herceptin leading to dramatic and durable disease responses. Finally, in triple-negative disease, we are starting to see the results of trials using immunotherapy. Across all the treatment groups, we have seen the emergence of new therapies that are specifically designed to target the molecular drivers of an individual's cancer, regardless of hormone or HER2 positivity. This is paving the way for a more personalized approach to breast cancer treatment.