AKT1 Mutations Research Articles

BMAL1 knockdown triggers different colon carcinoma cell fates by altering the delicate equilibrium between AKT/mTOR and P53/P21 pathways.

Dysregulation of the circadian timing system (CTS) frequently appears during colorectal cancer (CRC) progression. In order to better understand the role of the circadian clock in CRC progression, this study evaluated in vitro how knockdown of a core circadian protein BMAL1 (BMAL1-KD) influenced the behavior of two primary human CRC cell lines (HCT116 and SW480) and a metastatic CRC cell line (SW620).Unexpectedly, BMAL1-KD induced CRC cell-type specific responses rather than the same phenomenon throughout. First, BMAL1-KD increased AKT/mTOR activation in each CRC cell line, but to different extents. Second, BMAL1-KD-induced P53 activation varied with cell context. In a wild type P53 background, HCT116 BMAL1-KD cells quickly underwent apoptosis after shBMAL1 lentivirus transduction, while surviving cells showed less P53 but increased AKT/mTOR activation, which ultimately caused higher proliferation. In the presence of a partially functional mutant P53, SW480 BMAL1-KD cells showed moderate P53 and mTOR activation simultaneously with cell senescence. With a moderate increased AKT but unchanged mutant P53 activation, SW620 BMAL1-KD cells grew faster.Thus, under different CRC cellular pathological contexts, BMAL1 knockdown induced relatively equal effects on AKT/mTOR activation but different effects on P53 activation, which finally triggered different CRC cell fates.

Role of PI3K/AKT/mTOR Pathway Associated Oxidative Stress and Cardiac Dysfunction in Takotsubo Syndrome.

Takotsubo syndrome (TTS) is a stress-induced cardiomyopathy, but the accurate cause of this syndrome is still unknown. β-adrenergic agonist isoproterenol (ISO) is used to establish the TTS rats model. TTS rats were treated with or without LY294002 or Rapamycin. The rat cardiomyoblast cell line H9C2 was subjected to infect with constitutively active Akt (myr-Akt) or dominant-negative mutant Akt (dn-Akt) and then, treated with ISO. Cell apoptosis was assessed using the Bax/ Bcl-2 ratio. In addition, reactive oxygen species (ROS) levels were measured using dihydroethidium (DHE). Mitochondrial superoxide generation and membrane potential were assayed by MitoSOX and JC-1 fluorescence intensity. ISO might induce the erratic acute cardiac dysfunction and overexpression of PI3K/AKT/mTOR. Moreover, it also increased the oxidative stress and apoptosis in TTS rats. The Akt inhibitor significantly reversed the cardiac injury effect, which triggered by ISO treatment. In H9C2 cells, the inhibition of Akt provides a protective role against ISO-induced injury by reducing oxidative stress, apoptosis and mitochondrial dysfunction. This study provided new insight into the protective effects of myocardial dysfunction in TTS rats via chronic inhibition of the PI3K/AKT/mTOR expression, which could reduce mitochondrial ROS and oxidative stress-induced apoptosis. PI3K/AKT/mTOR inhibitor could be a therapeutic target to treat cardiovascular dysfunction induced by stress cardiomyopathy.

I-BET726 suppresses human skin squamous cell carcinoma cell growth in vitro and in vivo

Bromodomain-containing protein 4 (BRD4) is a potential therapeutic target of skin squamous cell carcinoma (SCC). I-BET726 is a novel BRD4 inhibitor. Its potential effect in skin SCC cells was tested in the present study. We show that I-BET726 potently inhibited survival, proliferation, cell cycle progression, and migration in established (A431/SCC-9/SCC-12/SCC-13 lines) and primary human skin SCC cells. I-BET726 induced significant apoptosis activation in skin SCC cells. It was more efficient in inhibiting skin SCC cells than known BRD4 inhibitors (JQ1, CPI203, and AZD5153). I-BET726 not only downregulated BRD4-regulated proteins (c-Myc, Bcl-2, and cyclin D1), but also inhibited sphingosine kinase 1 (SphK1) and Akt signalings in SCC cells. Restoring Akt activation, by a constitutively active S473D mutant Akt1 (“caAkt1”), partially inhibited I-BET726-induced cytotoxicity in A431 cells. In vivo, I-BET726 oral administration potently inhibited A431 xenograft growth in severe combined immunodeficient mice. Downregulation of BRD4-regulated proteins and inhibition of the SphK1-Akt signaling were detected in I-BET726-treated A431 xenograft tumor tissues. Together, I-BET726 inhibits skin SCC cell growth in vitro and in vivo.

Ovarian mucinous tumors with mural nodules: immunohistochemical and molecular analysis of 3 cases

BackgroundPrimary ovarian mucinous tumors with mural nodules are very rare. The histogenesis of the mural nodules remains unclear.MethodsWe investigated the clincopathological and molecular features in 3 cases with mural nodules.ResultsPatient 1 was diagnosed as mucinous carcinoma with mural nodules of anaplastic carcinoma that was composed of CK+ and CK7+ spindled cells and polygonal cells with marked pleomorphism. Aberrant p53 staining was found in the mural nodules rather than in the mucinous components. A concordant KRAS mutation (c.35G > A p.G12A) was identified in both mucinous tumors and mural nodules. She died of disease at 44 months. The mural nodule in patient 2 was interpreted as a sarcoma, no other specified. The uniform short spindle cells were separated by abundant myxoid matrix. They were CD10 + , CCND1-, SMA-, and negative for break-apart BCOR, PHF1, and JAZF1 FISH assay. The adenocarcinomatous component harbored LOH at D18S51 and FGA loci while the sarcomatous component had LOH at D19S433. She had lung metastasis at 18 months and was alive without evidence of disease for 40 months. Patient 3 harbored multiple mural nodules that were composed of vimentin+, focal CK+, atypical spindle cells. A diagnosis of sarcoma-like mural nodules was rendered. She was alive with no evidence of disease for 13 months. No hotspot mutant AKT1, KRAS, HRAS, and PI3KCA alleles were found in patients 2 and 3.ConclusionsMural nodules with anaplastic carcinoma or with true sarcomas may represent the dedifferentiation form of mucinous tumors or collision tumors, respectively. The worrisome histology in sarcoma-like mural nodules necessitates meticulous treatment for these patients.

In This Issue

See article, [p. 526][1] . </br>![][2]</img> Approximately 7% of estrogen receptor–positive (ER+) breast cancers harbor mutations in AKT1 (most often E17K) that cause PI3K-pathway activation. However, due to the rarity of this genomically defined breast cancer subgroup, the potential

PD39-05 PREVALENCE AND LANDSCAPE OF ACTIONABLE GENOMIC ALTERATIONS IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Advanced (including Drug Therapy) II (PD39)1 Apr 2020PD39-05 PREVALENCE AND LANDSCAPE OF ACTIONABLE GENOMIC ALTERATIONS IN RENAL CELL CARCINOMA Kyrollis Attalla*, Renzo G. DiNatale, Eduard Reznik, Christopher Fong, Francisco Sanchez-Vega, Andrew W. Silagy, Stanley Weng, Jonathan Coleman, Chung-Han Lee, Maria I. Carlo, Paul Russo, Timothy A. Chan, Nikolaus D. Schultz, Martin H. Voss, and A. Ari Hakimi Kyrollis Attalla*Kyrollis Attalla* More articles by this author , Renzo G. DiNataleRenzo G. DiNatale More articles by this author , Eduard ReznikEduard Reznik More articles by this author , Christopher FongChristopher Fong More articles by this author , Francisco Sanchez-VegaFrancisco Sanchez-Vega More articles by this author , Andrew W. SilagyAndrew W. Silagy More articles by this author , Stanley WengStanley Weng More articles by this author , Jonathan ColemanJonathan Coleman More articles by this author , Chung-Han LeeChung-Han Lee More articles by this author , Maria I. CarloMaria I. Carlo More articles by this author , Paul RussoPaul Russo More articles by this author , Timothy A. ChanTimothy A. Chan More articles by this author , Nikolaus D. SchultzNikolaus D. Schultz More articles by this author , Martin H. VossMartin H. Voss More articles by this author , and A. Ari HakimiA. Ari Hakimi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000918.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We report our experience with next-generation sequencing to characterize the prevalence and genomic landscape of actionable genomic alterations in renal cell carcinoma (RCC). METHODS: A query of our institutional clinical sequencing database was performed to include tumor samples sequenced across all cancers. Actionable alterations with clinical or biologic evidence supporting an association with response to targeted therapy were stratified by level of evidence using an oncology knowledge database (OncoKB). RESULTS: Data from 35,668 patients was included, and the 15 cancer types with the highest prevalence of actionable alterations were selected for subsequent analysis (28,027 patients). Of these cancers, RCC ranked 13th in prevalence of actionable alterations. Of 708 RCC samples included, 259 (37%) were from metastatic sites. However, of the remaining 449 primary samples, 208 (29%) belonged to patients who had metastatic disease at the time of sequencing. Although 81% of patients with any RCC harbored at least one known oncogenic mutation, only 90/687 (13%) harbored alterations for which compelling clinical data currently exist to justify the use of a standard or an investigational agent (levels of evidence 1 to 3B). This represents an increase from the previously reported prevalence (5% in 2017). The most common histologic subtype, clear cell RCC harbored the vast majority of actionable alterations, with 52/421 (12%) of clear cell RCC patients harboring at least one actionable alteration. Regarding the specific genes that harbor these alterations, 31 level 2A alterations were identified, all of which were TSC1/TSC2 mutations; 30 level 2B alterations were identified, of which PIK3CA (22) and BRCA1/BRCA2 (5) mutations were most common; 11 level 3A alterations, all of which were MTOR mutations, and 8 level 3B alterations, of which AKT1 (4) mutation was most common. CONCLUSIONS: Although the prevalence of actionable mutations in RCC seems to have doubled in recent years, the role of genetic testing in identifying candidates for targeted therapy in RCC is currently limited relative to other cancer types, emphasizing the need for additional research in this area to further inform targeted therapy decisions. Source of Funding: The Sidney Kimmel Center for Prostate and Urologic Cancers and in part through the NIH/NCI Cancer Center Support Grant P30 CA008748 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e810-e810 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kyrollis Attalla* More articles by this author Renzo G. DiNatale More articles by this author Eduard Reznik More articles by this author Christopher Fong More articles by this author Francisco Sanchez-Vega More articles by this author Andrew W. Silagy More articles by this author Stanley Weng More articles by this author Jonathan Coleman More articles by this author Chung-Han Lee More articles by this author Maria I. Carlo More articles by this author Paul Russo More articles by this author Timothy A. Chan More articles by this author Nikolaus D. Schultz More articles by this author Martin H. Voss More articles by this author A. Ari Hakimi More articles by this author Expand All Advertisement PDF downloadLoading ...

Pancreatic and duodenal homeobox-1 (PDX1) contributes to β-cell mass expansion and proliferation induced by Akt/PKB pathway

ABSTRACT Maintenance of pancreatic β-cell mass and function is fundamental to glucose homeostasis and to prevent diabetes. The PI3 K-Akt-mTORC1 pathway is critical for β-cells mass and function, while PDX1 has been implicated in β-cell development, maturation, and function. Here we tested whether Akt signaling requires PDX1 expression to regulate β-cell mass, proliferation, and glucose homeostasis. In order to address that, we crossed a mouse model overexpressing constitutively active Akt mutant in β-cells (β-caAkt) with mice lacking one allele of PDX1gene (β-caAkt/pdx1+/-). While the β-caAkt mice exhibit higher plasma insulin levels, greater β-cell mass and improved glucose tolerance compared to control mice, the β-caAkt/pdx1+/- mice are hyperglycemic and intolerant to glucose. The changes in glucose homeostasis in β-caAkt/pdx1+/- were associated with a 60% reduction in β-cell mass compared to β-caAkt mice. The impaired β-cell mass in the β-caAkt/pdx1+/- mice can be explained by a lesser β-cell proliferation measured by the number of Ki67 positive β-cells. We did not observe any differences in apoptosis between β-caAkt/pdx1+/- and β-caAkt mice. In conclusion, PDX1 contributes to β-cell mass expansion and glucose metabolism induced by activation of Akt signaling.

Novel pathogenic alterations in pediatric and adult desmoid-type fibromatosis \u2013 A systematic analysis of 204 cases

Desmoid-type fibromatosis (DTF, aggressive fibromatosis) is a non-metastasizing mesenchymal neoplasm of deep soft tissue with a tendency towards local recurrence. Genetic alterations affecting canonical Wnt/β-catenin signaling are reported in the majority of DTF. While most sporadic DTF harbor somatic mutations in CTNNB1, germline mutations in adenomatous polyposis coli (APC) are known to occur in hereditary DTF types (FAP, Gardner-Syndrome). Additional single nucleotide variants (SNVs) in AKT1 (E17K) and BRAF (V600E) were reported in pediatric DTF with potential clinical implications. We performed targeted next-generation sequencing (NGS) in a large cohort of 204 formalin-fixed DTF samples, comprising 22 pediatric cases (patients age ≤18 years). The mutational status was correlated with clinicopathological characteristics. Overall, deleterious CTNNB1 mutations were detected in 89% of DTF, most frequently affecting the serine/threonine phosphorylation sites T41 and S45 of β-catenin. While the T41A CTNNB1 mutation was significantly more often identified in the mesenterial localization, DTF originating from extra-intestinal sites more frequently harbored the S45P CTNNB1 alteration. Beyond common mutations in CTNNB1, additional SNVs were demonstrated in 7% of the DTF cohort and in 18% of the pediatric DTF subgroup. The mutational spectrum included deleterious mutations in AKT1 (G311S/D and T312I), ALK (R806H and G924S), AR (A159T), EGFR (P848L), ERBB2 (H174Y), IDH2 (H354Y), KIT (V559D), RET (T1038A), SDHA (R325M), and SDHD (R115W), as characterized by in silico prediction tools. In conclusion, our study indicates that DTF may harbor a broader mutational spectrum beyond CTNNB1 mutations, comprising targetable alterations including the herewith first reported imatinib-sensitive KIT V559D mutation in DTF.

Juvenile papillomatosis of the breast (Swiss cheese disease) has frequent associations with PIK3CA and/or AKT1 mutations

Juvenile papillomatosis (JP), the so-called Swiss cheese disease, is a rare benign breast disease of young adults. An association (up to 28%) with breast cancer within the family of affected patients has been reported. A multinodular cystic breast mass lesion and calcifications characterizes JP in imaging studies. The histological picture is diverse and comprises multiple intraductal papillomas, usual ductal hyperplasia, ductectasias, perifocal sclerosing adenosis, and calcification. Patients with complete excision of JP lesions have an excellent follow-up; breast cancer develops only on a very low subset of patients. Molecular background of JP has not been investigated until now. In this study, we addressed mutational analysis of JP cases and correlated these results with follow-up and family history in context with a comprehensive review of the JP literature. We identified 13 cases fulfilling the criteria of JP. All patients were women with a median age of 38 years (26-50 years). Follow-up information was available for 11 of 13 patients. Sufficient paraffin-embedded tissue and good DNA quality for next-generation sequencing (NGS) was available for 10 patients. Paraffin blocks were microdissected in the area of intraductal proliferative disease; the tissue cores underwent NGS analysis using the Oncomine Comprehensive Panel. In 5 of 10 patients, we found PIK3CA mutations; in 2 of 10 patients, we found AKT1 mutations in known hot spot regions. Further mutations in MET, FGFR3, PTEN, ATM, NF1, and GNAS genes were detected in individual patients. Some of these mutations were present at high allele frequencies suggesting germ line mutations. Two of 3 patients with positive family history had PIK3CA mutation; one patient with positive family history had an AKT1 mutation. One patient who subsequently developed invasive ductal carcinoma in the contralateral breast possibly had a germ line ATM mutation. Our results confirm hot spot mutations in PIK3CA and AKT1 genes in JP associated with positive family history for breast cancer, although these mutations are not specific for JP. The genetic link between JP, positive family history, and subsequent risk of breast cancerneeds to be analyzed in further studies.

A phase I dose-escalation study of enzalutamide in combination with the AKT inhibitor AZD5363 (capivasertib) in patients with metastatic castration-resistant prostate cancer

BackgroundActivation of the PI3K/AKT/mTOR pathway through loss of phosphatase and tensin homolog (PTEN) occurs in approximately 50% of patients with metastatic castration-resistant prostate cancer (mCRPC). Recent evidence suggests that combined inhibition of the androgen receptor (AR) and AKT may be beneficial in mCRPC with PTEN loss.Patients and methodsmCRPC patients who previously failed abiraterone and/or enzalutamide, received escalating doses of AZD5363 (capivasertib) starting at 320 mg twice daily (b.i.d.) given 4 days on and 3 days off, in combination with enzalutamide 160 mg daily. The co-primary endpoints were safety/tolerability and determining the maximum tolerated dose and recommended phase II dose; pharmacokinetics, antitumour activity, and exploratory biomarker analysis were also evaluated.ResultsSixteen patients were enrolled, 15 received study treatment and 13 were assessable for dose-limiting toxicities (DLTs). Patients were treated at 320, 400, and 480 mg b.i.d. dose levels of capivasertib. The recommended phase II dose identified for capivasertib was 400 mg b.i.d. with 1/6 patients experiencing a DLT (maculopapular rash) at this level. The most common grade ≥3 adverse events were hyperglycemia (26.7%) and rash (20%). Concomitant administration of enzalutamide significantly decreased plasma exposure of capivasertib, though this did not appear to impact pharmacodynamics. Three patients met the criteria for response (defined as prostate-specific antigen decline ≥50%, circulating tumour cell conversion, and/or radiological response). Responses were seen in patients with PTEN loss or activating mutations in AKT, low or absent AR-V7 expression, as well as those with an increase in phosphorylated extracellular signal-regulated kinase (pERK) in post-exposure samples.ConclusionsThe combination of capivasertib and enzalutamide is tolerable and has antitumour activity, with all responding patients harbouring aberrations in the PI3K/AKT/mTOR pathway.Clinical Trial NumberNCT02525068

Comprehensive targeted gene profiling to determine the genomic signature likely to drive progression of high-grade nonmuscle invasive bladder cancer to muscle invasive bladder cancer.

568 Background: Bladder cancer is the fifth most common cancer and eighth leading cause of cancer related-death in North America. It can present as non-muscle invasive bladder cancer (NMIBC) and/or muscle invasive bladder (MIBC). Although genomic profiling studies have established that low-grade NMIBC and MIBC are genetically distinct, high-grade NMIBC can recur and progress to MIBC [ Knowles, M.A. and C.D. Hurst, 2015]. Low grade, non-invasive bladder cancers are characterized by activating mutations in fibroblast growth factor receptor 3 (FGFR3), HRAS or other pathways of receptor kinase activation. High-grade disease, which is often becomes invasive, is characterized by inactivation of TP53 and Rb pathways [Kim, J., et al.]. Finding a subtype of invasive carcinoma with FGFR3 mutation may suggest an alternate pathway by which low grade, non-invasive pathology could transform into invasive disease [Knowles, M.A. and C.D. Hurst, 2015]. Methods: In this study, using a total of 30 bladder cancer (NMIBC and MIBC) patient samples from Windsor Regional Hospital Cancer Program, we performed comprehensive targeted gene sequencing to identify single nucleotide variants, small insertions / deletions, copy number variants and splice variants in over 500 common tumor genes panel. Results: Preliminary data from our study correlates with previously published mutation landscape for NMIBC and MIBC, and includes mutations in EGFR, FGFR3, FGFR4, PIK3CA, CDK6, ALK, JAK, as well as RET. While mutations in AKT1, BRCA1, CCND1, ERBB2, FGFR1, FGFR2, HRAS, and MET appear to be prevalent in NMIBC, mutations in IDH1 and MAP2K2 appear to be more common in MIBC. Three of the samples used in the study are from patients who progressed from high-grade NMIBC to MIBC. Conclusions: Therefore, have the genomic profiling performed at these two stages, which provides a unique ability to identify the potential “genomic triggers” for the transition.

Prevalence and landscape of actionable genomic alterations in renal cell carcinoma.

616 Background: We report our experience with next-generation sequencing to characterize the prevalence and genomic landscape of actionable genomic alterations in renal cell carcinoma (RCC). Methods: A query of our institutional clinical sequencing database was performed to include tumor samples sequenced across all cancers. Actionable alterations with clinical or biologic evidence supporting an association with response to targeted therapy were stratified by level of evidence using an oncology knowledge database (OncoKB). Results: Data from 35,668 patients was included, and the 15 cancer types with the highest prevalence of actionable alterations were selected for subsequent analysis (28,027 patients). Of these cancers, RCC ranked 13th in prevalence of actionable alterations. Of 708 RCC samples included, 259 (37%) were from metastatic sites. However, of the remaining 449 primary samples, 208 (29%) belonged to patients who had metastatic disease at the time of sequencing. Although 69% of patients with any RCC harbored at least one known oncogenic mutation, only 90/687 (13%) harbored alterations for which compelling clinical data currently exist to justify the use of a standard or an investigational agent (levels of evidence 1 to 3B). This represents an increase from the previously reported prevalence (5% in 2017). The most common histologic subtype, clear cell RCC harbored the vast majority of actionable alterations (52/421; 12%). Regarding the specific genes that harbor these alterations, 31 level 2A alterations were identified, all of which were TSC1/TSC2 mutations; 30 level 2B alterations were identified, of which PIK3CA (22) and BRCA1/BRCA2 (5) mutations were most common; 11 level 3A alterations, all of which were MTOR mutations, and 8 level 3B alterations, of which AKT1 (4) mutation was most common. Conclusions: Although the prevalence of actionable mutations in RCC seems to have doubled in recent years, the role of genetic testing in identifying candidates for targeted therapy in RCC is currently limited relative to other cancer types, emphasizing the need for additional research in this area to further inform targeted therapy decisions.

The prognostic role of BRAF and WNT pathways activation in kinase inhibitors-naïve clinical stage III cutaneous melanoma.

The results of local-regional advanced melanoma (stage III) management are still not satisfactory. Particularly, there is no personalized treatment in stage III melanoma patients due to the lack of useful classical pathological markers for prognostication of indolent or aggressive course of the disease. The aim of this study was to explore melanoma genomic landscape by means of the mutational profiling of 50 genes influencing carcinogenesis pathways in the randomly selected 93 kinase inhibitor-naïve (KI-naïve) stage III patients. The genomic alterations were found in 27 out of 50 tested genes and at least one pathogenic variant was detected in 77 out of 93 cases (82.7%). Survival rate was negatively affected by the presence of the somatic mutations in AKT1, ATM, CDH1 and SMARCB1, while the BRAF+ status in KI-naïve stage III population correlated with the longer median overall survival. Genomic alterations in WNT pathway correlated with extranodal adipocyte tissue involvement (P = 0.027) and higher number of metastatic lymph nodes (P = 0.045). In terms of survival, the Cox model confirmed the worse prognosis in patients with mutation in the WNT pathway [hazard ratio (HR) = 2.9, P = 0.017], and better prognosis in cases with mutations in BRAF pathway (HR = 0.5, P = 0.004). WNT/β-catenin pathway alteration was associated with more advanced/aggressive disease. From this perspective, the concept of blocking the activity of the WNT pathway in selected cases appears promising and complementary to the BRAF inhibition therapeutic option for the future.

Abstract P4-05-08: Genomic landscape of breast cancer occurring in elderly individuals

Abstract Introduction: There is evidence to suggest that the biology of breast cancer (BC) might vary according to age, as BCs in elderly patients might be associated with accumulation of DNA damage and BCs in younger individuals might be enriched for genetic alterations affecting cancer predisposition genes. Whether the genetic landscape of BC in elderly individuals differs is yet to be determined. Here, we sought to describe the spectrum of somatic genetic alterations and mutational signatures in BC according to age. Materials and Methods: We conducted a re-analysis of two cohorts of BC: i) MSK-IMPACT targeted sequencing cohort (primary, n=918; metastatic, n=1,000) and ii) whole-exome sequencing data of primary BCs from The Cancer Genome Atlas (TCGA) BC study (n=1087). BCs of younger (&amp;lt;65 years old) and elderly individuals (≥ 65 years old) were compared, matched by estrogen receptor (ER)/HER2 status at a 2:1 ratio (MSK-IMPACT) or a 1:1 ratio (TCGA). Somatic single nucleotide variants and copy number alterations (CNAs) were determined using a validated bioinformatics pipeline, and mutational signatures were defined using DeconstructSigs and SigMA. Two-tailed Fisher’s exact test was performed. Results: Primary BCs in elderly and younger patients, matched by ER/HER2 status, from the MSK-IMPACT (n=220 and n=440) and TCGA (n=290, each) cohorts, and metastatic BCs from elderly (n=98) and younger (n=196) patients from the MSK-IMPACT cohort, matched by ER/HER2 status, were compared. In both cohorts of primary BCs, PIK3CA, TP53 and CDH1 were the most frequently mutated genes. Compared to younger patients, primary BCs in elderly patients from the MSK-IMPACT cohort displayed a lower frequency of mutations in TP53 (23% vs 34%, P&amp;lt;0.01), AKT1 (2% vs 6%, P&amp;lt;0.05) and MAP2K4 (0.4% vs 4%, P&amp;lt;0.01). In the TCGA cohort, somatic mutations in AKT1 were found to be less frequent in elderly than in younger patients (1% vs 5%, P&amp;lt;0.05), whereas NF1 (2% vs 6%, P&amp;lt;0.05) mutations were more frequent in elderly than in younger patients with primary BCs. Our analyses of BC metastases subjected to MSK-IMPACT revealed that elderly patients harbored a higher frequency of mutations in NCOR1 (11% vs 4%, P&amp;lt;0.05) and RUNX1 (9% vs 2%, P&amp;lt;0.05) than younger patients. The frequency of bi-allelic inactivation of homologous recombination DNA repair deficiency (HRD) genes (i.e. BRCA1, BRCA2, PALB2, RAD51C and RAD51D) in primary BCs of elderly and younger individuals in the TCGA cohort was 2% and 5%, respectively. No differences in the frequency of CNAs were observed between the two groups. The most frequent mutational signatures found in primary BCs of elderly and younger patients were aging, APOBEC and HRD in the MSK-IMPACT cohort, and aging, APOBEC and HRD in primary BCs of the TCGA cohort, with APOBEC being enriched in younger patients compared to elderly patients. The most frequent mutational signatures in metastatic BCs in elderly and younger individuals of the MSK-IMPACT cohort were APOBEC followed by aging and HRD. Conclusion: Our findings show that BCs in elderly patients harbor a lower frequency of somatic mutations in TP53, AKT1 and MAP2K4 than younger individuals. In contrast, BCs in elderly patients more frequently harbor somatic mutations in the transcription factors RUNX1 and NCOR1. Bi-allelic alterations affecting HRD-related genes was numerically more frequent in younger than in older patients. As expected, BCs in elderly patients show a numerically higher incidence of the aging signature, whereas primary BCs affecting younger patients displayed a higher frequency of the APOBEC signature than primary BCs from elderly individuals. Citation Format: Pier Selenica, Fresia Pareja, Audree Tadros, Lorenzo Ferrando, David N Brown, Hong Zhang, Pedram Razavi, Diana Mandelker, Mark E Robson, Sarat Chandarlapaty, Britta Weigelt, Jorge S Reis-Filho. Genomic landscape of breast cancer occurring in elderly individuals [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 P4-05-08.

Abstract P1-19-14: 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 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.

Abstract P6-10-08: AKT1 alterations following exposure to endocrine-based therapy in patients with hormone-receptor positive (HR+) metastatic breast cancer (MBC): Clinical and functional implications

Abstract Background: The AKT1 oncogene represents a central node in the well-characterized PI3K/AKT/mTOR signal transduction pathway involved in the regulation of cellular proliferation as well as therapeutic resistance. With the emergence of multiple targeted therapies for HR+ MBC, we sought to explore the prevalence of AKT1 alterations, the spectrum of co-occurring mutations, and any potential impact on response to endocrine-based therapy, including CDK 4/6 and PIK3CA inhibitors. Methods: Presence or absence of AKT1 mutation was interrogated via cell-free DNA (cfDNA) analysis (Guardant assay) among patients with HR+/HER2- MBC receiving treatment at the Massachusetts General Hospital who underwent routine clinical cfDNA testing. Genomic sequencing results were then compared to detailed clinical annotation based upon retrospective chart review. In the laboratory, HR+/HER2- T47D breast cancer cells were transfected with AKT1, under the control of a doxycycline-inducible promoter, to induce AKT1 overexpression. Sensitivity to escalating doses of fulvestrant and palbociclib were interrogated via cell-titer-glo viability assay in vitro. Results: Among 272 patients with HR+/HER2- MBC, we identified 13 patients with AKT1 mutation (4.7%). 12/13 (92%) patients had a canonical AKT1E17K alteration, one patient (8%) had an AKT1 E49K alteration. AKT1 mutations occurred concurrently with alterations in ESR1 (31%), PIK3CA (23%), TP53 (23%), ERBB2 (15%), and RB1 (8%). All patients had been exposed to some form of anti-estrogen prior to sequencing, either via (neo)adjuvant therapy and/or in the metastatic setting; 5/13 (38%) had exposure to a CDK4/6 inhibitor prior to cfDNA sequencing. Following the identification of an AKT1 alteration, examples of rapid clinical progression on fulvestrant, CDK4/6i, and/or everolimus were identified, however the presence of an AKT1 alteration was not sufficient to predict pan-resistance to these agents, as counter-examples of clinical benefit were also identified. In an index patient with an AKT1 mutation and prior progression on letrozole and palbociclib, subsequent treatment with fulvestrant and abemaciclib did not yield clinical benefit, highlighting the potential role of AKT1 in mediating clinical resistance to endocrine therapy and CDK4/6i. In the pre-clinical model, HR+/HER2- breast cancer cells demonstrated resistance to both fulvestrant and palbociclib following upregulation of exogenous AKT1 expression. Updated clinical data with additional patients and response to regimens incorporating an anti-estrogen, a CDK4/6 inhibitor, an mTOR inhibitor, and/or a PIK3CA inhibitor will be presented at the meeting. Conclusions: Activating AKT1 mutations can be identified via targeted sequencing of cfDNA in patients with HR+/HER2- MBC, and may play an important role in mediating resistance to anti-estrogens, CDK4/6 inhibitors, and other emerging targeted therapies. Heterogeneity in response to endocrine-based therapy following the identification of an AKT1 alteration may be related to tumor-extrinsic factors, the AKT1 allelic fraction, or cooperativity between multiple resistance drivers. Further research is needed to confirm these findings and guide optimal therapeutic sequencing strategies for patients with HR+/HER2- AKT1 mutant MBC. Citation Format: Seth Wander, Pingping Mao, Maxwell Lloyd, Kailey Kowalski, Gabriela N. Johnson, Giuliana Malvarosa, Beverly Moy, Leif W. Ellisen, John Iafrate, Nikhil Wagle, Aditya Bardia. AKT1 alterations following exposure to endocrine-based therapy in patients with hormone-receptor positive (HR+) metastatic breast cancer (MBC): Clinical and functional implications [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 P6-10-08.

Abstract P4-10-11: PIK3CA status as a predictor of benefit from drugs targeting the PI3K/AKT/mTOR pathway: A meta-analysis of randomized trials

Abstract Introduction: A number of different drugs have been developed to target the PI3 kinase/AKT/mTOR pathway. Most randomized trials in breast cancer have been performed in patients unselected for pathway activation although aberrations of components of this pathway are common. Here we explore the predictive value of PIK3CA and AKT mutations in randomized trials of drugs targeting this signaling route. Methods: We searched PubMed for randomized trials of drugs targeting the PI3 kinase/AKT/mTOR pathway in metastatic breast cancer. The search was supplemented by a manual search of recent ASCO and ESMO meetings. Eligible studies needed to report efficacy data based on presence or absence of PIK3CA or AKT status measured either in tumor specimens or in circulating DNA. Hazard ratios (HR) for progression-free survival (PFS) in PIK3CA or AKT mutant and wild-type groups were extracted and pooled in a meta-analysis using generic inverse variance and random effects modeling. Sensitivity analyses were performed based on the mechanism of action of the experimental drug. Results: Of the 15 studies identified, 10 studies comprising of 3615 patients were eligible for analysis. Studies included pan-PI3kinase inhibitors (n=5), alpha-specific PI3kinase inhibitors (n=1), AKT inhibitors (n=2) and mTOR inhibitors (n=2). Approximately 38% of patients had either PIK3CA or AKT mutations. The addition of drugs targeting the PI3 kinase/AKT/mTOR pathway was associated with a significantly longer improvement in PFS in patients with PIK3CA or AKT mutations than in those with wild-type status (HR 0.61, 95% CI 0.50-0.75 versus HR 0.83, 95% CI 0.67-1.02, p for difference = 0.04). Similar results were observed in sensitivity analyses (see Table). Conclusion: Benefit from drugs targeting the PI3 kinase/AKT/mTOR pathway in breast cancer appears to occur exclusively in those with PIK3CA or AKT mutations. Future trials of these drugs should be designed only in biomarker selected patients. TableGroupPIK3CA or AKT mutationPIK3CA and AKT wildtypep for differenceHR95% CIHR95% CIExcluding AKT inhibitors0.640.52-0.790.810.63-1.030.16Excluding pan-PI3 kinase inhibitors0.580.47-0.710.710.51-1.150.24Excluding AKT and alpha-specific PI3 kinase inhibitors0.690.48-1.000.980.83-1.150.09Excluding mTOR inhibitors0.620.49-0.800.920.80-1.060.008 Citation Format: Fahad A Almugbel, Ramy Saleh, Alberto Ocana, Atanasio Pandiella, Eitan Amir. PIK3CA status as a predictor of benefit from drugs targeting the PI3K/AKT/mTOR pathway: A meta-analysis of randomized trials [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 P4-10-11.

Abstract P4-09-04: Prevalence of phosphatidylinositol-3-kinase (PI3K) pathway alterations and co-alteration of other markers in breast cancer

Abstract Background: PI3K/AKT signaling pathway is often activated in breast cancer (BC), through mutations in PIK3CA or AKT1, and alterations in PTEN, thus promoting cell survival. PI3K and AKT inhibitors have been shown to have significant activity against tumor progression and to overcome resistance in BC. We aim to explore the prevalence of PI3K pathway alterations and co-expression with other markers in different BC subtypes. This approach could allow identification of novel drug combinations that have the potential to elicit synergistic growth inhibition and be further explored in the clinical trial setting. Methods: Molecular profiles of 4895 female and male BC cases submitted to Caris Life Sciences were reviewed to identify pathogenic or presumed pathogenic mutations in PIK3CA, AKT1, PTEN, PIK3R1 and PIK3R2 using 592-gene next generation sequencing (NGS; average read depth 500 ×). Cases with loss of PTEN by IHC were also included. Co-mutation frequency with PIK3CA-AKT1-PTEN alterations were examined, including homologous recombination deficiency (HRD) genes and DNA damage response (DDR) pathways, markers of response to immune-oncology (IO) agents and RAS signaling pathway. Cases were classified in BC subtypes according to the ASCO-CAP guidelines. Results: The median age was 58 (range: 17-90). Gender frequency and primary/ metastatic site frequency were similar across BC subtypes. 3558 (72.7%) cases had at least one alteration in the PIK3CA-AKT1-PTEN pathway: 1472 (30.1%) cases harbored a PIK3CA mutation, 174 (3.6%) harbored an AKT1 mutation, and 2682 (54.8%) had PTEN alterations (PTEN mutation in 344, 7.0% and/or PTEN loss by IHC in 2516, 51.4% of cases). The most common hotspot mutations in PIK3CA were in the kinase domain (H1047R in 567 cases, 38.5% of all PIK3CA alterations) and in the helical domain (E545K in 304 cases, 20.7% of all PIK3CA alterations). Uncommon activating PIK3CA mutations were seen in 8.4% of breast tumors. The single hotspot mutation, E17K, was the most common AKT1 mutation (n=164, 94.3% of all AKT1 mutations). 81 (1.7%) tumors harbored a PIK3R1 mutation, with 66 unique alterations identified (4 pathogenic, 55 presumed pathogenic), and 4 (0.08%) cases harbored a PIK3R2 mutation (G373R mutation). With respect to the different BC subtypes, PIK3CA was the most frequent alteration in HER2 positive BC (present in 96.2% of mutated cases). Within HER2 negative subtypes, PTEN was most frequently altered and PTEN mutation or PTEN loss by IHC was present in 79.3% of mutated cases. Triple negative breast cancer (TNBC) was the subtype with the lowest frequency of PIK3CA mutations (18.0% in TNBC vs. 37% in other subtypes). The frequency of selected co-mutations with PIK3CA-AKT1-PTEN alterations is illustrated in table 1. Notable co-alterations in this cohort include increased PD-L1 expression and high tumor mutational burden (TMB). There were no significant increases in the frequency of mutations in DDR pathway mutations (not shown) in the PIK3CA-AKT1-PTEN altered cohort. Conclusions: We observed a high prevalence of expected hotspot mutations in PIK3CA and AKT1 across BC subtypes. There was a significant increase in PD-L1 expression in tumor cells, and high TMB in PIK3CA-AKT1-PTEN mutated cohorts. A similar association was seen with RAS signaling pathways. Further development of drugs that affect the PIK3CA-AKT1-PTEN pathway in all BC subtypes and combination with drugs that target the immune system may be of interest. Table 1. Selected co-alterations based on statistical significancePathwayGene/ ProteinAll subtypes (%)HR+ HER2+ (%)HR- HER2+ (%)HR+HER2- (%)TNBC (%)MTWTMTWTMTWTMTWTMTWTHomologous recombinationBRCA13.02.71.00.60.00.80.8*1.8*6.16.0BRCA24.15.41.02.53.62.34.1*7.9*4.43.0PALB20.7*1.3*0.00.00.00.00.5*2.5*0.90.3Possible predictors of IO benefitPD-149.850.038.552.266.766.737.541.766.058.1PD-L1 (SP142)6.9*4.2*3.30.78.14.93.33.011.68.1(Tumor Cells)PD-L1 (SP142)29.226.650.018.825.027.312.916.742.140.4(Immune Cells)MSI0.70.60.00.01.20.80.80.40.71.2TMB-High (≥ 10 mut/Mb)22.9*18.8*36.0*17.2*29.825.921.2*16.6*23.621.3Chromatin remodelingARID1A12.4*18.6*19.423.87.118.917.121.55.89.6ARID20.70.80.01.90.00.70.80.60.60.9RAS-RAF-MEK-ERK HRAS0.7*0.1*0.00.00.00.00.20.11.40.3KRAS2.0*1.1*1.00.00.00.01.91.22.22.0NRAS0.20.00.00.00.00.00.20.00.30.0BRAF0.50.11.00.01.20.00.60.30.40.0OthersTP5360.9*53.1*66.360.885.586.738.4*25.6*84.786.4CDH110.3*6.1*9.1*2.5*4.82.215.1*9.6*4.9*2.3*NF16.2*2.1*9.5*0.8*2.73.65.8*1.8*6.7*2.8*RB15.5*2.6*2.21.33.63.03.9*1.7*7.85.0ERBB22.3*3.4*3.03.13.56.73.23.51.0*2.3*Notes: MT = at least 1 pathogenic mutation in PIK3CA, AKT1, or PTEN or PTEN loss by IHC. WT = no pathogenic mutations in PIK3CA, AKT1, and PTEN.*statistically significant difference between MT and WT Citation Format: Katia Khoury, Antoinette Tan, Andrew Elliott, Joanne Xiu, Zoran Gatalica, Arielle L Heeke, Claudine Isaacs, Paula R Pohlmann, Lee S Schwartzberg, Michael Simon, Michael W Korn, Sandra M Swain, Filipa Lynce. Prevalence of phosphatidylinositol-3-kinase (PI3K) pathway alterations and co-alteration of other markers in breast cancer [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 P4-09-04.

Abstract P1-19-11: 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 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.

Abstract GS3-06: 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 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.