Circulating Tumor DNA and Biomarker Analyses From the LOTUS Randomized Trial of First-Line Ipatasertib and Paclitaxel for Metastatic Triple-Negative Breast Cancer.

PURPOSE: Combining the oral AKT inhibitor ipatasertib with paclitaxel as first-line therapy for metastatic triple-negative breast cancer significantly improved progression-free survival (PFS) in the placebo-controlled, randomized, phase II LOTUS trial, with a more pronounced effect in patients with PIK3CA/AKT1/PTEN-altered tumors. We report findings from the extensive translational research program. PATIENTS AND METHODS: Pretreatment plasma and tumor samples were evaluated for genetic alterations using FoundationACT and FoundationOne (Foundation Medicine, Cambridge, MA) hybrid capture next-generation sequencing assays, respectively. Prevalences of the most common mutations and PIK3CA/AKT1 mutation status were determined using both assays, and concordance was assessed. In longitudinal analyses, circulating tumor DNA (ctDNA) mutations were quantified in baseline and on-treatment (cycle 3, day 1 [C3D1]) samples. The relationship between outcomes and ctDNA fraction (CTF; highest variant allele frequency) and CTF ratio (C3D1 CTF to baseline CTF) was explored. RESULTS: Among 89 patients evaluable for ctDNA sequencing, 81 patients (91%) had 149 detectable mutations. There was high agreement between plasma- and tissue-based sequencing for known or likely short variant mutations but not amplifications. There was 100% concordance between ctDNA and tissue sequencing in patients with activating PIK3CA or AKT1 mutations. High baseline CTF was associated with shorter PFS in both treatment arms. Longitudinal analyses showed more favorable outcomes with lower absolute CTF at C3D1 and, to a lesser extent, greater CTF decreases. CONCLUSION: These results suggest that plasma ctDNA sequencing may allow reliable and convenient assessment of prognosis and identification of genetic markers associated with increased benefit from ipatasertib. On-treatment CTF showed a meaningful association with objective response and PFS.

Analytical Validation of a Hybrid Capture-Based Next-Generation Sequencing Clinical Assay for Genomic Profiling of Cell-Free Circulating Tumor DNA.

Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture-based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode-based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%-99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%-95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%-71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments-certified/College of American Pathologists-accredited/New York State-approved laboratory.

The clinical significance of K-ras mutation in endometrial "surface epithelial changes" and their associated endometrial adenocarcinoma.

OBJECTIVES: The entity of 'surface epithelial changes' (SECs) was first described in 1995 [1]. Morphologically, SECs usually arise from malignant glands at the superficial aspect of well differentiated (WD) endometrioid carcinomas (ECs) and impart the appearance of a 'maturational' phenomenon at the surface of the cancer. Exhibiting a paradoxically bland histologic appearance, SECs typically show morphologic features that mimic benign entities, particularly endocervical microglandular hyperplasia (MGH). SECs have been associated with approximately half of WD endometrioid carcinomas many of which showed focal mucinous differentiation. Despite their morphologically benign histology, some have questioned whether the presence of SECs represents a 'marker' for an underlying malignancy, especially in postmenopausal women with endocervical or MGH-type SECs in their endometrial sampling. Since the biologic nature of SECs is unknown, we aimed to study the prevalence of KRAS gene mutations in SECs and the underlying WD endometrioid adenocarcinomas (EC) from which they directly arise. METHODS: 24 cases with biopsy proven SECs and ECs in their subsequent hysterectomy were retrieved. Genomic DNA was extracted from formalin-fixed paraffin-embedded tissue. PCR amplification for KRAS codons 12 and 13 was performed, followed by sequencing using capillary electrophoresis. RESULTS: KRAS codons 12 and 13 mutations were detected in 19 of 24 (79%) SECs, and 19 of 24 (79%) ECs. All SECs had the same KRAS mutation as the underlying EC. CONCLUSIONS: Our results suggest that SECs are of neoplastic origin and that KRAS mutations play an important role in the tumorigenesis of ECs and SECs.

KRAS Mutations in Mucinous Lesions of the Uterus.

OBJECTIVES: The current study examined the KRAS mutation status in a spectrum of mucinous lesions of the uterus, including mucinous metaplasia (MM), atypical mucinous proliferation (AMP), endocervical mucosa, and microglandular hyperplasia (MGH). METHODS: Thirty-nine cases, including 15 AMPs, nine MMs, nine MGHs, and six normal endocervical mucosas, were selected from the departmental archive. All AMP cases with follow-up biopsies or hysterectomies were reviewed. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue and KRAS codons 12 and 13 sequence analyzed. RESULTS: KRAS codon 12 and 13 mutations were detected in 10 (67%) of the 15 AMP cases. No KRAS mutations were identified in MMs, MGHs, and endocervical mucosas (P = .002, AMP vs MM or MGH, Fisher exact test). Most women with AMP were postmenopausal (13/15 [86.7%]) and presented with dysfunctional uterine bleeding. Among the 10 cases of AMP harboring KRAS mutations, six (60%) cases were subsequently diagnosed with carcinoma, one with atypical complex hyperplasia, and two with AMP within endometrial polyps. CONCLUSIONS: The results suggest a possible association between KRAS mutations and mucinous differentiation in endometrial carcinogenesis. KRAS status can help in assessing benign from precursor or malignant mucinous lesions as well as differentiate endometrial lesions from those of cervical origin.

BRAF V600E Mutations in Endometrial Adenocarcinoma.

The BRAF V600E somatic mutation is recognized as an oncogenic driver of many human cancers involving the MAPK/ERK pathway. Colorectal and lung cancers associated with the BRAF V600E mutation often demonstrated mucinous morphology. This study hypothesized that the BRAF V600E mutation may be associated with mucinous morphology in endometrial cancer and aimed to investigate its prevalence in mucinous (endometrial) carcinoma (MC) and endometrioid adenocarcinoma with significant mucinous differentiations (ECMD) (>10% neoplastic cells). Twenty-eight cases of endometrial cancer were selected, including 17 (60.7%) cases of MC or ECMD. All patients were Caucasian with age ranging from 50 to 87 years old (median 65). Genomic DNA was extracted from formalin-fixed paraffin-embedded tumor tissue and subjected to both real-time mutant allele-specific amplification polymerase chain reaction (PCR) and PCR amplification, followed by direct sequencing. Three (3/28, 10.7%) BRAF V600E mutations were detected by real-time mutant allele-specific amplification PCR and confirmed by direct sequencing. Two of 3 cases positive for BRAF V600E mutation were ECMDs with "surface epithelial changes." KRAS mutations were found in 9 cases (32.1%), none with BRAF mutation. This is the first report of BRAF V600E mutation in endometrial cancer, indicating that it may contribute to tumorigenesis of endometrial cancer, although at a low frequency compared with KRAS mutations.