We describe here detailed protocols to design, optimize and validate in vitro phosphatase assays that we have utilized to conduct high-throughput screens for inhibitors of dual-specificity phosphatases: CDC25B, mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-3. We provide details of the critical steps that are needed to effectively miniaturize the assay into a 384-well, high-throughput format that is both reproducible and cost effective. In vitro phosphatase assays that are optimized according to these protocols should satisfy the assay performance criteria required for a robust high-throughput assay with Z-factors >0.5, and with low intra-plate, inter-plate and day-to-day variability (CV <20%). Assuming the availability of sufficient active phosphatase enzyme and access to appropriate liquid handling automation and detection instruments, a single investigator should be able to develop a 384-well format high-throughput assay in a period of 3-4 weeks.

ABSTRACT

Disorazoles comprise a family of 29 macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. The major fermentation product, disorazole A 1 , was found previously to irreversibly bind to tubulin and to have potent cytotoxic activity against tumor cells, possibly because of its highly electrophilic epoxide moiety. To test this hypothesis, we synthesized the epoxide-free disorazole C 1 and found it retained potent antiproliferative activity against tumor cells, causing prominent G 2 /M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C 1 produced disorganized microtubules at interphase, misaligned chromosomes during mitosis, apoptosis, and premature senescence in the surviving cell populations. Using a tubulin polymerization assay, we found disorazole C 1 inhibited purified bovine tubulin polymerization, with an IC 50 of 11.8 Ϯ 0.4 M, and inhibited [ 3 H]vinblastine binding noncompetitively, with a K i of 4.5 Ϯ 0.6 M. We also found noncompetitive inhibition of [ 3 H]dolastatin 10 binding by disorazole C 1 , with a K i of 10.6 Ϯ 1.5 M, indicating that disorazole C 1 bound tubulin uniquely among known antimitotic agents. Disorazole C 1 could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence.

Dual anti-HER blockade with neratinib and trastuzumab resulted in significant clinical benefit despite prior exposure to trastuzumab, lapatinib, T-DM1, a taxane, and multiple lines of chemotherapy. In selected populations, inhibiting multiple ErbB-family receptors may be more advantageous than single-agent inhibition. Based on favorable tolerance and efficacy, this three-drug combination will be further assessed in a randomized phase II neoadjuvant trial (NSABP FB-7:NCT01008150).

Key Points Question What is the comparative effectiveness of single-agent immune checkpoint inhibitors (ICIs) vs taxane chemotherapy in populations of patients with metastatic castration-resistant prostate cancer (mCRPC) defined by levels of tumor mutational burden (TMB)? Findings In this comparative effectiveness study of 741 patients with mCRPC, patients with TMB of 10 mutations per megabase (mt/Mb) or greater had significantly longer time to next treatment and overall survival with ICIs vs taxanes. Meaning These findings suggest that in scenarios where taxane use is considered, ICIs are a viable alternate treatment option for patients with mCRPC and TMB of 10 mt/Mb or greater.

RS distribution among N0, N1mi, and N+ patients is similar, suggesting a spectrum of biology and potential chemotherapy benefit exists among node-negative and node-positive ER+/HER2- breast cancer patients. If RxPONDER does not show a chemotherapy benefit in N+ patients with a low RS result, our findings indicate that substantial numbers of patients could be spared the burden of chemotherapy.

Background: Pembrolizumab recently received pan-tumor FDA approval for the treatment of patients with unresectable or metastatic solid tumors with tumor mutational burden (TMB) &gt;10 mutations per megabase (mut/Mb) and who have no satisfactory alternative treatment options. The KEYNOTE-119 and TAPUR trials have validated TMB as a predictive biomarker using TMB cutoffs of ≥ 10 mut/Mb and 9 mut/Mb, respectively, for benefit from single-agent pembrolizumab in subsets of patients with clinically advanced breast cancer (aBC). Limited data exist on which pathological subtypes of aBC are most likely to be TMB &gt;10 mut/Mb (TMB-High). This study analyzed the frequency of TMB-High tumors in aBC subtypes to evaluate variations between subtypes and correlation with additional immunotherapy biomarkers. Methods: Utilizing the Foundation Medicine breast cancer database, 5,475 samples with aBC and pathological subtype information were identified. Pathological subtypes included in this study were ER+/HER2-negative invasive ductal carcinoma (IDC), ER-negative/HER2+ IDC (HER2+), triple negative IDC (TNBC), invasive lobular, inflammatory, metaplastic, mucinous and papillary. TMB was determined on 0.8-1.1 Mb of sequenced DNA, and microsatellite instability (MSI) was assessed across 114 loci in 564 cases. PD-L1 was determined in a subset of cases by immunohistochemistry (Ventana SP142). Results: TMB-High (&gt;10 mut/Mb) was common in all breast cancer pathological subtypes except papillary carcinoma (Table 1). TMB-High was most often found in lobular, inflammatory and HER2+ carcinomas (16%, 14% and 12% of cases, respectively). Metaplastic, mucinous and papillary subtypes were least likely to be TMB-High (7%, 7% and 0% of cases, respectively). TMB-High was more common than MSI-High in all subtypes, excluding papillary. Emerging biomarkers that may play a role in mitigating immunotherapy response, such as MDM2 amplifications and STK11 alterations, were observed in most subtypes. The frequencies of these alterations, as well as alterations associated with FDA-approved therapies in breast cancer, are provided in Table 1 and will be further discussed. Conclusions: TMB-High (&gt;10 mut/Mb) is common in most pathological subtypes of clinically advanced breast cancer and can identify patients not identified by MSI or PD-L1 testing who may benefit from immunotherapy. Based on the high percentage of advanced breast cancer patients who are TMB-High or have genomic alterations in other biomarkers associated with FDA-approved targeted therapies, comprehensive genomic profiling including TMB and MSI should be considered for all pathological subtypes. Table 1Pathological subtypeInvasive ductal carcinoma (IDC)ER+, HER2-negativeER-negative, HER2+Triple-negative (TNBC)Invasive Lobular CarcinomaInflammatory MetaplasticMucinousPapillaryCases (n)1,2371,9536411,180353892911Age55 (23-89)55 (20-89)53 (20-85)62 (24-89)54 (28-87)59 (28-89)55 (30-80)61 (38-78)Tumor mutational burden (TMB)TMB &gt; 10 mut/Mb8%12%9%16%14%7%7%0%TMB &gt; 20 mut/Mb2%2%3%7%6%2%3%0%Additional immunotherapy biomarkersMSI-High0.2%0.1%0.4%0%0%0%0%0%PD-L1+ (&gt;1% Ventana SP142)N/AN/A47%N/AN/A26%0%N/AMDM2 amplification6%5%3%2%0%2%3%18%STK11 alteration1%1%2%1%3%3%3%0%Additional biomarkers associated with FDA-approved therapiesBRCA1/2 alteration3%/6%2%/3%7%/3%1%/4%6%/6%4%/3%3%/0%0%/9%ERBB2 amplification0%100%0%3%26%3%34%9%NTRK 1/2/3 rearrangement0%/0%/0%0%/0%/0%0%/0%/0%1%/0%/0%0%/0%/0%0%/0%/1%0%/0%/0%0%/0%/0%PIK3CA alteration38%38%19%56%34%38%21%36% Citation Format: Mason A Israel, Marni Tierno, Richard SP Huang, Kimberly McGregor, Ethan S Sokol, Prasanth Reddy, Jeffrey S Ross. High tumor mutational burden (&gt;=10 mut/Mb) is enriched in specific breast cancer pathological subtypes [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 PD9-09.

Background Liquid biopsy is a powerful tool that can enable treatment decisions for metastatic prostate cancer patients with difficult‐to‐biopsy tumors. However, the detection of genomic alterations via liquid biopsy is limited by the fraction (tumor fraction [TF]) of circulating tumor DNA (ctDNA) within the total cell‐free DNA content. While prior work has preliminarily correlated TF with clinical features of prostate cancer, we sought to validate and provide additional resolution, such that a clinical practitioner might anticipate the probability of successful liquid biopsy profiling leveraging commonly assessed clinical and laboratory features. Methods A total of 813 liquid biopsy specimens were assessable, with 545 associated with a PSA prostate specific antigen measurement, collected in standard‐of‐care settings across approximately 280 US academic or community‐based cancer clinics from September 2018 to July 2021. Deidentified data were captured into a real‐world clinico‐genomic database (CGDB). Comprehensive genomic profiling (CGP) was performed on extracted cell‐free DNA from liquid biopsy samples. Results In multivariable models, higher PSA level, lower hemoglobin, lower albumin, higher alkaline phosphatase (all p < 0.001), and collection of liquid biopsy blood draw within 60 days of new treatment initiation (p = 0.002) were the most strongly associated features with higher TF. At PSA levels of <5 ng/ml, 43% of patients had a TF of <1% indicating an increased likelihood of unevaluable results. Conversely, at PSA levels of >5 ng/ml, 78% of patients had a TF of at least 1% and 46% had a TF of ≥10%, suggesting improved sensitivity for detection of targetable alterations. Conclusions Universal genomic profiling of prostate cancers will require complementary use of liquid biopsy and tumor tissue profiling for suitable patients. The likelihood of adequate ctDNA shedding into plasma is one consideration when deciding whether to pursue CGP via liquid biopsy versus tumor profiling. Our real‐world data suggest that PSA < 5 ng/ml is associated with lower ctDNA yield on liquid biopsy, potentially increasing the incidence of negative results or a need for confirmation with tissue testing.