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Background In 2020, pembrolizumab was approved as a therapy for triple-negative breast cancer (TNBC) with the companion diagnostic DAKO 22C3 programmed death ligand-1 (PD-L1) immunohistochemistry assay. The study aimed to determine the landscape of PD-L1 expression as detected by the DAKO 22C3 PD-L1 assay in breast cancer subtypes and compare the clinicopathologic and genomic characteristics of PD-L1 positive and negative TNBC. Methods PD-L1 expression using the DAKO 22C3 antibody was scored using a combined positive score (CPS) and positive status was defined as CPS ≥10. Comprehensive genomic profiling was performed using the FoundationOne CDx assay. Results Of the 396 BC patients stained with DAKO 22C3, the majority were HR+/HER2− and TNBC (42% and 36%, respectively). Median PD-L1 expression and frequency of CPS ≥10 was highest in TNBC cases (median: 7.5, 50% CPS ≥10) and lowest in the HR+/HER2− group (median: 1.0, 15.5% CPS ≥10) (P Conclusions The subtypes of breast cancer have distinct patterns of PD-L1 expression, supporting that further research of immunotherapies may include specific evaluation of optimum cutoffs for non-TNBC patients. In TNBC, PD-L1 positivity is not associated with other clinicopathologic or genomic features and should be integrated into future studies of immunotherapy efficacy.

Anti-BRAF/EGFR therapy is approved for metastatic colorectal cancer (mCRC) with BRAFV600E mutations, although not all patients respond. Novel recent findings indicate the potential of RNF43 mutations to predict outcomes in patients with BRAF-mutated microsatellite stable (MSS) mCRC treated with anti-BRAF/EGFR therapy. This study aimed to independently and rapidly validate BRAFV600E/RNF43 co-mutations as predictive biomarkers of benefit to anti-EGFR/BRAF therapy. Clinical data were derived from electronic health record data from ~280 US cancer clinics between January 2011 and March 2022 from the Flatiron Health-Foundation Medicine real-world clinico-genomic mCRC database. Real-world cases of BRAFV600E-mutated mCRC, with patients receiving anti-BRAF/EGFR therapy (n = 49), were included. Patients who were MSS, with RNF43 mutations, had favorable progression-free survival (hazard ratio [HR] 0.29; 95% CI [CI], 0.13-0.65) and overall survival (HR 0.32, 95% CI, 0.12-0.84) compared with wild type. No difference in outcomes was observed between patient groups with RNF43-mutant versus wild-type receiving standard-of-care chemotherapy. BRAFV600E/RNF43 co-mutations predict mCRC anti-BRAF/EGFR outcomes in diverse clinical settings.

Purpose: Alpelisib is a PI3K alpha (PI3Kα)-selective inhibitor approved for the treatment of hormone receptor–positive/HER2-negative (HR+/HER2−) PIK3CA-mutated advanced breast cancer (ABC) based on the SOLAR-1 trial, which defined 11 substitutions in exons 7, 9, and 20 in PIK3CA (SOLAR1m). We report alpelisib effectiveness for ABC harboring SOLAR1m, as well as other pathogenic PIK3CA mutations (OTHERm) using comprehensive genomic profiling (CGP). Experimental Design: A total of 33,977 tissue and 1,587 liquid biopsies were analyzed using hybrid capture–based CGP covering the entire coding sequence of PIK3CA. Clinical characteristics and treatment history were available for 10,750 patients with ABC in the deidentified Flatiron Health-Foundation Medicine clinico-genomic database (FH-FMI CGDB). Results: PIK3CAm were detected in 11,767/33,977 (35%) of tissue biopsies, including 2,300 (7%) samples with OTHERm and no SOLAR1m. Liquid biopsy had 77% sensitivity detecting PIK3CAm, increasing to 95% with circulating tumor DNA fraction ≥2%. In patients with HR+/HER2− ABC and PIK3CAm receiving alpelisib/fulvestrant (ALP+FUL; n = 182) or fulvestrant alone (FUL; n = 119), median real-world progression-free survival (rwPFS) was 5.9 months on ALP+FUL [95% confidence interval (CI): 5.1–7.4] versus 3.1 months on FUL (95% CI: 2.7–3.7; P < 0.0001). In patients with OTHERm, median rwPFS was 4.0 months on ALP+FUL (95% CI: 2.8–10.1) versus 2.5 months on FUL (95% CI: 2.2–3.7; P = 0.0054). Conclusions: CGP detects diverse PIK3CAm in a greater number of patients with ABC than PCR hotspot testing; 20% of patients with PIK3CAm do not have SOLAR1m. These patients may derive benefit from alpelisib. See related commentary by Tau and Miller, p. 989

Patients with advanced gastroesophageal cancer (mEG) and tumor mutational burden ≥10 mut/Mb (TMB ≥ 10) have more favorable outcomes on immune checkpoint inhibitor (ICPI) monotherapy compared with chemotherapy in subgroup analyses of randomized controlled trials. We sought to evaluate the robustness of these associations in real-world settings where patients and practices are more diverse. A total of 362 2 L and 692 1 L patients, respectively received ICPI (n = 99, 33) or chemotherapy (n = 263, 659) across approximately 280 U.S. academic or community-based cancer clinics March 2014–July 2021. Deidentified data were captured into a real-world clinico-genomic database. All patients underwent Foundation Medicine testing. Time to next treatment (TTNT) and overall survival (OS) comparing ICPI versus chemotherapy were adjusted for treatment assignment imbalances using propensity scores. 2L: TMB ≥ 10 had more favorable TTNT [median 24 vs. 4.1 months; HR: 0.19; 95% confidence interval (CI): 0.09–0.44; P = 0.0001] and OS (median 43.1 vs. 6.2 months; HR: 0.24; 95% CI: 0.011–0.54; P = 0.0005), TMB < 10 did not (P > 0.05). 1L: TMB ≥ 10 had more favorable TTNT (not reached vs. median 4.1 months; HR: 0.13; 95% CI: 0.03–0.48; P = 0.0024) and OS (not reached vs. median 17.1 months; HR: 0.30; 95% CI: 0.08–1.14; P = 0.078), TMB < 10 had less favorable TTNT (median 2.8 vs. 6.5 months; HR: 2.36; 95% CI: 1.25–4.45; P = 0.008) and OS (median 4.5 vs. 13.1 months; HR: 1.82, 95% CI: 0.87–3.81; P = 0.11). TMB ≥ 10 robustly identifies patients with mEG with more favorable outcomes on 2 L ICPI monotherapy versus chemotherapy. 1 L data are more limited, but effects are consistent with 2L. Significance: Using real-world data, we sought to evaluate robustness of these clinical associations using the same assay platform and biomarker cut-off point used in both clinical trials and pan-tumor CDx approvals for later treatment lines. TMB ≥ 10 robustly identified patients with mEG with more favorable outcomes on ICPI monotherapy versus chemotherapy and suggests this subset of patients could be targeted for further trial development.

Unadjusted for known treatment assignment imbalances, patients receiving 1st line ICPI vs. chemotherapy have more favorable outcomes when TMB ≥ 10 but not TMB < 10. Kaplan-Meier curves are unadjusted for imbalances (propensity weights applied). TTNT is shown by drug class for (A) TMB < 10, and (B) TMB ≥ 10. OS is shown by drug class for (C) TMB < 10, and (D) TMB ≥ 10. X-axis is truncated at 36 months. Overall survival estimates are left truncated (see Methods) with at-risk tables adjusted accordingly. Interaction models can be found in Supplemental Figure 9.

Summary of TMB ranges per cohort and MSI (TMB10+ only). The median and interquartile range of TMB is shown per group within the cohorts, grouped by MSI status.

Patients with advanced gastroesophageal cancer (mEG) and tumor mutational burden ≥10 mut/Mb (TMB ≥ 10) have more favorable outcomes on immune checkpoint inhibitor (ICPI) monotherapy compared with chemotherapy in subgroup analyses of randomized controlled trials. We sought to evaluate the robustness of these associations in real-world settings where patients and practices are more diverse. A total of 362 2 L and 692 1 L patients, respectively received ICPI (n = 99, 33) or chemotherapy (n = 263, 659) across approximately 280 U.S. academic or community-based cancer clinics March 2014–July 2021. Deidentified data were captured into a real-world clinico-genomic database. All patients underwent Foundation Medicine testing. Time to next treatment (TTNT) and overall survival (OS) comparing ICPI versus chemotherapy were adjusted for treatment assignment imbalances using propensity scores. 2L: TMB ≥ 10 had more favorable TTNT [median 24 vs. 4.1 months; HR: 0.19; 95% confidence interval (CI): 0.09–0.44; P = 0.0001] and OS (median 43.1 vs. 6.2 months; HR: 0.24; 95% CI: 0.011–0.54; P = 0.0005), TMB < 10 did not (P > 0.05). 1L: TMB ≥ 10 had more favorable TTNT (not reached vs. median 4.1 months; HR: 0.13; 95% CI: 0.03–0.48; P = 0.0024) and OS (not reached vs. median 17.1 months; HR: 0.30; 95% CI: 0.08–1.14; P = 0.078), TMB < 10 had less favorable TTNT (median 2.8 vs. 6.5 months; HR: 2.36; 95% CI: 1.25–4.45; P = 0.008) and OS (median 4.5 vs. 13.1 months; HR: 1.82, 95% CI: 0.87–3.81; P = 0.11). TMB ≥ 10 robustly identifies patients with mEG with more favorable outcomes on 2 L ICPI monotherapy versus chemotherapy. 1 L data are more limited, but effects are consistent with 2L.Significance:Using real-world data, we sought to evaluate robustness of these clinical associations using the same assay platform and biomarker cut-off point used in both clinical trials and pan-tumor CDx approvals for later treatment lines. TMB ≥ 10 robustly identified patients with mEG with more favorable outcomes on ICPI monotherapy versus chemotherapy and suggests this subset of patients could be targeted for further trial development.

Supplementary Figure 4. Impact of ctDNA fraction on sensitivity of detection of PIK3CAmutations.

Purpose:Alpelisib is a PI3K alpha (PI3Kα)-selective inhibitor approved for the treatment of hormone receptor–positive/HER2-negative (HR+/HER2−) PIK3CA-mutated advanced breast cancer (ABC) based on the SOLAR-1 trial, which defined 11 substitutions in exons 7, 9, and 20 in PIK3CA (SOLAR1m). We report alpelisib effectiveness for ABC harboring SOLAR1m, as well as other pathogenic PIK3CA mutations (OTHERm) using comprehensive genomic profiling (CGP).Experimental Design:A total of 33,977 tissue and 1,587 liquid biopsies were analyzed using hybrid capture–based CGP covering the entire coding sequence of PIK3CA. Clinical characteristics and treatment history were available for 10,750 patients with ABC in the deidentified Flatiron Health-Foundation Medicine clinico-genomic database (FH-FMI CGDB).Results:PIK3CAm were detected in 11,767/33,977 (35%) of tissue biopsies, including 2,300 (7%) samples with OTHERm and no SOLAR1m. Liquid biopsy had 77% sensitivity detecting PIK3CAm, increasing to 95% with circulating tumor DNA fraction ≥2%. In patients with HR+/HER2− ABC and PIK3CAm receiving alpelisib/fulvestrant (ALP+FUL; n = 182) or fulvestrant alone (FUL; n = 119), median real-world progression-free survival (rwPFS) was 5.9 months on ALP+FUL [95% confidence interval (CI): 5.1–7.4] versus 3.1 months on FUL (95% CI: 2.7–3.7; P < 0.0001). In patients with OTHERm, median rwPFS was 4.0 months on ALP+FUL (95% CI: 2.8–10.1) versus 2.5 months on FUL (95% CI: 2.2–3.7; P = 0.0054).Conclusions:CGP detects diverse PIK3CAm in a greater number of patients with ABC than PCR hotspot testing; 20% of patients with PIK3CAm do not have SOLAR1m. These patients may derive benefit from alpelisib.See related commentary by Tau and Miller, p. 989

Supplementary Figure 9. Progression-free survival (PFS) among patients with SOLAR1mtreated with alpelisib+fulvestrant versus fulvestrant monotherapy.

Supplementary Figure 2. CONSORT diagrams for the cohorts with clinicogenomic data

Supplementary Figure 3. PIK3CA variants detected in paired tissue and liquid samples.

Supplementary Data from SPOP Mutations as a Predictive Biomarker for Androgen Receptor Axis–Targeted Therapy in De Novo Metastatic Castration-Sensitive Prostate Cancer

Supplementary Figure 1. CONSORT diagram of genomic analyses cohorts

Supplementary Figure 8. Overall survival among patients with PIK3CA-mutatedHR+HER2- breast cancer treated with alpelisib/fulvestrant versus fulvestrant alone.

Supplementary Figure from SPOP Mutations as a Predictive Biomarker for Androgen Receptor Axis–Targeted Therapy in De Novo Metastatic Castration-Sensitive Prostate Cancer

Supplementary Figure 6. OTHERm variants show a range of subclonality andindependent oncogenicity

Supplementary Figure 7. Individual covariate balance before and after inverse probabilityof treatment weighting.

Supplementary Figure 5. Comparison of performance of comprehensive genomicprofiling by next-generation sequencing (NGS) and PCR hotspot testing.

Ratio of detection rates in liquid versus tissue.

Purpose:Comprehensive genomic profiling (CGP) is of increasing value for patients with metastatic castration-resistant prostate cancer (mCRPC). mCRPC tends to metastasize to bone, making tissue biopsies challenging to obtain. We hypothesized CGP of cell-free circulating tumor DNA (ctDNA) could offer a minimally invasive alternative to detect targetable genomic alterations (GA) that inform clinical care.Experimental Design:Using plasma from 3,334 patients with mCRPC (including 1,674 screening samples from TRITON2/3), we evaluated the landscape of GAs detected in ctDNA and assessed concordance with tissue-based CGP.Results:A total of 3,129 patients (94%) had detectable ctDNA with a median ctDNA fraction of 7.5%; BRCA1/2 was mutated in 295 (8.8%). In concordance analysis, 72 of 837 patients had BRCA1/2 mutations detected in tissue, 67 (93%) of which were also identified using ctDNA, including 100% of predicted germline variants. ctDNA harbored some BRCA1/2 alterations not identified by tissue testing, and ctDNA was enriched in therapy resistance alterations, as well as possible clonal hematopoiesis mutations (e.g., in ATM and CHEK2). Potential androgen receptor resistance alterations were detected in 940 of 2,213 patients (42%), including amplifications, polyclonal and compound mutations, rearrangements, and novel deletions in exon 8.Conclusions:Genomic analysis of ctDNA from patients with mCRPC recapitulates the genomic landscape detected in tissue biopsies, with a high level of agreement in detection of BRCA1/2 mutations, but more acquired resistance alterations detected in ctDNA. CGP of ctDNA is a compelling clinical complement to tissue CGP, with reflex to tissue CGP if negative for actionable variants.See related commentary by Hawkey and Armstrong, p. 2961

Assessment of PD-L1 Assay Sensitivity and Specificity.

Figures and Tables

Purpose:Aggressive variant prostate cancer (AVPC) represents a clinical subset distinguished by therapy resistance and poor prognosis, linked to combined losses of the tumor suppressor genes (TSG) PTEN, RB1, and TP53. Circulating tumor cells (CTC) provide a minimally invasive opportunity for identification and molecular characterization of AVPC. We aimed to evaluate the incidence and clinical significance of compound (2+)TSG losses and genomic instability in prostate cancer CTC, and to expand the set genomic biomarkers relevant to AVPC.Experimental Design:Genomic analysis of chromosomal copy-number alterations (CNA) at single-cell resolution was performed in CTC from patients with and without AVPC before initiating chemotherapy with cabazitaxel or cabazitaxel and carboplatin. We evaluated associations between single-CTC genomics and clinical features, progression-free survival, and overall survival.Results:A total of 257 individual CTC were sequenced from 47 patients (1–22 CTC/patient). Twenty patients (42.6%) had concurrent 2+TSG losses in at least one CTC in association with poor survival and increased genomic instability, inferred by high large-scale transitions scores. Higher LST in CTC were independent of CTC enumerated, clinically more indicative of aggressive behavior than co-occurring TSG losses, and molecularly associated with gains in chromosomal regions including PTK2, Myc, and NCOA2; increased androgen receptor expression; and BRCA2 loss. In 57 patients with matched cell-free tumor DNA data, CTC were more frequently detectable and evaluable for CNA analysis (in 73.7% vs. 42.1%, respectively).Conclusions:Our findings suggest that genomic instability in CTC is a hallmark of advanced prostate cancer aggressiveness, and support single-CTC sequencing as a compelling tool to noninvasively characterize cancer heterogeneity.

Background: Lung cancer treatment has become increasingly dependent upon invasive biopsies to profile tumors for personalized therapy. Recently, tumor expression of programmed death-ligand 1 (PD-L1) has gained interest as a potential predictor of response to immunotherapy. Circulating biomarkers present an opportunity for tumor profiling without the risks of invasive procedures. We characterized PD-L1 expression within populations of nucleated cells in the peripheral blood of lung cancer patients in hopes of expanding the role of liquid biopsy in this setting.Methods: Peripheral blood samples from a multi-institutional prospective study of patients with clinical diagnosis of lung cancer were subjected to cytomorphometric and immunohistochemical evaluation using single-cell, automated slide-based, digital pathology. PD-L1 expression was determined by immunofluorescence.Results: PD-L1 expression was detected within peripheral circulating cells associated with malignancy (CCAM) in 26 of 112 (23%) non–small cell lung cancer patients. Two distinct populations of nucleated, nonhematolymphoid, PD-L1–expressing cells were identified; cytokeratin positive (CK+, PD-L1+, CD45−) and cytokeratin negative (CK−, PD-L1+, CD45−) cells, both with cytomorphometric features (size, nuclear-to-cytoplasm ratio) consistent with tumor cells. Patients with >1.1 PD-L1(+) cell/mL (n = 14/112) experienced worse overall survival than patients with ≤1.1 PD-L1(+) cell/mL (2-year OS: 31.2% vs. 78.8%, P = 0.00159). In a Cox model adjusting for stage, high PD-L1(+) cell burden remained a significant predictor of mortality (HR = 3.85; 95% confidence interval, 1.64–9.09; P = 0.002).Conclusions: PD-L1 expression is detectable in two distinct cell populations in the peripheral blood of lung cancer patients and is associated with worse survival.Impact: These findings could represent a step forward in the development of minimally invasive liquid biopsies for the profiling of tumors. Cancer Epidemiol Biomarkers Prev; 26(7); 1139–45. ©2017 AACR.

Figure S1: Examples of a decision boundary that separate class A (red) from class B (blue) in 1 dimension (figure 1A) and 2 dimensions (figure 1B); Figure S2: Schematic of the supervised learning process (A) and leave one out cross validation (B); Figure S3: Single iteration of Leave-One-Out Cross-Validation performed at the blood sample level; Figure S4: An example of concordance of AURKA amplification in tumor and CTCs; Figure S5: (A) Kernel Density Estimate (KDE) curves of the classifier output are plotted for each patient sample colored by their diagnosis: NEPC (red) and CRPC (blue); Figure S6: Cell-level NEPC classifier (A) Receiver-Operating-Characteristic (ROC) curve generated on the classifier's single-cell output after LOOCV; Figure S7: To address whether the CTC classifier is simply stochastic, a reflection of an overall higher CTC count, linearity was assessed with a Pearson's coefficient showing a weak relationship between frequency of NEPC CTCs and total cell count; Table S1: A summary of cell-level features utilized to train Random Forest cell-level classifiers in both the LOOCV and for the classification of CTCs in the test cohort; Table S2: Patient characteristics (discovery cohort) including prior systemic therapies, serum markers including PSA (ng/ml), Chromogranin (ng/ml), NSE (ng/ml) and CTC counts; Table S3: Liver metastases in NEPC vs. CRPC; Table S4: The median concentration of CK-negative and AR-negative CTCs in CRPC, atypical CRPC, and NEPC patients; Table S5: Confusion matrix for the ability of CD56 staining to discriminate patients diagnosed with Small Cell Carcinoma NEPC vs CRPC; Table S6: Example of results from a single tube of blood.

Positive percent agreement (PPA) between patient-matched cancer tissue and liquid biopsies.

The heterogeneity of an individual patient's tumor has been linked to treatment resistance, but quantitative biomarkers to rapidly and reproducibly evaluate heterogeneity in a clinical setting are currently lacking. Using established tools available in a College of American Pathologists–accredited and Clinical Laboratory Improvement Amendments–certified clinical laboratory, we quantified digital pathology features on 9,225 individual circulating tumor cells (CTC) from 179 unique metastatic castration-resistant prostate cancer (mCRPC) patients to define phenotypically distinct cell types. Heterogeneity was quantified on the basis of the diversity of cell types in individual patient samples using the Shannon index and associated with overall survival (OS) in the 145 specimens collected prior to initiation of the second or later lines of therapy. Low CTC phenotypic heterogeneity was associated with better OS in patients treated with androgen receptor signaling inhibitors (ARSI), whereas high heterogeneity was associated with better OS in patients treated with taxane chemotherapy. Overall, the results show that quantifying CTC phenotypic heterogeneity can help inform the choice between ARSI and taxanes in mCRPC patients. Cancer Res; 77(20); 5687–98. ©2017 AACR.

Purpose: The transition of prostate adenocarcinoma to a predominantly androgen receptor (AR) signaling independent phenotype can occur in the later stages of the disease and is associated with low AR expression +/− the development of small-cell or neuroendocrine tumor characteristics. As metastatic tumor biopsies are not always feasible and are difficult to repeat, we sought to evaluate noninvasive methods to identify patients transitioning toward a neuroendocrine phenotype (NEPC).Experimental Design: We prospectively studied a metastatic tumor biopsy, serum biomarkers, and circulating tumor cells (CTC, Epic Sciences) from patients with castration-resistant prostate cancer (CRPC) including those with pure or mixed NEPC histology present on biopsy. CTCs labeled with the patient's clinical status were used to learn features that discriminate NEPC patients, which was then applied to an independent cohort.Results: Twenty-seven patients with CRPC including 12 NEPC and 5 with atypical clinical features suggestive of NEPC transition were studied. CTCs from NEPC patients demonstrated frequent clusters, low or absent AR expression, lower cytokeratin expression, and smaller morphology relative to typical CRPC. A multivariate analysis of protein and morphologic variables enabled distinguishing CTCs of NEPC from CRPC. This CTC classifier was applied to an independent prospective cohort of 159 metastatic CRPC patients and identified in 17/159 (10.7%) of cases, enriched in patients with high CTC burden (P < 0.01) and visceral metastases (P = 0.04).Conclusions: CTCs from patients with NEPC have unique morphologic characteristics, which were also identified in a subset of CRPC patients with aggressive clinical features potentially undergoing NEPC transition. Clin Cancer Res; 22(6); 1510–9. ©2015 AACR.

Cox Proportional Hazards Model of Patients With Confirmed Lung Cancer.

Background In the current study, we examined the real-world prevalence of highly pigmented advanced melanomas (HPMel) and the clinicopathologic, genomic, and ICPI biomarker signatures of this class of tumors. Materials and Methods Our case archive of clinical melanoma samples for which the ordering physician requested testing for both PD-L1 immunohistochemistry (IHC) and comprehensive genomic profiling (CGP) was screened for HPMel cases, as well as for non-pigmented or lightly pigmented advanced melanoma cases (LPMel). Results Of the 1268 consecutive melanoma biopsies in our archive that had been submitted for PD-L1 IHC, 13.0% (165/1268) were HPMel and 87.0% (1103/1268) were LPMel. In the HPMel cohort, we saw a significantly lower tumor mutational burden (TMB, median 8.8 mutations/Mb) than in the LPMel group (11.4 mut/Mb), although there was substantial overlap. In examining characteristic secondary genomic alterations (GA), we found that the frequencies of GA in TERTp, CDKN2A, TP53, and PTEN were significantly lower in the HPMel cases than in LPMel. A higher rate of GA in CTNNB1, APC, PRKAR1A, and KIT was identified in the HPMel cohort compared with LPMel. Conclusions In this study, we quantified the failure rates of melanoma samples for PD-L1 testing due to high melanin pigmentation and showed that CGP can be used in these patients to identify biomarkers that can guide treatment decisions for HPMel patients. Using this practical clinical definition for tumor pigmentation, our results indicate that HPMel are frequent at 13% of melanoma samples, and in general appear molecularly less developed, with a lower TMB and less frequent secondary GA of melanoma progression.

Alpelisib is a phosphatidylinositol 3-kinase alpha (PI3Kα)-selective inhibitor approved for the treatment of hormone receptor positive/HER2 negative (HR+/HER2-) PIK3CA-mutated advanced breast cancer (ABC) based on the SOLAR-1 trial, which defined 11 substitutions in exons 7, 9, and 20 in PIK3CA (SOLAR1m). We report alpelisib effectiveness for ABC harboring SOLAR1m, as well as other pathogenic PIK3CA mutations (OTHERm) using comprehensive genomic profiling (CGP).33,977 tissue and 1,587 liquid biopsies were analyzed using hybrid-capture-based CGP covering the entire coding sequence of PIK3CA. Clinical characteristics and treatment history were available for 10,750 ABC patients in the de-identified Flatiron Health-Foundation Medicine clinico-genomic database (FH-FMI CGDB).PIK3CAm were detected in 11,767 / 33,977 (35%) of tissue biopsies, including 2,300 (7%) samples with OTHERm and no SOLAR1m. Liquid biopsy had 77% sensitivity detecting PIK3CAm, increasing to 95% with ctDNA fraction ≥2%. In patients with HR+/HER2- ABC and PIK3CAm receiving alpelisib/fulvestrant (ALP+FUL; n = 182) or fulvestrant alone (FUL; n = 119), median real-world progression free survival (rwPFS) was 5.9 months on ALP+FUL [95%CI: 5.1 - 7.4] versus 3.1 months on FUL [95%CI: 2.7 - 3.7] (p0.0001). In patients with OTHERm, median rwPFS was 4.0 months on ALP+FUL [95%CI: 2.8 - 10.1] versus 2.5 months on FUL [95%CI: 2.2 - 3.7] (p = 0.0054).CGP detects diverse PIK3CAm in a greater number of patients with ABC than PCR hotspot testing; 20% of patients with PIK3CAm do not have SOLAR1m. These patients may derive benefit from alpelisib.

BackgroundMultiple PD-L1 immunohistochemistry (IHC) assays, including DAKO 22C3, DAKO 28-8, and Ventana SP142 PD-L1 IHC assays, have been approved by the Food and Drug Administration as a companion diagnostic (CDx) for various antiprogrammed death-1 and antiprogrammed death ligand 1 (PD-L1) based cancer immunotherapies. Here we present 22C3, 28-8, and SP142 analysis of 418 tumor specimens encountered in routine clinical practice.MethodsAll specimens were tested with 22C3, 28-8, and SP142 assays following the manufacturer’s established staining protocols.ResultsThe same PD-L1 status (defined as tumor cell expression (TC) scores with all three assays ≥1% or all ConclusionsOur data suggest that 22C3 is the most sensitive PD-L1 IHC assay for tumor cell expression, followed by 28-8 and in turn by SP-142. These findings represent an additional factor for clinical teams to consider when deciding which PD-L1 IHC assay (and in turn which CDx-associated PD-L1 based immunotherapy) is most appropriate for each individual patient.

PURPOSE In real-world settings, patients with metastatic urothelial carcinoma (mUC) are often more frail than clinical trials, underscoring an unmet need to identify patients who might be spared first-line chemotherapy. We sought to determine whether tumor mutational burden (TMB) identifies patients with comparable or superior clinical benefit of first-line single-agent immune checkpoint inhibitors (ICPI) in real-world patients deemed cisplatin-unfit. METHODS Patients with mUC treated in first-line advanced setting (N = 401) received ICPI (n = 245) or carboplatin regiment without ICPI (n = 156) at physician's discretion in standard-of-care settings across approximately 280 US academic or community-based cancer clinics between March 2014 and July 2021. Deidentified data were captured into a real-world clinicogenomic database. All patients underwent testing using Foundation Medicine assays. Progression-free survival (PFS), time to next treatment (TTNT), and overall survival (OS) comparing ICPI versus chemotherapy were adjusted for known treatment assignment imbalances using propensity scores. RESULTS TMB ≥ 10 was detected in 122 of 401 (30.4%) patients. Among patients receiving ICPI, those with TMB ≥ 10 had more favorable PFS (HR, 0.59; 95% CI, 0.41 to 0.85), TTNT (HR, 0.59; 95% CI, 0.43 to 0.83), and OS (HR, 0.47; 95% CI, 0.32 to 0.68). Comparing ICPI versus carboplatin, adjusting for imbalances, patients with TMB ≥ 10 had more favorable PFS (HR, 0.51; 95% CI, 0.32 to 0.82), TTNT (HR, 0.56; 95% CI, 0.35 to 0.91), and OS (HR, 0.56; 95% CI, 0.29 to 1.08) on ICPI versus chemotherapy, but not TMB < 10. Comparisons unadjusted for imbalances had similar associations. CONCLUSIONS In real-world settings, mUC patients with TMB ≥ 10 have more favorable outcomes on first-line single-agent ICPI than carboplatin, adding clinical validity to TMB assessed by an existing US Food and Drug Administration–approved platform.

Purpose: Intensification of androgen deprivation therapy (ADT) with either docetaxel or androgen receptor axis–targeted therapies (ARAT) are the current standard of care for patients with metastatic castration-sensitive prostate cancer (mCSPC). However, biomarkers guiding treatment selection are lacking. We hypothesized that ADT intensification with ARAT, but not with docetaxel, would be associated with improved outcomes in patients with de novo (dn)-mCSPC harboring SPOP mutations. Experimental Design: Patient-level data from a deidentified nationwide (U.S.-based) prostate cancer clinico-genomic database between January 2011 and December 2021 were extracted. Eligibility criteria: diagnosis of metastatic disease within 30 days of original prostate cancer diagnosis, genomic profiling of a tissue biopsy collected within 90 days of original diagnosis, and initiation of ARAT or docetaxel within 120 days of initial diagnosis. The log-rank test and Cox proportional hazards models were used to compare time to castration-resistant prostate cancer (TTCRPC) and overall survival (OS) for patients with and without SPOP mutations undergoing ADT intensification with ARAT or docetaxel. Results: In the ARAT cohort, presence of SPOP mutation compared with wild-type was associated with more favorable TTCRPC [not reached (NR) vs. 16.7 months; adjusted HR (aHR), 0.20; 95% confidence interval (CI), 0.06–0.63; P = 0.006] and OS (NR vs. 27.2 months; aHR, 0.19; 95% CI, 0.05–0.79; P = 0.022). In contrast, SPOP mutation status was not associated with TTCRPC or OS in docetaxel-treated cohort. Conclusions: In real-world settings, SPOP mutations were associated with improved outcomes to ADT plus ARAT (but not ADT plus docetaxel) in patients with dn-mCSPC. This may serve as a predictive biomarker to guide treatment selection for patients with mCSPC.

PURPOSE To study the impact of standard-of-care hormonal therapies on metastatic prostate cancer (mPC) clinical genomic profiles in real-world practice, with a focus on homologous recombination-repair (HRR) genes. PATIENTS AND METHODS Targeted next-generation sequencing of 1,302 patients with mPC was pursued using the FoundationOne or FoundationOne CDx assays. Longitudinal clinical data for correlative analysis were curated via technology-enabled abstraction of electronic health records. Genomic biomarkers, including individual gene aberrations and genome-wide loss-of-heterozygosity (gLOH) scores, were compared according to biopsy location and time of sample acquisition (androgen deprivation therapy [ADT]-naïve, ADT-progression and post-ADT, and novel hormonal therapies [NHT]-progression), using chi-square and Wilcoxon rank-sum tests. Multivariable analysis used linear regression. False-discovery rate of 0.05 was applied to account for multiple comparisons. RESULTS Eight hundred forty (65%), 132 (10%), and 330 (25%) biopsies were ADT-naïve, ADT-progression, and NHT-progression, respectively. Later-stage samples were enriched for AR, MYC, TP53, PTEN, and RB1 aberrations (all adjusted P values < .05), but prevalence of HRR-related BRCA2, ATM, and CDK12 aberrations remained stable. Primary and metastatic ADT-naïve biopsies presented similar prevalence of TP53 (36% v 31%) and BRCA2 (8% v 7%) aberrations; 81% of ADT-naïve BRCA2-mutated samples presented BRCA2 biallelic loss. Higher gLOH scores were independently associated with HRR genes ( BRCA2, PALB2, and FANCA), TP53, and RB1 aberrations, and with prior exposure to hormonal therapies in multivariable analysis. CONCLUSION Prevalence of HRR-gene aberrations remains stable along mPC progression, supporting the use of diagnostic biopsies to guide poly (ADP-ribose) polymerase inhibitor treatment in metastatic castration-resistant prostate cancer. gLOH scores increase with emerging resistance to hormonal therapies, independently of individual HRR gene mutations.

Challenges with sequencing tissue samples from patients with prostate cancer have been reported in clinical trials. To assess the success rate of comprehensive genomic profiling (CGP) for prostate cancer patients, we analyzed a real-world cohort who underwent sequencing of their prostate tissue sample as well as a subset of patients with a reflex liquid biopsy. Overall, a significant majority (82%) of tissue prostate carcinoma samples yielded reportable CGP results. Of those samples that were unsuccessful, most (75%) were inadequate samples that did not meet pre-established criteria to advance into sequencing. For cases where liquid CGP was performed if tissue CGP was unsuccessful, mutations that were likely attributable to prostate carcinoma were observed in most cases and all cases were successful in generating a report. These results suggest that, for CGP testing, prostate cancer tissue is a reasonable matrix type and that liquid samples can be effectively used as an alternative to tissue.

The most useful biomarkers for clinical decision-making identify patients likely to have improved outcomes with one treatment vs another.To evaluate treatment class-specific outcomes of patients receiving immune checkpoint inhibitor (ICI) vs taxane chemotherapy by tumor mutational burden (TMB).This comparative effectiveness analysis of clinical variables and outcomes used prospectively defined biomarker-stratified genomic data from a deidentified clinicogenomic database. Data included men with previously treated metastatic castration-resistant prostate cancer (mCRPC) receiving ICI or single-agent taxane chemotherapy from January 2011 to April 2021 at approximately 280 US academic or community-based cancer clinics (approximately 800 sites of care). Data were analyzed from July to August 2021.Single-agent ICI or single-agent taxanes. Treatments were assigned at discretion of physician and patient without randomization. Imbalances of known factors between treatment groups were adjusted with propensity weighting.Prostate-specific antigen (PSA) response, time to next therapy (TTNT), and overall survival (OS).A total of 741 men (median [IQR], 70 [64-76] years) with mCRPC received comprehensive genomic profiling and were treated with ICI or single-agent taxane therapy. At baseline, the median (IQR) PSA level was 79.4 (19.0-254) ng/mL, 108 men (18.8%) had Eastern Cooperative Oncology Group Performance Status scores of 2 or greater, and 644 men (86.9%) had received prior systemic treatments for mCRPC. A total of 45 patients (6.1%) received ICI therapy and 696 patients (93.9%) received taxane therapy. Among patients with TMB of fewer than 10 mutations per megabase (mt/Mb) receiving ICI, compared with those receiving taxanes, had worse TTNT (median [IQR], 2.4 [1.1-3.2] months vs 4.1 [2.2-6.3] months; hazard ratio [HR], 2.65; 95% CI, 1.78-3.95; P .001). In contrast, for patients with TMB of 10 mt/Mb or greater, use of ICIs, compared with use taxanes, was associated with more favorable TTNT (median [IQR], 8.0 [3.4 to unknown] months vs 2.4 [2.4-7.3] months; HR, 0.37, 95% CI, 0.15-0.87; P = .02) and OS (median 19.9 [8.06 to unknown] months vs 4.2 [2.69 - 6.12] months; HR, 0.23; 95% CI, 0.10-0.57; P = .001). Among all 741 patients, 44 (5.9%) had TMB of 10 mt/Mb or greater, 22 (3.0%) had high microsatellite instability, and 20 (2.7%) had both. Treatment interactions with TMB of 10 mt/Mb or greater (TTNT: HR, 0.10; 95% CI, 0.32-0.31; P .001; OS: HR, 0.25; 95% CI, 0.076-0.81; P = .02) were stronger than high microsatellite instability alone (TTNT: HR, 0.12; 95% CI, 0.03-0.51; P = .004; OS: HR, 0.38; 95% CI, 0.13-1.12; P = .08).In this comparative effectiveness study, ICIs were more effective than taxanes in patients with mCRPC when TMB was 10 mt/Mb or greater but not when TMB was fewer than 10 mt/Mb. The results add validity to the existing TMB cutoff of 10 mt/Mb for ICI use in later lines of therapy, and suggest that ICIs may be a viable alternative to taxane chemotherapy for patients with mCRPC with high TMB.

Introduction: Here we describe the development of a protein immunofluorescent assay for the detection of nuclear-localized androgen receptor variant 7 (AR-V7) protein within circulating tumor cells (CTCs) identified in patient blood samples. Used in the clinic, the test result serves as a validated biomarker of futility for patients with progressing metastatic castration-resistant prostate cancer (mCRPC) who are treated with androgen receptor targeted therapies (AATT) in whom nuclear-localized AR-V7 CTCs are identified and have received level 2A evidence in the 2019 National Cancer Center Network (NCCN) guidelines (v1.0). Methods: Assay development was completed on the Epic Sciences rare cell detection platform using control cell lines of known AR-V7 status and clinical testing of mCRPC patient samples obtained at the decision point in management. Results and conclusions: Using these samples, all assay parameters, scoring criteria, and clinical cutoffs for positivity were prospectively selected and locked. After assay lock, blinded clinical validation testing was initiated on multiple, independent, clinical cohorts as reported by Scher et al (JAMA Oncol. 2016;2:1441-1449; JAMA Oncol. 2018;4:1179-1186) and Armstrong et al (J Clin Oncol. 2019;37:1120-1129).

Purpose: Aggressive variant prostate cancer (AVPC) represents a clinical subset distinguished by therapy resistance and poor prognosis, linked to combined losses of the tumor suppressor genes (TSG) PTEN, RB1, and TP53. Circulating tumor cells (CTC) provide a minimally invasive opportunity for identification and molecular characterization of AVPC. We aimed to evaluate the incidence and clinical significance of compound (2+)TSG losses and genomic instability in prostate cancer CTC, and to expand the set genomic biomarkers relevant to AVPC. Experimental Design: Genomic analysis of chromosomal copy-number alterations (CNA) at single-cell resolution was performed in CTC from patients with and without AVPC before initiating chemotherapy with cabazitaxel or cabazitaxel and carboplatin. We evaluated associations between single-CTC genomics and clinical features, progression-free survival, and overall survival. Results: A total of 257 individual CTC were sequenced from 47 patients (1–22 CTC/patient). Twenty patients (42.6%) had concurrent 2+TSG losses in at least one CTC in association with poor survival and increased genomic instability, inferred by high large-scale transitions scores. Higher LST in CTC were independent of CTC enumerated, clinically more indicative of aggressive behavior than co-occurring TSG losses, and molecularly associated with gains in chromosomal regions including PTK2, Myc, and NCOA2; increased androgen receptor expression; and BRCA2 loss. In 57 patients with matched cell-free tumor DNA data, CTC were more frequently detectable and evaluable for CNA analysis (in 73.7% vs. 42.1%, respectively). Conclusions: Our findings suggest that genomic instability in CTC is a hallmark of advanced prostate cancer aggressiveness, and support single-CTC sequencing as a compelling tool to noninvasively characterize cancer heterogeneity.

Background Proof of the clinical utility of a biomarker is when its use informs a management decision and improves patient outcomes relative to when it is not used. Objective To model the clinical benefit of the nuclear-localized androgen receptor splice variant 7 (AR-V7) test for men with progressing metastatic castration-resistant prostate cancer (mCRPC) at the second line of therapy or greater to inform the choice of an androgen receptor signaling inhibitor (ARSI) or a taxane. Design, setting, and participants The study population was a cross-sectional cohort of 193 unique patients with progressing mCRPC from whom 255 samples were drawn at the time of the second line or later treatment decision who then received an ARSI or taxane, with up to 3 yr of additional follow-up Circulating tumor cells (CTCs) were identified from blood samples and tested for AR-V7. Physicians were blinded to AR-V7 status and the testing laboratory was blinded to outcomes. Outcome measurements and statistical analyses We measured physician propensity for choosing an ARSI or taxane based on patient prognosis. We also measured overall survival (OS) adjusted for physician propensity by drug class; OS data were analyzed both without and with knowledge of nuclear-localized AR-V7 status. Results and limitations Treating physicians had a propensity for choosing a taxane over an ARSI for patients with more advanced disease or who received an ARSI as the immediate prior therapy. After adjusting for physician propensity, discernible OS differences were not observed between taxane- and ARSI-treated patients (median 15.6 vs 14.4 mo; p =0.11). Patients with detectable nuclear-localized AR-V7 in CTCs had superior survival with taxanes over ARSIs (median 9.8 vs 5.7 mo; p = 0.041). AR-V7–negative patients had superior survival on ARSIs over taxanes (p = 0.033) but overlapping curves limit the interpretation. Mutivariable models showed a robust interaction between AR-V7 status and drug, and a lower risk of death on taxanes for AR-V7–positive men. Conclusions Use of the nuclear-localized AR-V7 CTC test to inform treatment choice can improve patient outcomes relative to decisions based solely on standard-of-care measures. Patient summary Men with metastatic prostate cancer who test positive for AR-V7 protein in circulating tumor cells are likely to live longer if taxane chemotherapy is used.

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.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.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 of5 ng/ml, 43% of patients had a TF of1% indicating an increased likelihood of unevaluable results. Conversely, at PSA levels of5 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.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.

ImportanceThe KEYNOTE-177 trial demonstrated that patients with metastatic colorectal cancer (MCRC) with high microsatellite instability (MSI-H) and/or mismatch repair deficiency (DMMR) have better outcomes when receiving first-line immune checkpoint inhibitors (ICIs) compared with chemotherapy. Data on performance of ICIs in patients with MCRC in standard practice settings remain limited, and direct MMR vs MSI outcome association comparisons are lacking.ObjectiveTo validate MSI (determined by next-generation sequencing [NGS]) as a biomarker of ICI effectiveness among patients with MCRC in standard practice settings and examine the association of MSI assessed by NGS, DMMR by immunohistochemistry, and tumor mutational burden (cutoff, 10 mutations/megabase) with ICI outcomes.Design, Setting, and ParticipantsThis comparative effectiveness research study of outcomes in prospectively defined biomarker subgroups used data from a deidentified clinicogenomic database and included patients who received Foundation Medicine testing (FoundationOne or FoundationOne CDx) during routine clinical care at approximately 280 US academic or community-based cancer clinics between March 2014 and December 2021. The population included 1 cohort of patients with MSI-H MCRC who received first-line ICIs or chemotherapy and a second cohort who received ICIs in any line of therapy (LOT) for biomarker examination.ExposuresICI therapy or chemotherapy assigned at physician discretion without randomization.Main Outcomes and MeasuresThe main outcomes were time to next treatment (TTNT), progression-free survival (PFS), and overall survival (OS). Hazard ratios were adjusted for known prognostic imbalances. Comparisons of explanatory power used the likelihood ratio test.ResultsA total of 138 patients (median age, 67.0 years [IQR, 56.2-74.0 years]; 73 [52.9%] female) with MSI-H MCRC received first-line ICIs or chemotherapy. A total of 182 patients (median age, 64.5 years [IQR, 55.2-72.0]; 98 [53.8%] female) received ICIs in any LOT. Patients receiving first-line ICIs vs chemotherapy had longer TTNT (median, not reached [NR] vs 7.23 months [IQR, 6.21-9.72 months]; adjusted hazard ratio [AHR], 0.17; 95% CI, 0.08-0.35; P P P = .02). MSI added to DMMR better anticipated TTNT and PFS in patients receiving ICIs than DMMR alone. The same was not observed when DMMR evaluation was added to MSI.Conclusions and RelevanceIn this comparative effectiveness research study, MSI assessed by NGS robustly identified patients with favorable outcomes on first-line ICIs vs chemotherapy and appeared to better anticipate ICI outcomes compared with DMMR.