The IL6/JAK/STAT3 signaling axis is a therapeutic vulnerability in SMARCB1-deficient bladder cancer.

SMARCB1 loss has long been observed in many solid tumors. However, there is a need to elucidate targetable pathways driving growth and metastasis in SMARCB1-deficient tumors. Here, we demonstrate that SMARCB1 deficiency, defined as genomic SMARCB1 copy number loss associated with reduced mRNA, drives disease progression in patients with bladder cancer by engaging STAT3. SMARCB1 loss increases the chromatin accessibility of the STAT3 locus in vitro. Orthotopically implanted SMARCB1 knockout (KO) cell lines exhibit increased tumor growth and metastasis. SMARCB1-deficient tumors show an increased IL6/JAK/STAT3 signaling axis in in vivo models and patients. Furthermore, a pSTAT3 selective inhibitor, TTI-101, reduces tumor growth in SMARCB1 KO orthotopic cell line-derived xenografts and a SMARCB1-deficient patient derived xenograft model. We have identified a gene signature generated from SMARCB1 KO tumors that predicts SMARCB1 deficiency in patients. Overall, these findings support the clinical evaluation of STAT3 inhibitors for the treatment of SMARCB1-deficient bladder cancer.

Endocrine-Sensitive Disease Rate in Postmenopausal Patients With Estrogen Receptor-Rich/ERBB2-Negative Breast Cancer Receiving Neoadjuvant Anastrozole, Fulvestrant, or Their Combination: A Phase 3 Randomized Clinical Trial.

Importance: Adding fulvestrant to anastrozole (A+F) improved survival in postmenopausal women with advanced estrogen receptor (ER)-positive/ERBB2 (formerly HER2)-negative breast cancer. However, the combination has not been tested in early-stage disease. Objective: To determine whether neoadjuvant fulvestrant or A+F increases the rate of pathologic complete response or ypT1-2N0/N1mic/Ki67 2.7% or less residual disease (referred to as endocrine-sensitive disease) over anastrozole alone. Design, Setting, and Participants: A phase 3 randomized clinical trial assessing differences in clinical and correlative outcomes between each of the fulvestrant-containing arms and the anastrozole arm. Postmenopausal women with clinical stage II to III, ER-rich (Allred score 6-8 or >66%)/ERBB2-negative breast cancer were included. All analyses were based on data frozen on March 2, 2023. Interventions: Patients received anastrozole, fulvestrant, or a combination for 6 months preoperatively. Tumor Ki67 was assessed at week 4 and optionally at week 12, and if greater than 10% at either time point, the patient switched to neoadjuvant chemotherapy or immediate surgery. Main Outcomes and Measures: The primary outcome was the endocrine-sensitive disease rate (ESDR). A secondary outcome was the percentage change in Ki67 after 4 weeks of neoadjuvant endocrine therapy (NET) (week 4 Ki67 suppression). Results: Between February 2014 and November 2018, 1362 female patients (mean [SD] age, 65.0 [8.2] years) were enrolled. Among the 1298 evaluable patients, ESDRs were 18.7% (95% CI, 15.1%-22.7%), 22.8% (95% CI, 18.9%-27.1%), and 20.5% (95% CI, 16.8%-24.6%) with anastrozole, fulvestrant, and A+F, respectively. Compared to anastrozole, neither fulvestrant-containing regimen significantly improved ESDR or week 4 Ki67 suppression. The rate of week 4 or week 12 Ki67 greater than 10% was 25.1%, 24.2%, and 15.7% with anastrozole, fulvestrant, and A+F, respectively. Pathologic complete response/residual cancer burden class I occurred in 8 of 167 patients and 17 of 167 patients, respectively (15.0%; 95% CI, 9.9%-21.3%), after switching to neoadjuvant chemotherapy due to week 4 or week 12 Ki67 greater than 10%. PAM50 subtyping derived from RNA sequencing of baseline biopsies available for 753 patients (58%) identified 394 luminal A, 304 luminal B, and 55 nonluminal tumors. A+F led to a greater week 4 Ki67 suppression than anastrozole alone in luminal B tumors (median [IQR], -90.4% [-95.2 to -81.9%] vs -76.7% [-89.0 to -55.6%]; P < .001), but not luminal A tumors. Thirty-six nonluminal tumors (65.5%) had a week 4 or week 12 Ki67 greater than 10%. Conclusions and Relevance: In this randomized clinical trial, neither fulvestrant nor A+F significantly improved the 6-month ESDR over anastrozole in ER-rich/ERBB2-negative breast cancer. Aromatase inhibition remains the standard-of-care NET. Differential NET response by PAM50 subtype in exploratory analyses warrants further investigation. Trial Registration: ClinicalTrials.gov Identifier: NCT01953588.

Kinase inhibitor pulldown assay (KiP) for clinical proteomics.

Protein kinases are frequently dysregulated and/or mutated in cancer and represent essential targets for therapy. Accurate quantification is essential. For breast cancer treatment, the identification and quantification of the protein kinase ERBB2 is critical for therapeutic decisions. While immunohistochemistry (IHC) is the current clinical diagnostic approach, it is only semiquantitative. Mass spectrometry-based proteomics offers quantitative assays that, unlike IHC, can be used to accurately evaluate hundreds of kinases simultaneously. The enrichment of less abundant kinase targets for quantification, along with depletion of interfering proteins, improves sensitivity and thus promotes more effective downstream analyses. Multiple kinase inhibitors were therefore deployed as a capture matrix for kinase inhibitor pulldown (KiP) assays designed to profile the human protein kinome as broadly as possible. Optimized assays were initially evaluated in 16 patient derived xenograft models (PDX) where KiP identified multiple differentially expressed and biologically relevant kinases. From these analyses, an optimized single-shot parallel reaction monitoring (PRM) method was developed to improve quantitative fidelity. The PRM KiP approach was then reapplied to low quantities of proteins typical of yields from core needle biopsies of human cancers. The initial prototype targeting 100 kinases recapitulated intrinsic subtyping of PDX models obtained from comprehensive proteomic and transcriptomic profiling. Luminal and HER2 enriched OCT-frozen patient biopsies subsequently analyzed through KiP-PRM also clustered by subtype. Finally, stable isotope labeled peptide standards were developed to define a prototype clinical method. Data are available via ProteomeXchange with identifiers PXD044655 and PXD046169.

The uncharted role of HER2 mutant alleles in breast cancer.

Somatic HER2 mutations are a novel class of therapeutic targets across different cancer types. Treatment with the tyrosine kinase inhibitor (TKI) neratinib as a single agent continues to be evaluated in HER2-mutant metastatic disease. However, responses are heterogeneous, with frequent early progression. Herein, we discuss the under-explored effects of individual HER2 mutant alleles on therapeutic response, a role for HER2 mutation in metastatic propensity, and differences in patient outcomes in ER+ invasive lobular carcinoma (ILC) versus invasive ductal carcinoma (IDC). The preclinical efficacy of additional agents is also discussed, particularly the pan-HER inhibitor poziotinib.

Mass spectrometry quantifies target engagement for a KRASG12C inhibitor in FFPE tumor tissue.

BACKGROUND: Quantification of drug-target binding is critical for confirming that drugs reach their intended protein targets, understanding the mechanism of action, and interpreting dose-response relationships. For covalent inhibitors, target engagement can be inferred by free target levels before and after treatment. Targeted mass spectrometry assays offer precise protein quantification in complex biological samples and have been routinely applied in pre-clinical studies to quantify target engagement in frozen tumor tissues for oncology drug development. However, frozen tissues are often not available from clinical trials so it is critical that assays are applicable to formalin-fixed, paraffin-embedded (FFPE) tissues in order to extend mass spectrometry-based target engagement studies into clinical settings. METHODS: Wild-type RAS and RASG12C was quantified in FFPE tissues by a highly optimized targeted mass spectrometry assay that couples high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) with internal standards. In a subset of samples, technical reproducibility was evaluated by analyzing consecutive tissue sections from the same tumor block and biological variation was accessed among adjacent tumor regions in the same tissue section. RESULTS: Wild-type RAS protein was measured in 32 clinical non-small cell lung cancer tumors (622-2525 amol/µg) as measured by FAIMS-PRM mass spectrometry. Tumors with a known KRASG12C mutation (n = 17) expressed a wide range of RASG12C mutant protein (127-2012 amol/µg). The variation in wild-type RAS and RASG12C measurements ranged 0-18% CV across consecutive tissue sections and 5-20% CV among adjacent tissue regions. Quantitative target engagement was then demonstrated in FFPE tissues from 2 xenograft models (MIA PaCa-2 and NCI-H2122) treated with a RASG12C inhibitor (AZD4625). CONCLUSIONS: This work illustrates the potential to expand mass spectrometry-based proteomics in preclinical and clinical oncology drug development through analysis of FFPE tumor biopsies.

Real-world Experiences in the Transplantation of Hepatitis C-NAAT-positive Organs.

Background: Hepatitis C virus (HCV) nucleic acid amplification test (NAAT)-positive donors have increased the organ pool. Direct-acting antivirals (DAAs) have led to high rates of treatment success and sustained virologic response (SVR) in recipients with donor-derived HCV infection without significant adverse effects, although variability remains in the timing and duration of antivirals. Methods: This retrospective study analyzed all adult HCV-NAAT-negative transplant recipients who received an organ from HCV-NAAT-positive donors from November 24, 2018, to March 31, 2022, at Duke University Medical Center with protocolized delay of DAA initiation until after hospital discharge, with at least 180-d follow-up on all patients. Transplant and HCV-related outcomes were analyzed. Results: Two hundred eleven transplants (111 kidneys, 41 livers, 34 hearts, and 25 lungs) were performed from HCV-NAAT-positive donors to HCV-NAAT-negative recipients. Ninety percent of recipients became viremic within 7 d posttransplant. Ninety-nine percent of recipients were initiated on pangenotypic DAAs in the outpatient setting a median of 52 d posttransplant, most commonly with 12-wk courses of sofosbuvir-velpatasvir (lungs) and glecaprevir-pibrentasvir (heart, kidney, and liver). Ninety-seven percent of recipients had SVR after a first-line DAA; all ultimately achieved SVR at 12 wk after subsequent treatment courses. The median peak HCV RNA for all organ systems was 2 436 512 IU/mL; the median time from antiviral to undetectable RNA was 48 d, although differences were noted between organ groups. No patient deaths or graft losses were directly attributable to HCV infection. Conclusions: One hundred percent of transplant recipients of HCV-NAAT-positive organs ultimately developed SVR without significant adverse effects when HCV antivirals were initiated in the outpatient setting after transplant hospitalization, suggesting that this real-world treatment pathway is a viable option.

Supervised Risk Predictor of Breast Cancer Based on Intrinsic Subtypes.

PURPOSE: To improve on current standards for breast cancer prognosis and prediction of chemotherapy benefit by developing a risk model that incorporates the gene expression-based "intrinsic" subtypes luminal A, luminal B, HER2-enriched, and basal-like. METHODS: A 50-gene subtype predictor was developed using microarray and quantitative reverse transcriptase polymerase chain reaction data from 189 prototype samples. Test sets from 761 patients (no systemic therapy) were evaluated for prognosis, and 133 patients were evaluated for prediction of pathologic complete response (pCR) to a taxane and anthracycline regimen. RESULTS: The intrinsic subtypes as discrete entities showed prognostic significance (P = 2.26E-12) and remained significant in multivariable analyses that incorporated standard parameters (estrogen receptor status, histologic grade, tumor size, and node status). A prognostic model for node-negative breast cancer was built using intrinsic subtype and clinical information. The C-index estimate for the combined model (subtype and tumor size) was a significant improvement on either the clinicopathologic model or subtype model alone. The intrinsic subtype model predicted neoadjuvant chemotherapy efficacy with a negative predictive value for pCR of 97%. CONCLUSION: Diagnosis by intrinsic subtype adds significant prognostic and predictive information to standard parameters for patients with breast cancer. The prognostic properties of the continuous risk score will be of value for the management of node-negative breast cancers. The subtypes and risk score can also be used to assess the likelihood of efficacy from neoadjuvant chemotherapy.

Modulation of the proteostasis network promotes tumor resistance to oncogenic KRAS inhibitors.

Despite substantial advances in targeting mutant KRAS, tumor resistance to KRAS inhibitors (KRASi) remains a major barrier to progress. Here, we report proteostasis reprogramming as a key convergence point of multiple KRASi-resistance mechanisms. Inactivation of oncogenic KRAS down-regulated both the heat shock response and the inositol-requiring enzyme 1α (IRE1α) branch of the unfolded protein response, causing severe proteostasis disturbances. However, IRE1α was selectively reactivated in an ER stress-independent manner in acquired KRASi-resistant tumors, restoring proteostasis. Oncogenic KRAS promoted IRE1α protein stability through extracellular signal-regulated kinase (ERK)-dependent phosphorylation of IRE1α, leading to IRE1α disassociation from 3-hydroxy-3-methylglutaryl reductase degradation (HRD1) E3-ligase. In KRASi-resistant tumors, both reactivated ERK and hyperactivated AKT restored IRE1α phosphorylation and stability. Suppression of IRE1α overcame resistance to KRASi. This study reveals a druggable mechanism that leads to proteostasis reprogramming and facilitates KRASi resistance.

Kinase Inhibitor Pulldown Assay Identifies a Chemotherapy Response Signature in Triple-negative Breast Cancer Based on Purine-binding Proteins.

Triple-negative breast cancer (TNBC) constitutes 10%-15% of all breast tumors. The current standard of care is multiagent chemotherapy, which is effective in only a subset of patients. The original objective of this study was to deploy a mass spectrometry (MS)-based kinase inhibitor pulldown assay (KIPA) to identify kinases elevated in non-pCR (pathologic complete response) cases for therapeutic targeting. Frozen optimal cutting temperature compound-embedded core needle biopsies were obtained from 43 patients with TNBC before docetaxel- and carboplatin-based neoadjuvant chemotherapy. KIPA was applied to the native tumor lysates that were extracted from samples with high tumor content. Seven percent of all identified proteins were kinases, and none were significantly associated with lack of pCR. However, among a large population of "off-target" purine-binding proteins (PBP) identified, seven were enriched in pCR-associated samples (P < 0.01). In orthogonal mRNA-based TNBC datasets, this seven-gene "PBP signature" was associated with chemotherapy sensitivity and favorable clinical outcomes. Functional annotation demonstrated IFN gamma response, nuclear import of DNA repair proteins, and cell death associations. Comparisons with standard tandem mass tagged-based discovery proteomics performed on the same samples demonstrated that KIPA-nominated pCR biomarkers were unique to the platform. KIPA is a novel biomarker discovery tool with unexpected utility for the identification of PBPs related to cytotoxic drug response. The PBP signature has the potential to contribute to clinical trials designed to either escalate or de-escalate therapy based on pCR probability. Significance: The identification of pretreatment predictive biomarkers for pCR in response to neoadjuvant chemotherapy would advance precision treatment for TNBC. To complement standard proteogenomic discovery profiling, a KIPA was deployed and unexpectedly identified a seven-member non-kinase PBP pCR-associated signature. Individual members served diverse pathways including IFN gamma response, nuclear import of DNA repair proteins, and cell death.

Proteogenomic Approaches for the Identification of NF1/Neurofibromin-depleted Estrogen Receptor-positive Breast Cancers for Targeted Treatment.

NF1 is a key tumor suppressor that represses both RAS and estrogen receptor-α (ER) signaling in breast cancer. Blocking both pathways by fulvestrant (F), a selective ER degrader, together with binimetinib (B), a MEK inhibitor, promotes tumor regression in NF1-depleted ER+ models. We aimed to establish approaches to determine how NF1 protein levels impact B+F treatment response to improve our ability to identify B+F sensitive tumors. We examined a panel of ER+ patient-derived xenograft (PDX) models by DNA and mRNA sequencing and found that more than half of these models carried an NF1 shallow deletion and generally have low mRNA levels. Consistent with RAS and ER activation, RET and MEK levels in NF1-depleted tumors were elevated when profiled by mass spectrometry (MS) after kinase inhibitor bead pulldown. MS showed that NF1 can also directly and selectively bind to palbociclib-conjugated beads, aiding quantification. An IHC assay was also established to measure NF1, but the MS-based approach was more quantitative. Combined IHC and MS analysis defined a threshold of NF1 protein loss in ER+ breast PDX, below which tumors regressed upon treatment with B+F. These results suggest that we now have a MS-verified NF1 IHC assay that can be used for patient selection as a complement to somatic genomic analysis. Significance: A major challenge for targeting the consequence of tumor suppressor disruption is the accurate assessment of protein functional inactivation. NF1 can repress both RAS and ER signaling, and a ComboMATCH trial is underway to treat the patients with binimetinib and fulvestrant. Herein we report a MS-verified NF1 IHC assay that can determine a threshold for NF1 loss to predict treatment response. These approaches may be used to identify and expand the eligible patient population.

The Breast Cancer Proteome and Precision Oncology.

The goal of precision oncology is to translate the molecular features of cancer into predictive and prognostic tests that can be used to individualize treatment leading to improved outcomes and decreased toxicity. Success for this strategy in breast cancer is exemplified by efficacy of trastuzumab in tumors overexpressing ERBB2 and endocrine therapy for tumors that are estrogen receptor positive. However, other effective treatments, including chemotherapy, immune checkpoint inhibitors, and CDK4/6 inhibitors are not associated with strong predictive biomarkers. Proteomics promises another tier of information that, when added to genomic and transcriptomic features (proteogenomics), may create new opportunities to improve both treatment precision and therapeutic hypotheses. Here, we review both mass spectrometry-based and antibody-dependent proteomics as complementary approaches. We highlight how these methods have contributed toward a more complete understanding of breast cancer and describe the potential to guide diagnosis and treatment more accurately.

Kinome Reprogramming Is a Targetable Vulnerability in ESR1 Fusion-Driven Breast Cancer.

Transcriptionally active ESR1 fusions (ESR1-TAF) are a potent cause of breast cancer endocrine therapy (ET) resistance. ESR1-TAFs are not directly druggable because the C-terminal estrogen/anti-estrogen-binding domain is replaced with translocated in-frame partner gene sequences that confer constitutive transactivation. To discover alternative treatments, a mass spectrometry (MS)-based kinase inhibitor pulldown assay (KIPA) was deployed to identify druggable kinases that are upregulated by diverse ESR1-TAFs. Subsequent explorations of drug sensitivity validated RET kinase as a common therapeutic vulnerability despite remarkable ESR1-TAF C-terminal sequence and structural diversity. Organoids and xenografts from a pan-ET-resistant patient-derived xenograft model that harbors the ESR1-e6>YAP1 TAF were concordantly inhibited by the selective RET inhibitor pralsetinib to a similar extent as the CDK4/6 inhibitor palbociclib. Together, these findings provide preclinical rationale for clinical evaluation of RET inhibition for the treatment of ESR1-TAF-driven ET-resistant breast cancer. SIGNIFICANCE: Kinome analysis of ESR1 translocated and mutated breast tumors using drug bead-based mass spectrometry followed by drug-sensitivity studies nominates RET as a therapeutic target. See related commentary by Wu and Subbiah, p. 3159.

MyKidneyCoach, Patient Activation, and Clinical Outcomes in Diverse Kidney Transplant Recipients: A Randomized Control Pilot Trial.

Kidney transplant (KT) recipients who are not actively engaged in their care and lack self-management skills have poor transplant outcomes, which are disproportionately observed among Black KT recipients. This pilot study aimed to determine whether the MyKidneyCoach app, an mHealth intervention that provides self-management monitoring and coaching, improved patient activation, engagement, and nutritional behaviors in a diverse KT population. Methods: This was a randomized, age-stratified, parallel-group, attention-control, pilot study in post-KT patients. Participants were randomized into the attention-control with access to MyKidneyCoach for education and self-management (n = 9) or the intervention with additional tailored nurse coaching (n = 7). Feasibility, acceptability, and clinical outcomes were assessed. Results: The acceptability of MyKidneyCoach by System Usability Scale was 67.5 (95% confidence interval [CI], 59.1-75.9). Completion rates based on actively using MyKidneyCoach were 81% (95% CI, 57%-93%) and study retention rate of 73%. Patient activation measure significantly increased overall by a mean of 11 points (95% CI, 3.2-18.8). Additionally, Black patients (n = 7) had higher nutrition self-efficacy scores of 80.5 (95% CI, 74.4-86.7) compared with 75.6 (95% CI, 71.1-80.1) in non-Black patients (n = 9) but lower patient activation measure scores of 69.3 (95% CI, 56.3-82.3) compared with 71.8 (95% CI, 62.5-81) in non-Black patients after 3 mo. Conclusions: MyKidneyCoach was easy to use and readily accepted with low attrition, and improvements were demonstrated in patient-reported outcomes. Both Black and non-Black participants using MyKidneyCoach showed improvement in self-management competencies; thus, this intervention may help reduce healthcare inequities in KT.

Adjuvant paclitaxel and trastuzumab for node-negative, HER2-positive breast cancer: final 10-year analysis of the open-label, single-arm, phase 2 APT trial.

BACKGROUND: We aimed to report on long-term outcomes of patients with small, node-negative, HER2-positive breast cancer treated with adjuvant paclitaxel and trastuzumab and to establish potential biomarkers to predict prognosis. METHODS: In this open-label, single-arm, phase 2 study, patients aged 18 years or older, with small (≤3 cm), node-negative, HER2-positive breast cancer, and an Eastern Cooperative Oncology Group performance status of 0-1, were recruited from 16 institutions in 13 cities in the USA. Eligible patients were given intravenous paclitaxel (80 mg/m2) with intravenous trastuzumab (loading dose of 4 mg/kg, subsequent doses 2 mg/kg) weekly for 12 weeks, followed by trastuzumab (weekly at 2 mg/kg or once every 3 weeks at 6 mg/kg) for 40 weeks to complete a full year of trastuzumab. The primary endpoint was 3-year invasive disease-free survival. Here, we report 10-year survival outcomes, assessed in all participants who received protocol-defined treatment, with exploratory analyses using the HER2DX genomic tool. This study is registered on ClinicalTrials.gov, NCT00542451, and is closed to accrual. FINDINGS: Between Oct 29, 2007, and Sept 3, 2010, 410 patients were enrolled and 406 were given adjuvant paclitaxel and trastuzumab and included in the analysis. Mean age at enrolment was 55 years (SD 10·5), 405 (99·8%) of 406 patients were female and one (0·2%) was male, 350 (86·2%) were White, 28 (6·9%) were Black or African American, and 272 (67·0%) had hormone receptor-positive disease. After a median follow-up of 10·8 years (IQR 7·1-11·4), among 406 patients included in the analysis population, we observed 31 invasive disease-free survival events, of which six (19·4%) were locoregional ipsilateral recurrences, nine (29·0%) were new contralateral breast cancers, six (19·4%) were distant recurrences, and ten (32·3%) were all-cause deaths. 10-year invasive disease-free survival was 91·3% (95% CI 88·3-94·4), 10-year recurrence-free interval was 96·3% (95% CI 94·3-98·3), 10-year overall survival was 94·3% (95% CI 91·8-96·8), and 10-year breast cancer-specific survival was 98·8% (95% CI 97·6-100). HER2DX risk score as a continuous variable was significantly associated with invasive disease-free survival (hazard ratio [HR] per 10-unit increment 1·24 [95% CI 1·00-1·52]; p=0·047) and recurrence-free interval (1·45 [1·09-1·93]; p=0·011). INTERPRETATION: Adjuvant paclitaxel and trastuzumab is a reasonable treatment standard for patients with small, node-negative, HER2-positive breast cancer. The HER2DX genomic tool might help to refine the prognosis for this population. FUNDING: Genentech.

Identifying biomarkers of differential chemotherapy response in TNBC patient-derived xenografts with a CTD/WGCNA approach.

Although systemic chemotherapy remains the standard of care for TNBC, even combination chemotherapy is often ineffective. The identification of biomarkers for differential chemotherapy response would allow for the selection of responsive patients, thus maximizing efficacy and minimizing toxicities. Here, we leverage TNBC PDXs to identify biomarkers of response. To demonstrate their ability to function as a preclinical cohort, PDXs were characterized using DNA sequencing, transcriptomics, and proteomics to show consistency with clinical samples. We then developed a network-based approach (CTD/WGCNA) to identify biomarkers of response to carboplatin (MSI1, TMSB15A, ARHGDIB, GGT1, SV2A, SEC14L2, SERPINI1, ADAMTS20, DGKQ) and docetaxel (c, MAGED4, CERS1, ST8SIA2, KIF24, PARPBP). CTD/WGCNA multigene biomarkers are predictive in PDX datasets (RNAseq and Affymetrix) for both taxane- (docetaxel or paclitaxel) and platinum-based (carboplatin or cisplatin) response, thereby demonstrating cross-expression platform and cross-drug class robustness. These biomarkers were also predictive in clinical datasets, thus demonstrating translational potential.

Single-cell analysis reveals prognostic fibroblast subpopulations linked to molecular and immunological subtypes of lung cancer.

Fibroblasts are poorly characterised cells that variably impact tumour progression. Here, we use single cell RNA-sequencing, multiplexed immunohistochemistry and digital cytometry (CIBERSORTx) to identify and characterise three major fibroblast subpopulations in human non-small cell lung cancer: adventitial, alveolar and myofibroblasts. Alveolar and adventitial fibroblasts (enriched in control tissue samples) localise to discrete spatial niches in histologically normal lung tissue and indicate improved overall survival rates when present in lung adenocarcinomas (LUAD). Trajectory inference identifies three phases of control tissue fibroblast activation, leading to myofibroblast enrichment in tumour samples: initial upregulation of inflammatory cytokines, followed by stress-response signalling and ultimately increased expression of fibrillar collagens. Myofibroblasts correlate with poor overall survival rates in LUAD, associated with loss of epithelial differentiation, TP53 mutations, proximal molecular subtypes and myeloid cell recruitment. In squamous carcinomas myofibroblasts were not prognostic despite being transcriptomically equivalent. These findings have important implications for developing fibroblast-targeting strategies for cancer therapy.

Local-Regional Recurrence After Neoadjuvant Endocrine Therapy: Data from ACOSOG Z1031 (Alliance), a Randomized Phase 2 Neoadjuvant Comparison Between Letrozole, Anastrozole, and Exemestane for Postmenopausal Women with Estrogen Receptor-Positive Clinical Stage 2 or 3 Breast Cancer.

BACKGROUND: The ACOSOG Z1031 trial addressed the ability of three neoadjuvant aromatase inhibitors (NAIs) to reduce residual disease (cohort A) and to assess whether switching to neoadjuvant chemotherapy (NCT) after 4 weeks of receiving NAI with Ki67 greater than 10% increases pathologic complete response (pCR) in postmenopausal women with estrogen receptor-enriched (Allred score 6-8) breast cancer (BC). METHODS: The study enrolled 622 women with clinical stage 2 or 3 estrogen receptor-positive (ER+) BC. Cohort A comprised 377 patients, and cohort B had 245 patients. The analysis cohort consisted of 509 patients after exclusion of patients who did not meet the trial eligibility criteria, switched to NCT or surgery due to 4-week Ki67 greater than 10%, or withdrew before surgery. Distribution of time to local-regional recurrence (LRR) was estimated using the competing-risk approach, in which distant recurrence and second primaries were considered to be competing-risk events. Patients who died without LRR, distant recurrence, or a second primary were censored at the last evaluation. RESULTS: Of the 509 patients, 342 (67.2%) had breast-conserving surgery (BCS). Of 221 patients thought to require mastectomy at presentation, 50% were able to have BCS. Five (1%) patients had no residual disease in the breast or nodes at surgery. Among 382 women alive at this writing, 90% have been followed longer than 5 years. The 5-year cumulative incidence rate for LRR is estimated to be 1.53% (95% confidence interval 0.7-3.0%). CONCLUSIONS: Rarely does NAI result in pCR for patients with stage 2 or 3 ER+ BC. However, a significant proportion will have downstaged to allow for BCS. Local-regional recurrence after surgery is uncommon (1.5% at 5 years), supporting the use of BCS after NAI.

ATM Regulates Differentiation of Myofibroblastic Cancer-Associated Fibroblasts and Can Be Targeted to Overcome Immunotherapy Resistance.

Myofibroblastic cancer-associated fibroblast (myoCAF)-rich tumors generally contain few T cells and respond poorly to immune-checkpoint blockade. Although myoCAFs are associated with poor outcome in most solid tumors, the molecular mechanisms regulating myoCAF accumulation remain unclear, limiting the potential for therapeutic intervention. Here, we identify ataxia-telangiectasia mutated (ATM) as a central regulator of the myoCAF phenotype. Differentiating myofibroblasts in vitro and myoCAFs cultured ex vivo display activated ATM signaling, and targeting ATM genetically or pharmacologically could suppress and reverse differentiation. ATM activation was regulated by the reactive oxygen species-producing enzyme NOX4, both through DNA damage and increased oxidative stress. Targeting fibroblast ATM in vivo suppressed myoCAF-rich tumor growth, promoted intratumoral CD8 T-cell infiltration, and potentiated the response to anti-PD-1 blockade and antitumor vaccination. This work identifies a novel pathway regulating myoCAF differentiation and provides a rationale for using ATM inhibitors to overcome CAF-mediated immunotherapy resistance. SIGNIFICANCE: ATM signaling supports the differentiation of myoCAFs to suppress T-cell infiltration and antitumor immunity, supporting the potential clinical use of ATM inhibitors in combination with checkpoint inhibition in myoCAF-rich, immune-cold tumors.

Personalized ctDNA micro-panels can monitor and predict clinical outcomes for patients with triple-negative breast cancer.

Circulating tumor DNA (ctDNA) in peripheral blood has been used to predict prognosis and therapeutic response for triple-negative breast cancer (TNBC) patients. However, previous approaches typically use large comprehensive panels of genes commonly mutated across all breast cancers. Given the reduction in sequencing costs and decreased turnaround times associated with panel generation, the objective of this study was to assess the use of custom micro-panels for tracking disease and predicting clinical outcomes for patients with TNBC. Paired tumor-normal samples from patients with TNBC were obtained at diagnosis (T0) and whole exome sequencing (WES) was performed to identify somatic variants associated with individual tumors. Custom micro-panels of 4-6 variants were created for each individual enrolled in the study. Peripheral blood was obtained at baseline, during Cycle 1 Day 3, at time of surgery, and in 3-6 month intervals after surgery to assess variant allele fraction (VAF) at different timepoints during disease course. The VAF was compared to clinical outcomes to evaluate the ability of custom micro-panels to predict pathological response, disease-free intervals, and patient relapse. A cohort of 50 individuals were evaluated for up to 48 months post-diagnosis of TNBC. In total, there were 33 patients who did not achieve pathological complete response (pCR) and seven patients developed clinical relapse. For all patients who developed clinical relapse and had peripheral blood obtained ≤ 6 months prior to relapse (n = 4), the custom ctDNA micro-panels identified molecular relapse at an average of 4.3 months prior to clinical relapse. The custom ctDNA panel results were moderately associated with pCR such that during disease monitoring, only 11% of patients with pCR had a molecular relapse, whereas 47% of patients without pCR had a molecular relapse (Chi-Square; p-value = 0.10). In this study, we show that a custom micro-panel of 4-6 markers can be effectively used to predict outcomes and monitor remission for patients with TNBC. These custom micro-panels show high sensitivity for detecting molecular relapse in advance of clinical relapse. The use of these panels could improve patient outcomes through early detection of relapse with preemptive intervention prior to symptom onset.