Tumor-reactive T cell clonotype dynamics underlying clinical response to TIL therapy in melanoma.

Adoptive cell therapy (ACT) using in vitro expanded tumor-infiltrating lymphocytes (TILs) has inconsistent clinical responses. To better understand determinants of therapeutic success, we tracked TIL clonotypes from baseline tumors to ACT products and post-ACT blood and tumor samples in melanoma patients using single-cell RNA and T cell receptor (TCR) sequencing. Patients with clinical responses had baseline tumors enriched in tumor-reactive TILs, and these were more effectively mobilized upon in vitro expansion, yielding products enriched in tumor-specific CD8+ cells that preferentially infiltrated tumors post-ACT. Conversely, lack of clinical responses was associated with tumors devoid of tumor-reactive resident clonotypes and with cell products mostly composed of blood-borne clonotypes that persisted in blood but not in tumors post-ACT. Upon expansion, tumor-specific TILs lost tumor-associated transcriptional signatures, including exhaustion, and responders exhibited an intermediate exhausted effector state after TIL engraftment in the tumor, suggesting functional reinvigoration. Our findings provide insight into the nature and dynamics of tumor-specific clonotypes associated with clinical response to TIL-ACT, with implications for treatment optimization.

The impact of erythropoiesis-stimulating agents administration concomitantly with adjuvant anti-HER2 treatments on the outcomes of patients with early breast cancer: a sub-analysis of the ALTTO study.

PURPOSE: To assess whether erythropoiesis-stimulating agents (ESA) administration impacts the outcomes of patients with HER2-positive early breast cancer (EBC). METHODS: ALTTO (NCT00490139) patients were categorized by ESA use during adjuvant anti-HER2 treatment. Disease-free-survival (DFS), overall survival (OS), and time-to-distant recurrence (TTDR) were analyzed by ESA administration, with subgroup analyses according to prognostic factors. Log-rank tests and Cox modeling were performed. Adverse events (AEs) of ESA-interest were compared. RESULTS: Among 8381 patients recruited in ALTTO, 123 (1.5%) received ESA concomitantly with study treatment. The median age of patients receiving ESA was 54 years, 39.0% premenopausal, most had tumor size > 2 cm (56.9%), node-positive (58.5%), and positive estrogen receptor expression (61.8%). Median follow-up was shorter in the ESA group [6.1 years (IQR 5.3-7.0) vs. 6.9 years (6.0-7.1); p < 0.001]. There was no DFS difference by ESA administration (log-rank p = 0.70), with 3- and 7-year DFS of 89.2% (95% CI 81.8-93.8%) and 81.6% (71.4-88.5%) in ESA group vs. 88.3% (87.6-89.0%) and 80.0% (79.1-80.9%) in No-ESA group. In subgroup analyses, the interaction of ESA administration with menopausal status was statistically significant (unadjusted p = 0.024; stratified p = 0.033), favoring premenopausal women receiving ESA. We observed no significant association of ESA administration with OS (log-rank p = 0.57; 7-year OS in ESA 88.6% vs. 90.0% in non-ESA) or TTDR. ESA-interest AEs were experienced by eight (6.5%) patients receiving ESA and 417 (5.1%) in the No-ESA group (p = 0.41). CONCLUSION: ESA administration to patients receiving adjuvant anti-HER2 treatment for HER2-positive EBC was safe and not associated with a negative impact on survival outcomes.

Essential medicines list in national cancer control plans: a secondary analysis from a global study.

With the advent of innovative therapeutics for and the rising costs of cancer management, low-income and middle-income countries face increasing challenges to deliver effective and sustainable health care. Understanding of how countries are selecting and prioritising essential cancer interventions is poor, including in the formulation of policies for essential medicines. We did an in-depth subanalysis from a global dataset of national cancer control plans (NCCPs), aiming to identify possible determinants of inclusion of policies related to essential medicines in the NCCP. The results showed poor global comprehensiveness of NCCPs, and substantial deficits in policies for financial hardships due to cancer care, specifically for access to cancer medicines. Specification of budget allocations, policy of protection from catastrophic health expenditure, and national treatment guidelines in the NCCPs contributed to more consistent policies on essential cancer medicines. The bedrock to deliver effective cancer programmes resides in the assurance of comprehensive, consistent, and coherent policy formulation, to orient resource selection and health investments, ultimately delivering equitable health for all.

Pembrolizumab versus placebo as adjuvant therapy for completely resected stage IB-IIIA non-small-cell lung cancer (PEARLS/KEYNOTE-091): an interim analysis of a randomised, triple-blind, phase 3 trial.

BACKGROUND: Pembrolizumab is a standard-of-care for advanced non-small-cell lung cancer (NSCLC). We assessed pembrolizumab as adjuvant therapy for completely resected stage IB-IIIA NSCLC. METHODS: In this randomised, triple-blind, phase 3 trial (PEARLS/KEYNOTE-091), patients were recruited from 196 medical centres in 29 countries. Eligible patients were aged 18 years or older, with completely resected, pathologically confirmed stage IB (tumours of ≥4 cm in diameter), II, or IIIA NSCLC per the American Joint Committee on Cancer staging system (7th edition) of any histology or PD-L1 expression level, and an Eastern Cooperative Oncology Group performance status of 0 or 1; adjuvant chemotherapy was to be considered for stage IB disease and was strongly recommended for stage II and IIIA disease, according to national and local guidelines. Using a central interactive voice-response system, eligible participants were randomly assigned (1:1), using a minimisation technique and stratified by disease stage, previous adjuvant chemotherapy, PD-L1 expression, and geographical region, to pembrolizumab 200 mg or placebo, both administered intravenously every 3 weeks for up to 18 cycles. Participants, investigators, and analysts were masked to treatment assignment. Dual primary endpoints were disease-free survival in the overall population and in the population with PD-L1 tumour proportion score (TPS) of 50% or greater. Efficacy was assessed in the intention-to-treat (ITT) population (ie, all participants randomly assigned to a treatment group). Safety was assessed in all participants randomly assigned to treatment who received at least one dose of study treatment. Here we report results of the second interim analysis, prespecified to occur when approximately 118 disease-free survival events had occurred in the PD-L1 TPS of 50% or greater population. This study is registered with ClinicalTrials.gov, NCT02504372, and is active but not recruiting. FINDINGS: Between Jan 20, 2016, and May 6, 2020, 1177 (60%) of 1955 screened participants were randomly assigned to pembrolizumab (n=590, including n=168 with PD-L1 TPS of ≥50%) or placebo (n=587; including n=165 with PD-L1 TPS of ≥50%) and included in the ITT population. Median follow-up as of data cutoff (Sept 20, 2021) for this interim analysis was 35·6 months (IQR 27·1-45·5). In the overall population, median disease-free survival was 53·6 months (95% CI 39·2 to not reached) in the pembrolizumab group versus 42·0 months (31·3 to not reached) in the placebo group (HR 0·76 [95% CI 0·63-0·91], p=0·0014). In the PD-L1 TPS of 50% or greater population, median disease-free survival was not reached in either the pembrolizumab group (95% CI 44·3 to not reached) or the placebo group (95% CI 35·8 to not reached; HR 0·82 [95% CI 0·57-1·18]; p=0·14). Grade 3 or worse adverse events occurred in 198 (34%) of 580 participants who received pembrolizumab and 150 (26%) of 581 participants who received placebo. Grade 3 or worse events that occurred in at least ten participants in either treatment group were hypertension (35 [6%]) and pneumonia (12 [2%]) with pembrolizumab and hypertension (32 [6%]) with placebo. Serious adverse events occurred in 142 (24%) participants in the pembrolizumab group and 90 (15%) in the placebo group; serious adverse events that occurred in more than 1% of participants were pneumonia (13 [2%]), pneumonitis (12 [2%]), and diarrhoea (seven [1%]) with pembrolizumab and pneumonia (nine [2%]) with placebo. Treatment-related adverse events led to death in four (1%) participants treated with pembrolizumab (one due to both cardiogenic shock and myocarditis, one due to both septic shock and myocarditis, one due to pneumonia, and one due to sudden death) and in no participants treated with placebo. INTERPRETATION: Pembrolizumab significantly improved disease-free survival compared with placebo and was not associated with new safety signals in completely resected, PD-L1-unselected, stage IB-IIIA NSCLC. Pembrolizumab is potentially a new treatment option for stage IB-IIIA NSCLC after complete resection and, when recommended, adjuvant chemotherapy, regardless of PD-L1 expression. FUNDING: Merck Sharp & Dohme, a subsidiary of Merck & Co.

Outcomes of patients with small and node-negative HER2-positive early breast cancer treated with adjuvant chemotherapy and anti-HER2 therapy-a sub-analysis of the ALTTO study.

BACKGROUND: Patients with small node-negative HER2-positive breast cancer are commonly treated with paclitaxel and 1 year of adjuvant trastuzumab. We performed a sub-analysis of the ALTTO trial to explore the long-term outcomes of patients with small node-negative tumours. METHODS: The ALTTO trial randomised 8381 patients with early HER2-positive BC treated with adjuvant chemotherapy (anthracycline/taxane- or taxane/carboplatin-based), to trastuzumab (T), lapatinib (L), their sequence (T → L) or their combination (L + T). Patients with tumours ≤3 cm and node-negative were included in this sub-analysis. RESULTS: A total of 2821 patients were analysed (median follow-up of 7 years). The median age was 52 years, and most patients had tumours ≤2 cm (64.3%). The 7-year disease-free survival (DFS) was 88.1% (95% CI: 86.7-89.3%). DFS was similar for arms T, T + L and T⟶L and significantly lower for arm L (stratified log-rank P = 0.031). The 7-year overall survival rate was 95.9% (95% CI: [95.0-96.6%) and the 7-year time-to-distant recurrence was 93.4% (95% CI: 92.3-94.4%). CONCLUSION: With most patients treated with anthracycline-based regimens, ALTTO shows that patients with small tumours treated with trastuzumab and concomitant chemotherapy have excellent long-term outcomes, similar to those of the APT trial. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT00490139.

Programmed death-ligand 1 expression influenced by tissue sample size. Scoring based on tissue microarrays' and cross-validation with resections, in patients with, stage I-III, non-small cell lung carcinoma of the European Thoracic Oncology Platform Lungscape cohort.

PD-L1, as assessed by immunohistochemistry, is a predictive biomarker for immuno-oncology treatment in lung cancer. Different scoring methods have been used to assess its status, resulting in a wide range of positivity rates. We use the European Thoracic Oncology Platform Lungscape non-small cell lung carcinoma cohort to explore this issue. PD-L1 expression was assessed via immunohistochemistry on tissue microarrays (up to four cores per case), using the DAKO 28-8 immunohistochemistry assay, following a two-round external quality assessment procedure. All samples were analyzed under the same protocol. Cross-validation of scoring between tissue microarray and whole sections was performed in 10% randomly selected samples. Cutoff points considered: ≥1, 50 (primarily), and 25%. At the two external quality assessment rounds, tissue microarray scoring agreement rates between pathologists were: 73% and 81%. There were 2008 cases with valid immunohistochemistry tissue microarray results (50% all cores evaluable). Concordant cases at 1, 25, and 50% were: 85, 91, and 93%. Tissue microarray core results were identical for 70% of cases. Sensitivity of the tissue microarray method for 1, 25, and 50% was: 80, 78, and 79% (specificity: 90, 95, 98%). Complete agreement between tissue microarrays and whole sections was achieved for 60% of the cases. Highest sensitivity rates for 1% and 50% cutoffs were detected for higher number of cores. Underestimation of PD-L1 expression on small samples is more common than overestimation. We demonstrated that classification of PD-L1 on small biopsy samples does not represent the overall expression of PD-L1 in all non-small cell cancer carcinoma cases, although the majority of cases are 'correctly' classified. In future studies, sampling more and larger biopsies, recording the biopsy size and tumor load may permit further refinement, increasing predictive accuracy.

A Phase I/II trial comparing autologous dendritic cell vaccine pulsed either with personalized peptides (PEP-DC) or with tumor lysate (OC-DC) in patients with advanced high-grade ovarian serous carcinoma.

BACKGROUND: Most ovarian cancer patients are diagnosed at a late stage with 85% of them relapsing after surgery and standard chemotherapy; for this reason, new treatments are urgently needed. Ovarian cancer has become a candidate for immunotherapy by reason of their expression of shared tumor-associated antigens (TAAs) and private mutated neoantigens (NeoAgs) and the recognition of the tumor by the immune system. Additionally, the presence of intraepithelial tumor infiltrating lymphocytes (TILs) is associated with improved progression-free and overall survival of patients with ovarian cancer. The aim of active immunotherapy, including vaccination, is to generate a new anti-tumor response and amplify an existing immune response. Recently developed NeoAgs-based cancer vaccines have the advantage of being more tumor specific, reducing the potential for immunological tolerance, and inducing robust immunogenicity. METHODS: We propose a randomized phase I/II study in patients with advanced ovarian cancer to compare the immunogenicity and to assess safety and feasibility of two personalized DC vaccines. After standard of care surgery and chemotherapy, patients will receive either a novel vaccine consisting of autologous DCs pulsed with up to ten peptides (PEP-DC), selected using an agnostic, yet personalized, epitope discovery algorithm, or a sequential combination of a DC vaccine loaded with autologous oxidized tumor lysate (OC-DC) prior to an equivalent PEP-DC vaccine. All vaccines will be administered in combination with low-dose cyclophosphamide. This study is the first attempt to compare the two approaches and to use NeoAgs-based vaccines in ovarian cancer in the adjuvant setting. DISCUSSION: The proposed treatment takes advantage of the beneficial effects of pre-treatment with OC-DC prior to PEP-DC vaccination, prompting immune response induction against a wide range of patient-specific antigens, and amplification of pre-existing NeoAgs-specific T cell clones. Trial registration This trial is already approved by Swissmedic (Ref.: 2019TpP1004) and will be registered at http://www.clinicaltrials.gov before enrollment opens.

Dose-dense sequential adjuvant chemotherapy followed, as indicated, by trastuzumab for one year in patients with early breast cancer: first report at 5-year median follow-up of a Hellenic Cooperative Oncology Group randomized phase III trial.

BACKGROUND: Dose-dense sequential chemotherapy including anthracyclines and taxanes has been established in the adjuvant setting of high-risk operable breast cancer. However, the preferable taxane and optimal schedule of administration in a dose-dense regimen have not been defined yet. METHODS: From July 2005 to November 2008, 1001 patients (990 eligible) were randomized to receive, every 2 weeks, 3 cycles of epirubicin 110 mg/m2 followed by 3 cycles of paclitaxel 200 mg/m2 followed by 3 cycles of intensified CMF (Arm A; 333 patients), or 3 cycles of epirubicin followed by 3 cycles of CMF, as in Arm A, followed 3 weeks later by 9 weekly cycles of docetaxel 35 mg/m2 (Arm B; 331), or 9 weekly cycles of paclitaxel 80 mg/m2 (Arm C; 326). Trastuzumab was administered for one year to HER2-positive patients post-radiation. RESULTS: At a median follow-up of 60.5 months, the 3-year disease-free survival (DFS) rate was 86%, 90% and 88%, for Arms A, B and C, respectively, while the 3-year overall survival (OS) rate was 96% in all arms. No differences were found in DFS or OS between the combined B and C Arms versus Arm A (DFS: HR = 0.81, 95% CI: 0.59-1.11, P = 0.20; OS: HR = 0.84, 95% CI: 0.55-1.30, P = 0.43). Among the 255 patients who received trastuzumab, 189 patients (74%) completed 1 year of treatment uneventfully. In all arms, the most frequently reported severe adverse events were neutropenia (30% vs. 27% vs. 26%) and leucopenia (12% vs. 13% vs. 12%), while febrile neutropenia occurred in fifty-one patients (6% vs. 4% vs. 5%). Patients in Arm A experienced more often severe pain (P = 0.002), neurological complications (P = 0.004) and allergic reactions (P = 0.004), while patients in Arm B suffered more often from severe skin reactions (P = 0.020). CONCLUSIONS: No significant differences in survival between the regimens were found in the present phase III trial. Taxane scheduling influenced the type of severe toxicities. HER2-positive patients demonstrated comparable 3-year DFS and OS rates with those reported in other similar studies. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12610000151033.

Phase II design with sequential testing of hypotheses within each stage.

The main goal of a Phase II clinical trial is to decide, whether a particular therapeutic regimen is effective enough to warrant further study. The hypothesis tested by Fleming's Phase II design (Fleming, 1982) is [Formula: see text] versus [Formula: see text], with level [Formula: see text] and with a power [Formula: see text] at [Formula: see text], where [Formula: see text] is chosen to represent the response probability achievable with standard treatment and [Formula: see text] is chosen such that the difference [Formula: see text] represents a targeted improvement with the new treatment. This hypothesis creates a misinterpretation mainly among clinicians that rejection of the null hypothesis is tantamount to accepting the alternative, and vice versa. As mentioned by Storer (1992), this introduces ambiguity in the evaluation of type I and II errors and the choice of the appropriate decision at the end of the study. Instead of testing this hypothesis, an alternative class of designs is proposed in which two hypotheses are tested sequentially. The hypothesis [Formula: see text] versus [Formula: see text] is tested first. If this null hypothesis is rejected, the hypothesis [Formula: see text] versus [Formula: see text] is tested next, in order to examine whether the therapy is effective enough to consider further testing in a Phase III study. For the derivation of the proposed design the exact binomial distribution is used to calculate the decision cut-points. The optimal design parameters are chosen, so as to minimize the average sample number (ASN) under specific upper bounds for error levels. The optimal values for the design were found using a simulated annealing method.

Characteristics and Survival Outcomes of Patients With Metastatic RET Fusion-Positive Solid Tumors Receiving Non-RET Inhibitor Standards of Care in a Real-World Setting.

PURPOSE: RET fusions are oncogenic drivers across different solid tumors. However, the genomic landscape and natural history of patients with RET fusion-positive solid tumors are not well known. We describe the clinical characteristics of RET tyrosine kinase inhibitor (TKI)-naïve patients with RET fusion-positive solid tumors (excluding non-small-cell lung cancer [NSCLC]), treated in a real-world setting and assess the prognostic effect of RET fusions. METHODS: Data for RET TKI-naïve patients with metastatic solid tumors (excluding NSCLC) who had ≥one Foundation Medicine comprehensive genomic profiling test (January 1, 2011-March 31, 2022) were obtained from a deidentified nationwide (US-based) clinicogenomic database. The primary objective of this study was to compare the overall survival (OS) of patients with RET fusion-positive tumors versus matched patients with RET wild-type (RET-WT) tumors. Patients with RET-WT solid tumors were matched (4:1) to patients with RET fusion-positive tumors on the basis of preselected covariates. RESULTS: The study population included 26 patients in the RET fusion-positive cohort, 7,220 patients in the RET-WT cohort (before matching), and 104 patients in the matched RET-WT cohort. Co-occurring genomic alterations were rare in the RET fusion-positive cohort. Median OS was consistently lower in patients with RET fusion-positive tumors versus those with RET-WT tumors, using three different analyses (hazard ratios, 2.0, 1.7, and 2.2). CONCLUSION: These data suggest that RET fusions represent a negative prognostic factor in patients with metastatic solid tumors and highlight the need for wider genomic testing and use of RET-specific TKIs that could improve patient outcomes. Our study also highlights the value of real-world data when studying rare cancers or cancers with rare genomic alterations.

Randomized phase II selection trial of FLASH and conventional radiotherapy for patients with localized cutaneous squamous cell carcinoma or basal cell carcinoma: A study protocol.

Background: Cutaneous basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the most prevalent skin cancers in western countries. Surgery is the standard of care for these cancers and conventional external radiotherapy (CONV-RT) with conventional dose rate (0.03-0.06 Gy/sec) represents a good alternative when the patients or tumors are not amenable to surgery but routinely generates skin side effects. Low energy electron FLASH radiotherapy (FLASH-RT) is a new form of radiotherapy exploiting the biological advantage of the FLASH effect, which consists in delivering radiation dose in milliseconds instead of minutes in CONV-RT. In pre-clinical studies, when compared to CONV-RT, FLASH-RT induced a robust, reproducible and remarkable sparing of the normal healthy tissues, while the efficacy on tumors was preserved. In this context, we aim to prospectively evaluate FLASH-RT versus CONV-RT with regards to toxicity and oncological outcome in localized cutaneous BCC and SCC. Methods: This is a randomized selection, non-comparative, phase II study of curative FLASH-RT versus CONV-RT in patients with T1-T2 N0 M0 cutaneous BCC and SCC. Patients will be randomly allocated to low energy electron FLASH-RT (dose rate: 220-270 Gy/s) or to CONV-RT arm. Small lesions (T1) will receive a single dose of 22 Gy and large lesions (T2) will receive 30 Gy in 5 fractions of 6 Gy over two weeks.The primary endpoint evaluates safety at 6 weeks after RT through grade ≥ 3 toxicity and efficacy through local control rate at 12 months. Approximately 60 patients in total will be randomized, considering on average 1-2 lesions and a maximum of 3 lesions per patients corresponding to the total of 96 lesions required. FLASH-RT will be performed using the Mobetron® (IntraOp, USA) with high dose rate functionality.LANCE (NCT05724875) is the first randomized trial evaluating FLASH-RT and CONV-RT in a curative setting.

ctDNA dynamics and mechanisms of acquired resistance in patients treated with osimertinib with or without bevacizumab from the randomised phase II ETOP-BOOSTER trial.

BACKGROUND: ETOP 10-16 BOOSTER study was a randomised phase II trial of osimertinib and bevacizumab versus osimertinib in patients with an acquired EGFR T790M mutation. The mechanisms of acquired resistance to osimertinib and bevacizumab have not been described previously. METHODS: Next generation sequencing (Guardant360®) was conducted in serial plasma samples. The association between ctDNA and efficacy outcomes was explored and molecular alterations at progression were described. RESULTS: 136 patients (88% of 155 randomised) had plasma samples at baseline (68 per arm), 110 (71%) at week 9 and 65 (42%) at progression. In a multivariable model for progression-free survival (PFS), the treatment effect was found different by smoking status (interaction p=0.046), with the effect of smoking also different by baseline EGFR T790M (interaction p=0.033), while both TP53 at baseline and tissue EGFR Exon 21 L858R mutation were significantly associated with worse PFS outcome. Smokers (current/former) without baseline EGFR T790M showed a significant improvement in PFS under combination treatment, albeit with small numbers (p=0.015). Week-9 EGFR T790M clearance was associated with improved PFS in the osimertinib arm (p=0.0097). Acquired EGFR C797S mutations were detected in 22% and 13% of patients in the combination and osimertinib arm, respectively. CONCLUSIONS: The differential effect of treatment by smoking was not explained by TP53 mutation or other molecular alterations examined. Molecular mechanisms of acquired resistance were detected but no novel molecular alterations were identified in the combination arm.

The Role of Medical Societies and the Relevance of Clinical Perspective in the Evolving EU HTA Process: Insights Generated at the 2023 Fall Convention and Survey of the European Access Academy.

BACKGROUND: This work aimed to determine the role and action points for the involvement of medical societies in the European Health Technology Assessment (EU HTA) Methods: An online pre-convention survey was developed addressing four areas related to the EU HTA: (i) medical societies' role; (ii) role of clinical guidelines; (iii) interface with the European Society for Medical Oncology Magnitude of Clinical Benefit Scale (ESMO-MCBS); and (iv) approaching 'best-available evidence' (BAE). A descriptive analysis of questionnaire outcomes was conducted to inform the European Access Academy (EAA) Fall Convention 2023. Within the working groups (WGs), action points were identified and prioritised. RESULTS: A total of 57 experts from 15 countries responded to the survey. The WGs were attended by (i) 11, (ii) 10, (iii) 12, and (iv) 12 experts, respectively, representing a variety of national backgrounds and stakeholder profiles. The most relevant action points identified were as follows: (i) incorporation of clinical context into population, intervention, comparator, outcomes (PICO) schemes, (ii) timely provision of up-to-date therapeutic guidelines, (iii) ensuring the inclusion of MCBS insights into the EU HTA process, and (iv) considering randomized controlled trials (RCTs) as the gold standard and leveraging regulatory insights if development programs only include single-arm trials. CONCLUSIONS: The involvement of medical societies is a critical success factor for the EU HTA. The identified key action points foster the involvement of patient associations and medical societies.

ROS1 fusions in resected stage I-III adenocarcinoma: Results from the European Thoracic Oncology Platform Lungscape project.

BACKGROUND: ROS1 fusion is a relatively low prevalence (0.6-2.0%) but targetable driver in lung adenocarcinoma (LUAD). Robust and low-cost tests, such as immunohistochemistry (IHC), are desirable to screen for patients potentially harboring this fusion. The aim was to investigate the prevalence of ROS1 fusions in a clinically annotated European stage I-III LUAD cohort using IHC screening with the in vitro diagnostics (IVD)-marked clone SP384, followed by confirmatory molecular analysis in pre-defined subsets. METHODS: Resected LUADs constructed in tissue microarrays, were immunostained for ROS1 expression using SP384 clone in a ready-to-use kit and Ventana immunostainers. After external quality control, analysis was performed by trained pathologists. Staining intensity of at least 2+ (any percentage of tumor cells) was considered IHC positive (ROS1 IHC + ). Subsequently, ROS1 IHC + cases were 1:1:1 matched with IHC0 and IHC1 + cases and subjected to orthogonal ROS1 FISH and RNA-based testing. RESULTS: The prevalence of positive ROS1 expression (ROS1 IHC + ), defined as IHC 2+/3+, was 4 % (35 of 866 LUADs). Twenty-eight ROS1 IHC + cases were analyzed by FISH/RNA-based testing, with only two harboring a confirmed ROS1 gene fusion, corresponding to a lower limit for the prevalence of ROS1 gene fusion of 0.23 %. They represent a 7 % probability of identifying a fusion among ROS1 IHC + cases. Both confirmed cases were among the only four with sufficient material and H-score ≥ 200, leading to a 50 % probability of identifying a ROS1 gene fusion in cases with an H-score considered strongly positive. All matched ROS1 IHC- (IHC0 and IHC1 + ) cases were also found negative by FISH/RNA-based testing, leading to a 100 % probability of lack of ROS1 fusion for ROS1 IHC- cases. CONCLUSIONS: The prevalence of ROS1 fusion in an LUAD stage I-III European cohort was relatively low. ROS1 IHC using SP384 clone is useful for exclusion of ROS1 gene fusion negative cases.

Response to tumor-infiltrating lymphocyte adoptive therapy is associated with preexisting CD8+ T-myeloid cell networks in melanoma.

Adoptive cell therapy (ACT) using ex vivo-expanded tumor-infiltrating lymphocytes (TILs) can eliminate or shrink metastatic melanoma, but its long-term efficacy remains limited to a fraction of patients. Using longitudinal samples from 13 patients with metastatic melanoma treated with TIL-ACT in a phase 1 clinical study, we interrogated cellular states within the tumor microenvironment (TME) and their interactions. We performed bulk and single-cell RNA sequencing, whole-exome sequencing, and spatial proteomic analyses in pre- and post-ACT tumor tissues, finding that ACT responders exhibited higher basal tumor cell-intrinsic immunogenicity and mutational burden. Compared with nonresponders, CD8+ TILs exhibited increased cytotoxicity, exhaustion, and costimulation, whereas myeloid cells had increased type I interferon signaling in responders. Cell-cell interaction prediction analyses corroborated by spatial neighborhood analyses revealed that responders had rich baseline intratumoral and stromal tumor-reactive T cell networks with activated myeloid populations. Successful TIL-ACT therapy further reprogrammed the myeloid compartment and increased TIL-myeloid networks. Our systematic target discovery study identifies potential T-myeloid cell network-based biomarkers that could improve patient selection and guide the design of ACT clinical trials.

Evaluation of the prognostic role of centromere 17 gain and HER2/topoisomerase II alpha gene status and protein expression in patients with breast cancer treated with anthracycline-containing adjuvant chemotherapy: pooled analysis of two Hellenic Cooperative Oncology Group (HeCOG) phase III trials.

BACKGROUND: The HER2 gene has been established as a valid biological marker for the treatment of breast cancer patients with trastuzumab and probably other agents, such as paclitaxel and anthracyclines. The TOP2A gene has been associated with response to anthracyclines. Limited information exists on the relationship of HER2/TOP2A gene status in the presence of centromere 17 (CEP17) gain with outcome of patients treated with anthracycline-containing adjuvant chemotherapy. METHODS: Formalin-fixed paraffin-embedded tumor tissue samples from 1031 patients with high-risk operable breast cancer, enrolled in two consecutive phase III trials, were assessed in a central laboratory by fluorescence in situ hybridization for HER2/TOP2A gene amplification and CEP17 gain (CEP17 probe). Amplification of HER2 and TOP2A were defined as a gene/CEP17 ratio of >2.2 and ≥2.0, respectively, or gene copy number higher than 6. Additionally, HER2, TopoIIa, ER/PgR and Ki67 protein expression was assessed by immunohistochemistry (IHC) and patients were classified according to their IHC phenotype. Treatment consisted of epirubicin-based adjuvant chemotherapy followed by hormonal therapy and radiation, as indicated. RESULTS: HER2 amplification was found in 23.7% of the patients and TOP2A amplification in 10.1%. In total, 41.8% of HER2-amplified tumors demonstrated TOP2A co-amplification. The median (range) of HER2, TOP2A and CEP17 gain was 2.55 (0.70-45.15), 2.20 (0.70-26.15) and 2.00 (0.70-26.55), respectively. Forty percent of the tumors had CEP17 gain (51% of those with HER2 amplification). Adjusting for treatment groups in the Cox model, HER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with time to relapse or time to death. CONCLUSION: HER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with outcome in high-risk breast cancer patients treated with anthracycline-based adjuvant chemotherapy. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12611000506998 and ACTRN12609001036202.

Assessment of RANK/RANK-L prevalence and clinical significance in NSCLC European Thoracic Oncology Platform Lungscape cohort and SPLENDOUR randomized clinical trial.

BACKGROUND: The primary objective of this study is to evaluate the clinical significance of RANK/L expression, in both a retrospective cohort of surgically resected stage I-III NSCLC (Lungscape) and a randomized clinical trial-cohort (SPLENDOUR) of advanced NSCLC treated with chemotherapy alone or in combination with denosumab. METHODS: RANK-L expression was assessed on tissue microarrays (TMAs) in Lungscape and whole sections in SPLENDOUR, using immunohistochemistry, with H-scores values > 0 indicating positivity. Prevalence of RANK positivity and its association with clinicopathological characteristics, and patient outcome was explored in a subset of the ETOP Lungscape cohort and in SPLENDOUR. Also investigated were the prevalence of RANK overexpression (proportion of positive cancer cells ≥ 50%) in the Lungscape cohort, and RANK-L in the SPLENDOUR trial. RESULTS: In the Lungscape cohort, RANK expression was assessed at a median follow-up of 46 months (N = 488 patients; 4 centers); 35% were female, 44/49/6% adenocarcinomas (AC)/squamous cell carcinomas (SCC)/other, 48/27/25% with stage I/II/III. Median RFS/TTR/OS were 58/Not reached/74 months. Prevalence of RANK expression was 31% (95%CI:27%-35%); significantly higher in AC: 50% (95%CI:43%-57%) vs SCC: 12% (95%CI:8%-16%) (p < 0.001); more frequent in females (42% vs 25%, p < 0.001) and tumors ≤ 4 cm (35.3% vs 23.3%, p = 0.0065). No association with outcome was found. In the SPLENDOUR trial (463 patients), the prevalence of membranous and cytoplasmic RANK positivity was 34% (95%CI:30%-38%) and 9% (95%CI:7%-12%), respectively, while prevalence for RANK-L was 5% (95%CI:3%-7%) and 36% (95%CI:31%-40%), respectively. Cytoplasmic RANK-L positivity was more common among females (47% vs 31%, p = 0.001) and in non-SCC histology (45% vs 10%, p < 0.0001). At the pre-specified 1% significance level, no prognostic or predictive effect was found. CONCLUSIONS: Both cohorts indicate that RANK expression is more common in adenocarcinoma/non-squamous NSCLC and in female patients. No prognostic effect is found, and in the clinical trial involving addition of denosumab to chemotherapy no predictive effect is detected.

A phase 1 trial of adoptive transfer of vaccine-primed autologous circulating T cells in ovarian cancer.

We have previously shown that vaccination with tumor-pulsed dendritic cells amplifies neoantigen recognition in ovarian cancer. Here, in a phase 1 clinical study ( NCT01312376 /UPCC26810) including 19 patients, we show that such responses are further reinvigorated by subsequent adoptive transfer of vaccine-primed, ex vivo-expanded autologous peripheral blood T cells. The treatment is safe, and epitope spreading with novel neopeptide reactivities was observed after cell infusion in patients who experienced clinical benefit, suggesting reinvigoration of tumor-sculpting immunity.

PD-1-expressing macrophages and CD8 T cells are independent predictors of clinical benefit from PD-1 inhibition in advanced mesothelioma.

BACKGROUND: Few tissue biomarkers exist to date that could enrich patient with cancer populations to benefit from immune checkpoint blockade by programmed cell death protein 1/ligand-1 (PD-/L-1) inhibitors. PD-L1 expression has value in this context in some tumor types but is an imperfect predictor of clinical benefit. In malignant pleural mesothelioma, PD-L1 expression is not predictive of the benefit from PD-1 blockade. We aimed to identify novel markers in malignant pleural mesothelioma to select patients better. METHODS: We performed a multiplex-immune histochemistry analysis of tumor samples from the phase III PROMISE-meso study, which randomized 144 pretreated patients to receive either pembrolizumab or standard second-line chemotherapy. Our panel focused on CD8+T cell, CD68+macrophages, and the expression of PD-1 and PD-L1 on these and cancer cells. We analyzed single and double positive cells within cancer tissues (infiltrating immune cells) and in the stroma. In addition, we performed cell neighborhood analysis. The cell counts were compared with clinical outcomes, including responses, progression-free and overall survivals. RESULTS: We confirmed the absence of predictive value for PD-L1 in this cohort of patients. Furthermore, total CD8 T cells, CD68+macrophages, or inflammatory subtypes (desert, excluded, inflamed) did not predict outcomes. In contrast, PD-1-expressing CD8+T cells (exhausted T cells) and PD-1-expressing CD68+macrophages were both independent predictors of progression-free survival benefit from pembrolizumab. Patients with tumors simultaneously harboring PD1+T cells and PD-1+macrophages benefited the most from immune therapy. CONCLUSION: We analyzed a large cohort of patients within a phase III study and found that not only PD-1+CD8 T cells but also PD-1+CD68+ macrophages are predictive. This data provides evidence for the first time for the existence of PD-1+macrophages in mesothelioma and their clinical relevance for immune checkpoint blockade.

European Epidemiology of Pleural Mesothelioma-Real-Life Data From a Joint Analysis of the Mesoscape Database of the European Thoracic Oncology Platform and the European Society of Thoracic Surgery Mesothelioma Database.

INTRODUCTION: Pleural mesothelioma (PM) is an aggressive malignancy with increasing prevalence and poor prognosis. Real-life data are a unique approach to reflect the reality of PM epidemiology, treatment, and prognosis in Europe. METHODS: A joint analysis of the European Thoracic Oncology Platform Mesoscape and the European Society of Thoracic Surgeons (ESTS) databases was performed to better understand the characteristics and epidemiology of PM, including histologic subtype, staging, and treatment. Overall survival (OS) was assessed, adjusting for parameters of clinical interest. RESULTS: The analysis included 2766 patients (Mesoscape: 497/10 centers/ESTS: 2269/77 centers). The primary histologic subtype was epithelioid (71%), with 57% patients on stages III to IV. Within Mesoscape, the patients received either multimodality (59%) or palliative intention treatment (41%). The median follow-up was 47.2 months, on the basis of 1103 patients (Mesoscape: 491/ESTS: 612), with 823 deaths, and median OS was 17.4 months. In multivariable analysis, female sex, epithelioid subtype, and lower stage were associated with longer OS, when stratifying by cohort, age, and Eastern Cooperative Oncology Group Performance Status. Within Mesoscape, multimodality treatment including surgery was predictive of longer OS (hazard ratio = 0.56, 95% confidence interval: 0.45-0.69), adjusting for sex, histologic subtype, and Eastern Cooperative Oncology Group Performance Status. Overall, surgical candidates with a macroscopic complete resection had a significantly longer median OS compared with patients with R2 (25.2 m versus 16.4 m; log-rank p < 0.001). CONCLUSIONS: This combined European Thoracic Oncology Platform/ESTS database analysis offers one of the largest databases with detailed clinical and pathologic outcome. Our finding reflects a benefit for selected patients that undergo multimodality treatment, including macroscopic complete resection, and represents a valuable resource to inform the epidemiology and treatment options for individual patients.