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Key Points ctDNA technology is useful for noninvasive monitoring of lymphoma treated with targeted agents in the clinical trial setting.An early >2-log reduction of ctDNA levels after 2 treatment cycles was a confirmed threshold for molecular response., Visual Abstract, To advance the use of circulating tumor DNA (ctDNA) applications, their broad clinical validity must be tested in different treatment settings, including targeted therapies. Using the prespecified longitudinal systematic collection of plasma samples in the phase 1/2a LYM1002 trial (registered on www.clinicaltrials.gov as NCT02329847), we tested the clinical validity of ctDNA for baseline mutation profiling, residual tumor load quantification, and acquisition of resistance mutations in patients with lymphoma treated with ibrutinib+nivolumab. Inclusion criterion for this ancillary biological study was the availability of blood collected at baseline and cycle 3, day 1. Overall, 172 ctDNA samples from 67 patients were analyzed by the LyV4.0 ctDNA Cancer Personalized Profiling Deep Sequencing Assay. Among baseline variants in ctDNA, only TP53 mutations (detected in 25.4% of patients) were associated with shorter progression-free survival; clones harboring baseline TP53 mutations did not disappear during treatment. Molecular response, defined as a >2-log reduction in ctDNA levels after 2 cycles of therapy (28 days), was achieved in 28.6% of patients with relapsed diffuse large B-cell lymphoma who had ≥1 baseline variant and was associated with best response and improved progression-free survival. Clonal evolution occurred frequently during treatment, and 10.3% new mutations were identified after 2 treatment cycles in nonresponders. PLCG2 was the topmost among genes that acquired new mutations. No patients acquired the C481S BTK mutation implicated in resistance to ibrutinib in CLL. Collectively, our results provide the proof of concept that ctDNA is useful for noninvasive monitoring of lymphoma treated with targeted agents in the clinical trial setting.

Splenic marginal zone B-cell lymphoma (SMZL) is a heterogeneous clinico-biological entity. The clinical course is variable, multiple genes are mutated with no unifying mechanism, and essential regulatory pathways and surrounding microenvironments are diverse. We sought to clarify the heterogeneity of SMZL by resolving different subgroups and their underlying genomic abnormalities, pathway signatures, and microenvironment compositions to uncover biomarkers and therapeutic vulnerabilities. We studied 303 SMZL spleen samples collected through the IELSG46 multicenter international study (NCT02945319) by using a multiplatform approach. We carried out genetic and phenotypic analyses, defined self-organized signatures, validated the findings in independent primary tumor metadata and determined correlations with outcome data. We identified 2 prominent genetic clusters in SMZL, termed NNK (58% of cases, harboring NF-κB, NOTCH, and KLF2 modules) and DMT (32% of cases, with DNA-damage response, MAPK, and TLR modules). Genetic aberrations in multiple genes as well as cytogenetic and immunogenetic features distinguished NNK- from DMT-SMZLs. These genetic clusters not only have distinct underpinning biology, as judged by differences in gene-expression signatures, but also different outcomes, with inferior survival in NNK-SMZLs. Digital cytometry and in situ profiling segregated 2 basic types of SMZL immune microenvironments termed immune-suppressive SMZL (50% of cases, associated with inflammatory cells and immune checkpoint activation) and immune-silent SMZL (50% of cases, associated with an immune-excluded phenotype) with distinct mutational and clinical connotations. In summary, we propose a nosology of SMZL that can implement its classification and also aid in the development of rationally targeted treatments.

Context: Ibrutinib inhibits BTK with the most typical response in CLL being partial remission (PR) with measurable minimal residual disease (MRD) in blood. Remission is usually maintained until development of genetically-driven resistance. Objective: Mechanism of adaptation and survival in MRD. Patients and Methods: Pre-treatment CLL cells and under-treatment MRD were systematically collected from 33 high-risk CLL patients. Samples were characterized by high-dimensional, single-cell flow cytometry; bulk genomic, transcriptomic, and chromatin accessibility profiling; and single-cell RNA-seq profiling. Results: The genetic composition of MRD remained constant across timepoints, indicating that ibrutinib did not have any impact in shaping its genomic diversity. Ibrutinib induced chromatin dynamics in MRD resulting in a predominantly closed state. Chromatin regions that became more accessible under ibrutinib were enriched of binding sites for transcription factor that lay downstream ERK signaling. Regions that turned into a less accessible state were enriched for the binding sites of TF emanating from the BCR. At the transcriptomic level, most of the differentially expressed genes between pre- and post-treatment samples were downregulated in MRD, while only a fraction was upregulated. IL4, NF-kB, and metabolism and proliferation signatures were downregulated in MRD, while MAPK and RAS signatures were upregulated. CLL spleen samples from TCL1 mice under ibrutinib treatment showed an upregulation of MAPK pathway genes compared to untreated mice. Single-cell transcriptomes revealed a distinct post-treatment cell population driven by MAPK activity that functionally pre-existed in a fraction of CLL cells of baseline samples before ibrutinib start. At the signaling level, ibrutinib had no effect on the integrity of RAS-BRAF-MAPK-ERK pathway protein expression upon crosslinking of the BCR with anti-IgM but not after stimulation of TLR9 or CD40. Functional validation on primary samples by western blotting shows that RAS/ERK can be activated by BCR stimulation, irrespective of ibrutinib treatment in ex vivo experiments. Pharmacological inhibition of MEK (trametinib) and ERK (ulixertinib) synergized with ibrutinib, leading to decreased cell viability. Conclusions: MRD under ibrutinib adapts its phenotype in an epigenetic way to maintain functional competence of BCR signaling via the MAPK pathway, which turned to be a vulnerability of MRD persisting under ibrutinib.

Introduction Progression of disease within 2 years after starting rituximab-chemotherapy (POD24) has been identified as a convincing adverse prognostic factor for follicular lymphoma (FL), and can serve as a clinical endpoint to identify patients at high risk of early lymphoma-related mortality. However, POD24 is not accessible at diagnosis. Several stratification models adopting baseline variables have been developed for predicting outcomes in FL patients. The Follicular Lymphoma International Prognostic Index (FLIPI) and PRIMA Prognostic-Index (PRIMA-PI) include clinical characteristics, while m7- Follicular Lymphoma International Prognostic Index (m7-FLIPI) and POD24 Prognostic Index (POD24-PI) integrate gene mutations with clinical features. Moreover, the mutation status of specific genes (including TP53, EZH2, TNFRSF14, and BCL2) has been reported to be relevant to early progression. It has been reported that m7-FLIPI and POD24-PI are predictive for POD24. Here we compare the diagnostic accuracy for POD24 of various stratification models and gene mutations in an institutional cohort of FL. Methods Consecutive patients diagnosed as FL grades 1-3a were enrolled from the Oncology Institute of Southern Switzerland (n=75), the University of Eastern Piedmont (n=118), and the Hematology of the AUSL IRCCS of Reggio Emilia (n=59). DNA was extracted from FFPE tissue specimen obtained at diagnosis. We performed CAPP-seq to detect the mutation status of the genes included in m7-FLIPI and POD24 PI (EZH2,ARID1A, MEF2B, EP300, FOXO1, CREBBP, and CARD11), as well as BCL2, TNFRSF14 and TP53. POD24 was the primary endpoint of this research. Sensitivity, specificity, predictive values and balanced accuracy of every stratification model were calculated to estimate the prognostic efficacy. Results With FLIPI score, 34% of patients were classified as low risk, 22% as intermediate risk, and 41% as high risk. With PRIMA-PI, the fractions for low risk, intermediate risk and high risk group were 24%, 21% and 49%, respectively. With m7-FLIPI, 82% of patients were low risk, and 15% high risk. For POD24-PI, 65% of patients were low risk, and 32% high risk. The mutation frequencies of TP53, TNFRSF14, EZH2 and BCL2 were 8%, 28%, 27% and 38%, respectively. We validated the prognostic utility of FLIPI, PRIMA-PI, m7-FLIPI and POD24-PI for progression free survival (PFS) and OS. However, no statistically significant relevance with PFS or OS has been found for TP53, TNFRSF14, EZH2 or BCL2 mutations. POD24 was calculated in the 142 patients who received systemic treatment (immunotherapy or chemoimmunotherapy) within 6 months from diagnosis: 18% of them were POD24-positive. POD24 positivity associated with shorter overall survival (OS) (p=0.0138). After adjusting for multiple comparisons, no high-risk groups identified by TNFRSF14, EZH2 or BCL2 gene mutations, FLIPI, PRIMA-PI, m7-FLIPI or POD24-PI were associated with POD24. In terms of diagnostic performance for POD24, PRIMA-PI showed the highest accuracy (57%), FLIPI had the highest positive predictive value (64%), and m7-FLIPI had the highest negative predictive value (82%). Conclusion The mutation status of specific single genes were not related to POD24, PFS nor OS, indicating that the mutation of single gene may not be sufficient to identify high-risk FL patients. Though retaining their prognostic value, the integration of mutations onto the clinical biomarker-based prognostic scores has a limited discrimination capacity for POD24. Our results prompt the investigation of: i) POD discrimination capacity of prognostic models based on molecular phenotypes (i.e. gene expression) reflecting the tumor-microenvironment milieu; ii) new models based on a combination of biomarkers capturing the most informative clinical, genetic and phenotypic features. Figure 1 Disclosures Moccia: Takeda: Consultancy, Other: Advisory Boards: Roche, Janssen, Takeda; Roche: Consultancy, Other: Advisory Boards: Roche, Janssen, Takeda; Janssen: Consultancy, Other: Advisory Boards: Roche, Janssen, Takeda. Stathis:ADC Therapeutcis: Other, Research Funding; Abbvie: Other: Travel Grant; MEI Pharma: Other, Research Funding; Novartis: Other, Research Funding; Roche: Other, Research Funding; Pfizer: Other, Research Funding; PharmaMar: Other: Travel Grant; Cellestia: Research Funding; Loxo: Honoraria, Other, Research Funding; Member of the steering committee of the trial of this abstract: Other; Bayer: Other, Research Funding; Merck: Other, Research Funding. Gerber:Alnylam: Other: funding for accredited continuing medical education; Axonlab: Other: funding for accredited continuing medical education program ; Bayer: Other: funding for accredited continuing medical education program ; Bristol Myers Squibb: Other: funding for accredited continuing medical education program ; Daiichi-Sankyo: Other: funding for accredited continuing medical education program ; Janssen: Other: funding for accredited continuing medical education program ; Mitsubishi Tanabe Pharma: Other: funding for accredited continuing medical education program ; NovoNordisk: Other: funding for accredited continuing medical education program ; Octapharma: Other; Takeda: Other; Sanofi: Other; SOBI: Other; Thermo Fishe: Other; Axonlab: Other; Pfizer: Other: personal fees ; Sanofi: Other: funding for accredited continuing medical education. Gaidano:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astrazeneca: Membership on an entity's Board of Directors or advisory committees; Sunesys: Membership on an entity's Board of Directors or advisory committees. Rossi:Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding. Zucca:Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants, Research Funding; Celltrion Healthcare: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Abbvie: Other: Travel Grants; Beigene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding.

In the search for new anti-breast cancer compounds a series of diastereomeric aqua[1-(2,6-dichloro-4-hydroxyphenyl)-2-phenylethylenediamine]sulfato platinum(II) complexes was tested on the hormone-sensitive MXT-M-3.2 breast cancer of the mouse. By simultaneous determination of tumor and uterine weights at the end of the experiment we obtained an insight into the mode of action. Changes in the uterine weights indicate whether the anti-breast cancer effect of the test substance is caused either by its capability to reduce the endogenous estrogen level via inhibition of estrogen biosynthesis (mechanism B), or by its estrogenic side effects which enhance the immune defense in the host animals (mechanism C). Studies on the [3H]thymidine incorporation into DNA of MDAMB-231 breast cancer cells showed that all test compounds inhibit deoxyribonucleic acid synthesis, like cisplatin (mechanism A). However, in comparison to the standard cisplatin, their activities were low. The three most effective test compounds threo-2-PtSO4 (2-phenyl-residue), threo-5-PtSO4 (2-(4-hydroxyphenyl)-residue), and threo-8-PtSO4 (2-(2-fluoro-4-hydroxyphenyl)-residue) seem to exert their anti-breast cancer effect by mechanism B. Moreover, threo-5-PtSO4 was moderately active on the hormone- insensitive MXT-M-3.2 (Ovex) breast cancer of the mouse. Aqua[erythro-1-(2,6-dichloro-4-hydroxyphenyl)-2-(2-chloro-4-hydroxypheny l) ethylenediamine]sulfato-platinum(II) (erythro-9-PtSO4) was the only breast cancer inhibiting compound of the series acting mainly according to mechanism C. A minor contribution of mechanism A, impairment of the DNA function in the tumor cell, to the anti-breast cancer activity of these aqua[1-(2,6-dichloro-4-hydroxyphenyl)-2-phenylethylenediamine]sulfato platinum(II) complexes cannot be excluded, since such effects are apparent in the MDA-MB-231 as well as in the MCF-7 breast cancer cell line at higher drug concentrations. In this test series which was performed with the crystal violet chemosensitivity assay, the MCF-7 cells proved to be somewhat more sensitive.

The Pt complex [meso-1,2-bis(2,6-dichloro-4-hydroxyphenyl)-ethylenediamine]diaqua -Pt(II) sulfate (meso-6-PtSO4) was designed with the concept of combining the cytotoxic cisplatin with an estrogen receptor (ER)-binding ligand for targeting to ER+ mammary tumor cells. This Pt complex selectively inhibits growth of ER+ mammary tumors in rodents. To study the cellular mechanisms of action, cultures of two human mammary tumor cell lines, MCF-7 (ER+) and MDA-MB231 (ER-), were used and the effects of estradiol, tamoxifen, and cis-Pt compared with those of meso-6-PtSO4. The relative binding affinity of the meso-6-PtSO4 to the ER in MCF-7 cells was 0.35 compared to estradiol (relative binding affinity, 100). Nevertheless, the Pt complex was able to induce ER processing and increase the level of the progesterone receptor at concentrations of 1-10 nM. Growth of MCF-7 cells was inhibited at concentrations of meso-6-PtSO4 greater than 10 microM. MDA-MB231 cells were inhibited likewise by the Pt complex, indicating a lack of selectivity for the ER+ cells. The results show that meso-6-PtSO4 possesses both estrogen-like and cis-Pt-like properties. Since growth inhibition did not correlate with ER-mediated processes, these two properties are expressed independently at the cellular level. The selective growth inhibitory effect of meso-6-PtSO4 in vivo is suggested to involve endocrinological and/or immunological factors.