Immune-checkpoint inhibitors (ICIs) showed limited efficacy in mismatch repair proficient (MMRp) metastatic colorectal cancer (mCRC) to date. Many RAS/BRAF wildtype mCRCs respond to EGFR antibodies (cetuximab/panitumumab) and we previously showed that acquired resistance to these is characterised by an inflamed phenotype and PD-L1 and LAG3 upregulation. The single arm phase 2 iSCORE trial investigated whether RAS/BRAF wildtype MMRp mCRCs that had acquired resistance to prior chemotherapy and an EGFR antibody benefit from nivolumab (anti-PD1) and relatlimab (anti-LAG3) ICIs. Patients (pts) with RAS/BRAF wildtype MMRp mCRC that had responded to chemotherapy + EGFR antibody and subsequently progressed were recruited for treatment with nivolumab (480mg iv) and relatlimab (160mg iv) every 4 weeks. The primary endpoint was disease control rate at 6 months (DCR6) from treatment initiation. To detect an increase in DCR6 from 10% to 30%, with a two-sided 5% significance and power of 80%, 25 pts were needed. Secondary endpoints included duration of disease control, best objective response (ORR) during 6 months, progression free survival (PFS), overall survival (OS) and safety. Pre-treatment and on-treatment biopsies were obtained for biomarker analyses. 32 pts were registered. 25pts who received at least one dose of nivolumab/relatlimab were included in the primary endpoint analysis. 24% of pts had received ≥2 prior lines of systemic therapy. The median number of cycles administered was 2 (range: 1-12). 1pt remained on treatment (cycle 11) at the time of data cut-off (30/01/2023). Among 25 evaluable pts, best objective responses during 6 months by RECIST 1.1 were one CR, one PR, two SD and 20 PD; 1pt died without a follow-up scan. One PR and one SD were reported during treatment in two additional pts by iRECIST after pseudoprogression. Median duration of disease control for pts with clinical benefit (CR, PR, SD) by iRECIST was 9.0mo [range:1.8-11.1]. 4/7pts [57.1%] without liver metastases and 2/18pts [11.1%] with liver metastases achieved clinical benefit by iRECIST. The best ORR during 6 months was 8% [95% CI:1.0-26.0] by both RECIST 1.1 and iRECIST. DCR6 was 12% [95% CI:2.5-31.2] by RECIST 1.1 and 16% [95% CI:4.5-36.1] by iRECIST in all 25pts. At data cut-off, median PFS and OS were 1.6mo [95% CI:1.6-1.8] and 15.2mo [95% CI:6.4-18.5], respectively. Five grade 3 treatment related adverse events (TRAE) were reported across 5pts; no grade 4/5 TRAEs occurred. The most common TRAEs (any grade) were fatigue (24%) and acneiform rash (12%). In pre-treatment biopsies, the median PD-L1 combined positive score (CPS) was 0 [range:0-3] for pts without liver metastases and PD-L1 CPS was 0 for all four of these with clinical benefit. The median PD-L1 CPS was 1.5 [range:0-65] for pts with liver metastases, with PD-L1 CPS 6 and 12 in the 2pts with clinical benefit. The prespecified endpoint of 25% DCR6 was not met with nivolumab/relatlimab. However, the clinical benefit rate in pts without liver metastases and prolonged disease control in two pts with liver metastases are encouraging. Nivolumab/relatlimab was well tolerated. Biomarker analyses, including TMB and T-cell quantification, are ongoing and will be presented.

The somatic hypermutation (SHM) status of the clonotypic, rearranged immunoglobulin heavy variable (IGHV) gene is an established prognostic and predictive marker in chronic lymphocytic leukemia (CLL). Analysis of SHM is generally performed by polymerase chain reaction (PCR)-amplification of clonal IGHV-IGHD-IGHJ gene rearrangements followed by sequencing to identify IGHV gene sequences and germline identity. Targeted-hybridization next-generation sequencing (NGS) can simultaneously assess clonality and other genetic aberrations. However, it has limitations for SHM analysis due to sequence similarity between different IGHV genes and mutations introduced by SHM, which can affect alignment efficiency and accuracy. We developed a novel SHM assessment strategy using a targeted-hybridization NGS approach (EuroClonality- NDC assay) and applied it to 331 samples of lymphoproliferative disorder (LPD). Our strategy focuses on analyzing the sequence downstream to the clonotypic, rearranged IGHJ gene up to the IGHM enhancer (IGHJ-E) which provides more accurate alignment. Overall, 84/95 (88.4%) CLL cases with conventional SHM data showed concordant SHM status, increasing to 91.6% when excluding borderline cases. Additionally, IGHJ-E mutation analysis in a wide range of pre- and post-germinal center LPD showed significant correlation with differentiation and lineage status, suggesting that IGHJ-E analysis is a promising surrogate marker enabling SHM to be reported using NGS-capture strategies and whole genome sequencing.

Introduction: The evolution of metastatic cancers over time can be assessed in circulating tumor DNA (ctDNA) but sequencing and bioinformatics tools for ultra-deep ctDNA whole-exome sequencing (WES) analysis are lacking. Methods: We developed ctDNA WES that only requires 15ng DNA to achieve sequencing depths of 1000-2000x. Error correction with molecular barcodes and duplex DNA detection allowed calling of mutations ≥0.5% variant frequency (VF). This pilot study applied ctDNA WES to plasma from 20 EGA patients (pts) and standard WES to matched biopsies in order to assess ctDNA WES performance and whether clonal mutation burden (cMB), a critical immunotherapy biomarker and important for neoantigen vaccine designs, differed between ctDNA and biopsies. We furthermore established a mutation and copy number data analysis pipeline using Bayesian clustering to define subclones and track their evolution during therapy in 3pts. Results: The median age of pts was 71y, 95% had distant metastases and 5% locally advanced EGAs. At a median sequencing depth of ????x after de-duplication, VFs of mutations in pre-treatment ctDNA was low (<2% VF) in 8 pts, and intermediate (2-10%) to high (>10%) in 12. Whether cMB differed in biopsies vs ctDNA was assessed in 7 pts with high VFs in ctDNA and good cancer purity in matching biopsy WES. The median cMB was 82 in biopsies and 111 in ctDNA. The increase was driven by 3 cases with 30%, 35% and 59% higher cMB in ctDNA vs biopsies. In two pts, most mutations that only appeared clonal in ctDNA were subclonal rather than absent in the primary tumor, indicating the subclonal presence of the metastasis progenitor clone. Subclonal intermixing in primary tumors hence limits the accurate identification of mutations that are clonal in metastatic disease. Evolutionary dynamics analyses in 3 pts who had good responses to chemotherapy before progression showed a major clonal sweep in one, supporting monoclonal resistance, and evolution of small subclones in two, indicating polyclonal resistance. A MEK1 K57T mutation evolved at resistance in a HER2 amplified EGA treated with trastuzumab+chemotherapy, demonstrating the utility to identify mechanisms of acquired resistance. Conclusions: ctDNA WES can assess the genetics of entire metastatic cancer cell populations over time and deconvolute their evolutionary trajectories. 43% pts had higher cMB in ctDNA compared to biopsies. Liquid biopsy analyses by WES may be superior to tissue-based WES for mutation burden analysis and neoantigen vaccine designs. ctDNA WES identified distinct evolutionary modes of resistance. A larger cohort is being analyzed to define how these differ clinically and whether they can be predicted from pre-treatment ctDNA WES. Efforts to increase the ctDNA WES sensitivity are ongoing as 8 pts had low ctDNA VFs which increased false negative rates. Citation Format: Neil McCafferty, Caroline Fong, Louise J. Barber, Andrew Woolston, Dimitrios Kleftogiannis, Taqia Rana, Susan Cromarty, Shannon Kidd, Ruwaida Begum, Ian Chau, Naureen Starling, David Cunningham, Marco Gerlinger. Clonal mutation burden and evolutionary dynamics analysis in metastatic gastro-esophageal adenocarcinoma (GEA) by error corrected whole-exome circulating tumor DNA sequencing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5593.

We report a patient initially diagnosed with a triple hit high-grade B cell lymphoma (HGBL-TH), in which further morphologic, immunohistochemical, and next-generation sequencing studies of subsequent specimens disclosed it to be a germinal center diffuse large B cell lymphoma (GC-DLBCL) with BCL2/BCL6 gene translocations, PVT1-deletion, and gain of MYC genes evolving from a previous follicular lymphoma. However, fluorescence in situ hybridization (FISH) studies with the break-apart probe for MYC gene showed a fusion and two separated signals (red and green, respectively) leading to the interpretation of MYC gene translocation and a false diagnosis of a TH-lymphoma, according to the recent WHO classification. Nevertheless, PVT1 deletion plus MYC gain/amplification has been described as a cause of the double-hi transcription profile. These data highlight the need for new criteria to identify these highly aggressive lymphomas.

Current diagnostic standards for lymphoproliferative disorders include multiple tests for detection of clonal immunoglobulin (IG) and/or T-cell receptor (TCR) rearrangements, translocations, copy-number alterations (CNAs), and somatic mutations. The EuroClonality-NGS DNA Capture (EuroClonality-NDC) assay was designed as an integrated tool to characterize these alterations by capturing IGH switch regions along with variable, diversity, and joining genes of all IG and TCR loci in addition to clinically relevant genes for CNA and mutation analysis. Diagnostic performance against standard-of-care clinical testing was assessed in a cohort of 280 B- and T-cell malignancies from 10 European laboratories, including 88 formalin-fixed paraffin-embedded samples and 21 reactive lesions. DNA samples were subjected to the EuroClonality-NDC protocol in 7 EuroClonality-NGS laboratories and analyzed using a bespoke bioinformatic pipeline. The EuroClonality-NDC assay detected B-cell clonality in 191 (97%) of 197 B-cell malignancies and T-cell clonality in 71 (97%) of 73 T-cell malignancies. Limit of detection (LOD) for IG/TCR rearrangements was established at 5% using cell line blends. Chromosomal translocations were detected in 145 (95%) of 152 cases known to be positive. CNAs were validated for immunogenetic and oncogenetic regions, highlighting their novel role in confirming clonality in somatically hypermutated cases. Single-nucleotide variant LOD was determined as 4% allele frequency, and an orthogonal validation using 32 samples resulted in 98% concordance. The EuroClonality-NDC assay is a robust tool providing a single end-to-end workflow for simultaneous detection of B- and T-cell clonality, translocations, CNAs, and sequence variants.

In 2010, an 83-year-old woman presented with a 5-cm nodule on her right knee, diagnosed as primary cutaneous-diffuse-large-B-cell lymphomas-leg type (PCDLBCL-LT) that suffered complete remission (CR) after treatment with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP; six cycles). Eight years later three small nodules appeared at the same site (Figs 1A and 2A). Again, after surgery, chemotherapy (R-mini-CHOP×3) and local radiotherapy, CR was achieved without evidence of recurrence two years later. This clinical behaviour is surprising since the prognosis of most PCDLBCL-LT patients is usually poor, with multiple cutaneous/extracutaneous recurrences.1 Notwithstanding, spontaneous regression of some cases has been reported.2 Histologically, the 2010 cutaneous biopsy consisted of a dermis and subcutaneous tissue diffusely infiltrated by medium-sized round cells with scant cytoplasm, hyperchromatic nuclei and small nucleoli (Fig 1). Apoptotic cells and mitotic figures were found. In contrast, the 2018 lesion showed larger cells with abundant cytoplasm, round nuclei and rather prominent nucleoli (Fig 2). Epidermis was spared in both specimens. The immunophenotype of both samples was identical, showing neoplastic cells CD20, BCL6, CD10, BCL2 and MYC and were negative for CD30, Cyclin D1, SOX11, TdT, MUM1, p53 and EBV (EBER) (Figs 1 and 2). The proliferation activity (Ki-67) was high (almost 100%). Based on morphology and immunophenotype, the diagnosis of DLBCL of GC-phenotype (according to the Hans algorithm) was made in both instances. Given the double expression of MYC and BCL2, fluorescent in situ hybridization (FISH) studies for BCL6/BCL2 and MYC genes were performed. Translocations of both MYC/BCL6 genes were found. Interestingly, a biallelic and a monoallelic MYC gene rearrangement was detected in the first and second biopsy respectively (Figure S1). To the best of our knowledge, this finding has not been described so far in PCDLBCL-LT or systemic high-grade B-cell lymphomas with MYC/BCL2/BCL6 rearrangements (high grade B cell lymphoma [HGBCL]-DH/TH), although it has been reported for the DUSP22 gene in anaplastic large cell lymphomas.3 Next-generation sequencing (NGS) studies identified a BCL6–IGL rearrangement, with identical breakpoints on BCL6 and IGLJ1 in both samples. Rearrangements of BCL6 to IGL or to other non-IG partners probably have an influence on prognosis.4 Interestingly, a MYC–IGH translocation was identified in the 2010 sample but not at relapse. Moreover, a fusion (using a fusion MYC–IGH probe) was identified at first diagnosis, but not at relapse, confirming NGS results (Figure S2). It is well known that the majority of MYC rearrangements in HGBCL-DH/TH have non-IG partners.5 HGBCL-DH/TH are usually of GC phenotype6 while PCDLBCL-LT are typically of ABC phenotype. Of PCDLBCL-LT, 16% show CD10 expression, inversely correlated with the presence of MYC gene translocation.7 Over 90% of cases of PCDLBCL-LT express BCL2, and about two thirds of them are double expressors. Furthermore, MYC rearrangement has been reported in 32% of the cases,1 with only two cases described so far of double rearrangement.7 Both double expression of BCL2/MYC or MYC gene rearrangement in PCDLBCL-LT patients are related to poor outcome.7 The presence of a second hit involving the BCL6 gene did not seem to make any difference in cases with MYC gene rearrangements.7 Prognosis of HGBCL-DH/TH depends not only on the partner of MYC gene rearrangements but also on whether BCL2 or BCL6 gene is the second hit.8 Next-generation sequencing also showed the same rearrangements of IGH and IGK genes (IGHJ4–IGHD3-22, IGKJ2–IGKV1-39, intronRSS–Kde and IGLJ1–IGLV3-21) in both samples although they differed in their somatic mutation repertoire. Mutations of SMARCA4 and KMT2D genes were detected in the first biopsy but not in the second one, while FAT1 gene mutation appeared only in the recurrence. The remaining gene alterations (CD79B, TNFAIP3, HIST1H1E and PIM1) were present in both (Table SIII). The MYD88 p.L265P mutation, present in about 60% of PCDLBCL-LT cases, and related to poor prognosis,9 was not found here, neither by quantitative reverse transcription polymerase chain reaction (qPCR) nor NGS. Other genes previously reported in PCDLBCL-LT were present both in the first biopsy and at relapse (CD79B, HIST1H1E and PIM1).9 Mutations on the TNFAIP3 (A20) gene have not been previously reported in PCDLBCL-LT, even though deletions of this gene are frequent.9 Interestingly, in the first biopsy, mutation of genes related to germinal centre B-cell origin lymphomas, such as SMARCA4 or KMT2D (MLL2) was found. Significance of mutations in FAT1 is still not known in DLBCLs. Interestingly, biallelic CDKN2A gene deletions were identified in both samples. These data imply that the lymphoma diagnosed in 2018 was a bona fide recurrence of the one diagnosed in 2010, demonstrating that both tumours share a common clonal progenitor but were subjected to divergent evolution (Figure S3). Tumours such as PCDLBCL-LT are aggressive lymphomas characterized by a proliferation of immunoblastic-like large neoplastic B cells of ABC phenotype which characteristically show both MYD88 p.L265P mutations and CDKN2A gene deletions. Secondary skin involvement by a systemic HGBCL-DH/TH, Burkitt lymphoma, mantle cell lymphoma and systemic follicular lymphoma transforming into HGBCL-DH/TH could be ruled out both clinically and immunophenotypically. Nevertheless, primary cutaneous follicular lymphoma (PCFCL) with a diffuse pattern and predominance of large cells should be taken into consideration. These usually occur in the head and neck or the trunk. Histologically, a mixture of centrocytes and centroblasts with a large amount of small bystander T cells and scattered CD23- or CD21-positive residual follicular dendritic cells is seen. They are characteristically BCL2- and CD10-negative. BCL2-positive cases with CD10 expression and BCL2 gene translocation makes it advisable to rule out a systemic origin. Only mutations in the TNFRSF14 gene have been reported in PCFCL, usually in combination with 1p36 deletion.10 All these facts together allowed us to exclude the diagnosis of PCFCL with a diffuse pattern. In conclusion, we report a PCDLBCL-LT that showed peculiar immunophenotype, molecular background and a remarkably indolent clinical behaviour. These data suggest that the group of PCDLBCL-LT is more heterogeneous than previously thought. This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) of the Spanish Ministry of Economy and Competence (MINECO, RTICC ISCIII and CIBERONC) (SAF2013-47416-R, RD06/0020/0107, RD012/0036/0060 and Plan Nacional I+D+I: PI17/2172, PI16/01294 and PIE15/0081), AECC and the Madrid Autonomous Community. Informed written consent has been obtained. Studies have been performed according to the Declaration of Helsinki. The procedures have been approved by a local ethics committee. Date 08-11-2018; Approval number: PICO75-18_FJD; CEIm-FJD. Dr. Piris is sponsored by TAKEDA. The other authors have no conflict to declare. Table SI. Panel of antibodies investigated in this study Table SII. List of probes of the custom B-cell lymphoma panel. Table SIII. Somatic mutations found in biopsies taken in 2010 and 2018 respectively. Name of the gene, alteration and allele frequency of the change are indicated. Data S1. Material and methods. 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Introduction: Current diagnostic standards for lymphoproliferative disorders include detection of clonal immunoglobulin (IG) and/or T cell receptor (TR) rearrangements, translocations, copy number alterations (CNA) and somatic mutations. These analyses frequently require a series of separate tests such as clonality PCR, fluorescence in situ hybridisation and/or immunohistochemistry, MLPA or SNParrays and sequencing. The EuroClonality-NGS DNA capture (EuroClonality-NDC) panel, developed by the EuroClonality-NGS Working Group, was designed to characterise all these alterations by capturing variable, diversity and joining IG and TR genes along with additional clinically relevant genes for CNA and mutation analysis. Methods: Well characterised B and T cell lines (n=14) representing a diverse repertoire of IG/TR rearrangements were used as a proficiency assessment to ensure 7 testing EuroClonality centres achieved optimal sequencing performance using the EuroClonality-NDC optimised and standardised protocol. A set of 56 IG/TR rearrangements across the 14 cell lines were compiled based on detection by Sanger, amplicon-NGS and capture-NGS sequencing technologies. For clinical validation of the NGS panel, clinical samples representing both B and T cell malignancies (n=280), with ≥ 5% tumour infiltration were collected from 10 European laboratories, with 88 (31%) being formalin fixed paraffin-embedded samples. Samples were distributed to the 7 centres for library preparation, hybridisation with the EuroClonality-NDC panel and sequencing on a NextSeq 500, using the EuroClonality-NDC standard protocol. Sequencing data were analysed using a customised version of ARResT/Interrogate, with independent review of the results by 2 centres. All cases exhibiting discordance between the benchmark and capture NGS results were submitted to an internal review committee comprising members of all participating centres. Results: All 7 testing centres detected all 56 rearrangements of the proficiency assessment and continued through to the validation phase. A total of 10/280 (3.5%) samples were removed from the validation analysis due to NGS failures (n=1), tumour infiltration < 5% (n=7), and sample misidentification (n=2). The EuroClonality-NDC panel detected B cell clonality (i.e. detection of at least one clonal rearrangement at IGH, IGK or IGL loci) in 189/197 (96%) B cell malignancies. Seven of the 8 discordant cases were post-germinal centre malignancies exhibiting Ig somatic hypermutation. The EuroClonality-NDC panel detected T cell clonality (i.e. detection of at least one clonal rearrangement at TRA, TRB, TRD or TRG loci) in 70/73 (96%) T cell malignancies. In all 3 discordant cases analysis of benchmark PCR data was not able to detect clonality at any TR loci. Next, we examined whether the EuroClonality-NDC panel could detect clonality at each of the individual loci, resulting in sensitivity values of 95% or higher for all IG/TR loci, with the exception of those where limited benchmark data were available, i.e. IGL (n=3) and TRA (n=7). The specificity of the panel was assessed on benign reactive lesions (n=21) that did not contain clonal IG/TR rearrangements based on BIOMED-2/EuroClonality PCR results; no clonality was observed by EuroClonality-NDC in any of the 21 cases. Limit of detection (LOD) assessment to detect IG/TR rearrangements was performed using cell line blends with each of the 7 centres receiving blended cell lines diluted to 10%, 5.0%, 2.5% and 1.25%. Across all 7 centres the overall detection rate was 100%, 94.1%, 76.5% and 32.4% respectively, giving an overall LOD of 5%. Sufficient data were available in 239 samples for the analysis of translocations. The correct translocation was detected in 137 out of 145 cases, resulting in a sensitivity of 95%. Table 1 shows how translocations identified by the EuroClonality-NDC protocol were restricted to disease subtypes known to harbour those types of translocations. Analysis of CNA and somatic mutations in all samples is underway and will be presented at the meeting. Conclusions: The EuroClonality-NDC panel, with an optimised laboratory protocol and bioinformatics pipeline, detects IG and TR rearrangements and translocations with high sensitivity and specificity with a LOD ≤ 5% and provides a single end-to-end workflow for the simultaneous detection of IG/TR rearrangements, translocations, CNA and sequence variants. Table. Disclosures Stamatopoulos: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Klapper:Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Ferrero:Gilead: Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSA Pharma: Membership on an entity's Board of Directors or advisory committees; Servier: Speakers Bureau. van den Brand:Gilead: Speakers Bureau. Groenen:Gilead: Speakers Bureau. Brüggemann:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy. Langerak:Gilead: Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Research Funding; Genentech, Inc.: Research Funding; Janssen: Speakers Bureau. Gonzalez:Roche: Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau.