Somatic multigene analysis by next-generation sequencing (NGS) is routinely integrated in medical oncology for clinical decision-making. However, with the fast-growing number of recommended and required genes as well as pan-cancer biomarkers, small panels have become vastly insufficient. Comprehensive genomic profiling (CGP) is, thus, required to screen for clinically relevant markers. In this multicentric study, we report on an extensive analysis across seven centers comparing the results of the novel OncoDEEP CGP assay with the diagnostically validated TruSight Oncology 500 (TSO500) kit on 250 samples. Overall concordance was 90% for clinically relevant gene variants and >96% for more complex biomarkers. Agreement for fusion detection was 94% for the 11 overlapping clinically actionable driver genes. The higher coverage uniformity of OncoDEEP compared to TSO500 allows users to pool more samples per sequencing run. Tertiary data analysis, including reporting, is integrated in the OncoDEEP solution, whereas this is an add-on for TSO500. Finally, we showed that, analytically, the OncoDEEP panel performs well, thereby advocating its use for CGP of solid tumors in diagnostic laboratories, providing an all-in-one solution for optimal patient management.

Abstract Somatic multi-gene analysis by Next Generation Sequencing (NGS) becomes standardly integrated in medical oncology for clinical decision making. However, with the fast-growing number of recommended and required genomic biomarkers, small panels have become vastly insufficient for most tumor types. Comprehensive Genomic Profiling (CGP) is amenable to screen for single nucleotide variants (SNVs) and subtle insertions and deletions (indels) in several hundreds of genes. Moreover, it provides information on copy number variations (CNVs) and gene fusions of the most relevant genes for optimal patient management. Currently, most comprehensive panels also allow for screening of tumor-agnostic genomic biomarkers, including microsatellite instability (MSI), tumor mutation burden (TMB) and homologous recombination deficiency (HRD), which are implemented as prognostic and therapeutic signatures in a wide variety of solid tumors. So far, only a handful of CGP assays have been validated for their diagnostic utility in routine laboratory practice for the care of cancer patients. In this study we report on an extensive multicentric analysis across multiple centers in Belgium and The Netherlands comparing the novel OncoDEEP CGP assay (OncoDNA) with the diagnostically validated TSO500 assay (Illumina). We describe the technical differences between both assays as well as their outcome and shortcomings. Detection of clinically relevant SNVs, indels and CNVs was very similar and differences were often due to assay-specific settings. Similarly, TMB, MSI and HRD data were concordant for most samples but those with scores close to the cut-offs could deviate. For fusion detection, concordance was found for the limited number of 11 clinically-actionable driver genes covered by the OncoDEEP kit. The uniformity of coverage was much higher with the OncoDEEP assay thereby allowing to pool 2- to 3-times more samples per NGS run. For the diagnostic implementation of the OncoDEEP assay we then performed an extensive validation with clinical samples representing a wide variety of solid tumor types, as well as reference samples. All performance metrics passed the validation criteria. In conclusion, the OncoDEEP kit can be used for reliable diagnostic comprehensive profiling of solid tumors, but currently, extensive fusion analysis requires an additional screening method. Citation Format: Ernst-Jan M. Speel, Pieter-Jan Volders, Joni Van der Meulen, Aaron De Cock, Stefanie Vermeire, Jacques Van Huysse, Marie De Barsy, Gabriela Beniuga, Wendy de Leng, Anne Jansen, Sharon Thijssen, Hendrikus Jan Dubbink, Zeliha Ozgur, Wilfred van Ijcken, Brigitte Maes, Guy Froyen. Comprehensive genomic profiling of solid tumor patients with the OncoDEEP assay for broad analysis in clinical diagnostics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2938.

Background: Neoadjuvant association of immune checkpoints inhibitors (ICI) and dose dense chemotherapy is promising for triple negative breast cancers (TNBC). However, response rates vary from one study to another. Timing, best chemotherapy partner and efficacy in less immunogenic breast cancer (BC), like luminal B tumors, should be further investigated. This study evaluates for TNBC and luminal B HER2(-) BC the neoadjuvant treatment with paclitaxel followed by a short combination of an anti-PD-L1 antibody with anthracyclines. Method B-IMMUNE (NCT03356860), a multicentric phase Ib/II prospective trial, included patients with stage I to III luminal B HER2(-) or TNBC treated with paclitaxel 80mg/m2 weekly from week 1 to 12 followed by 4 cycles of epirubicine 90mg/m2 and cyclophosphamide 600 mg/m2 (EC) Q2W in a neoadjuvant setting. Phase Ib evaluated a single infusion of durvalumab (anti-PD-L1) combined with the 3rd cycle of EC. Phase II evaluated infusions of durvalumab with the 1st and 3rd EC cycles. Surgery was planned 3 weeks after the last EC cycle. Primary objectives were safety and pathological complete response (pCR) rate compared to a historical control. Secondary endpoint was the overall response rate (ORR) based on breast MRI. Eleven patients were enrolled in a control arm without durvalumab, exclusively for translational research purposes. Based on a 2-stage Simon design with an α = 0.1 and β = 0.1, 22 TNBC patients were needed in the phase II to test a null hypothesis of 30% pCR rate against a one-side alternative of 60%, and 24 luminal B BC patients to test a null hypothesis of 15% pCR rate against a one-side alternative of 40% (including an additional accrual margin of 10% for eventual dropouts). At least 9 pCRs had to be observed among the first 20 evaluable TNBC patients and 6 among the first 22 evaluable luminal B patients to rule out the null hypothesis. Results This analysis concerns the 50 patients treated with the experimental treatment, 3 from the phase Ib and 47 from the phase II part. Median age was 51 y-old (31 to 72y), tumor subtypes were 24 TNBC, 25 Luminal B and one sarcoma excluded from the efficacy analysis. Seven (14%) patients had a stage I tumor, 17 (34%) a stage IIA, 13 (26%) a stage IIB, 8 (16%) a stage IIIA, 4 (8%) a stage IIIB and 1 (2%) a stage IIIC. Concerning safety, 232 AEs were reported on 39/50 patients and 34 (14,6%) were graded ≥ 3. The 5 most frequent all-grade AEs were fatigue (8,2%), diarrhea (5,6%), neutropenia (5,2%), anemia and nausea (4,3%). Most frequent grade 3 AEs were anemia and neutropenia (14,7%). Among 4 immune-related adverse events, all were thyroid disorders. One patient died 10 months after the end of treatment due to progressive disease in the liver. Forty-six of the 47 phase II patients were evaluable for efficacy. pCR was reported in 12/22 TNBC patients (55%) and 8/24 luminal B HER2(-) patients (33%). Subgroup analyses based on PD-L1 expression and TILs score are planned. Conclusions The B-IMMUNE study met its primary objective showing a significant improvement in pCR versus the historical control in both TNBC and in Luminal B HER2(-) BC cohorts with the addition of only 2 doses of durvalumab to the anthracyclines. The safety profile is comparable to those previously described with reported immune related adverse events limited to thyroid endocrine disorders. Citation Format: Alix Devaux, Gabriela Beniuga, Claire Quaghebeur, Stéphanie Henry, Mieke Van Bockstal, Christine Galant, Paul Delrée, Jean-Luc Canon, Brigitte Honhon, Dominique Korman, Vincent Verschaeve, Christophe Lonchay, Sarah Lefevre, Lionel D’Hondt, Martine Berlière, Sophie Delmarcelle, Jean-Michel Mine, Timour Willems, Gebhard Müller, Nathalie Myant, Isabelle Bar, Sandy Haussy, Pierre G. Coulie, François P. Duhoux, Javier Carrasco. B-IMMUNE final analysis: a phase Ib/II study of durvalumab combined with dose-dense EC in a neoadjuvant setting for patients with locally advanced luminal B HER2(-) or triple negative breast cancers. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-16.

Mahiat, Cédric MD; Jacquerie, Alexandre MD; Beniuga, Gabriela MD; Weynand, Birgit MD, PhD; Dermesropian, François MD; Colinet, Benoît MD Author Information

Retrospective studies reported that preoperative oxaliplatin-based chemotherapy increased pathological response (PR) in patients resected for colorectal liver metastases (CRLM). This multicenter prospective randomized (1/1) phase II trial evaluated PR on resected CRLM after preoperative mFOLFOX6 (arm A) or FOLFIRI (arm B) + bevacizumab. The primary endpoint was the major pathological response rate (MPRR), defined as the percentage of patients presenting CRLMs with mean tumor regression grade (TRG) < 3. Secondary endpoints included safety, progression-free survival (PFS) and overall survival (OS). Out of 65 patients, 57 patients (28 and 29 in arm A/B) were resected for CRLM (one patient with lung metastases). Clinical and treatment characteristics were similar in both arms. One-month postoperative complications were 39.3%/31.0% in arm A/B (p = 0.585). MPRR and complete PR were 32.1%/20.7% (p = 0.379) and 14.3%/0.0% (p = 0.052) in arm A/B, respectively. PFS and OS were not different. Patients with PR among all CRLMs (max TRG ≤ 3; 43.8% of patients) had a lower risk of relapse (PFS: HR = 0.41, 95%CI = 0.204−0.840, p = 0.015) and a tendency towards better survival (OS: HR = 0.34, 95%CI = 0.104−1.114, p = 0.075). The homogeneity of PR was associated with improved PFS/OS. This trial fails to demonstrate a significant increase in MPRR in patients treated with mFOLFOX6-bevacizumab but confirms PR as an important prognostic factor.

Introduction: The neurotrophic tropomyosin-related kinase (NTRK) genes encode the tropomyosin receptor kinases (TRKs). Patients with solid tumors harboring an oncogenic NTRK fusion are eligible for treatment with TRK inhibitors. NTRK fusion is often associated with TRK overexpression. Pan-TRK immunohistochemistry (IHC) is used to screen for NTRK fusions, but immunoreactivity patterns are poorly defined. Methods: Data on pan-TRK immunoreactivity patterns in 2,669 solid tumors (comprising carcinomas, sarcomas, and melanocytic lesions) were retrospectively collected by nine laboratories and comprised tumor type, percentage of pan-TRK-positive tumor cells, staining intensity, cytoplasmic, membrane and/or nuclear staining pattern, and the presence or absence of NTRK fusion. Results: Overall, 2,457 tumors (92%) were pan-TRK negative and 212 neoplasms (8%) were pan-TRK positive. Twenty-two pan-TRK-positive tumors (0.8%) harbored an NTRK fusion, representing 10% of all pan-TRK-positive tumors. Cytoplasmic immunoreactivity was most often observed, followed by membrane immunoreactivity. Nuclear pan-TRK positivity was least frequent, but was most often (33%) associated with NTRK fusion. Conclusion: Pan-TRK IHC can be used to screen for NTRK fusions, especially in commonly diagnosed solid tumors with low NTRK fusion prevalence. In case of pan-TRK immunoreactivity, regardless of its intensity and tumor cell percentage, subsequent molecular tests should be performed to formally confirm the presence or absence of NTRK fusions.

A Belgian ring trial for pan-TRK immunohistochemistry (IHC) staining was organised to harmonise pan-TRK IHC staining protocols and interpretation. As a reference method, the VENTANA pan-TRK Assay (clone EPR17341) on the Benchmark Ultra platform was selected. Six samples were selected: 2 negative, 2 fusion positive and 2 samples with wild-type endogenous TRK expression. Each participating laboratory stained the slides using their routine pan-TRK IHC and reported their results. In addition, they were asked to return one TRK-stained slide from each case. The coordinating lab evaluated these slides, compared them with the reference method and scored them. Two clones were used during the ring trial: A7H6R (Cell Signaling) and EPR17341 (Abcam/Ventana). Seven protocols achieved a sufficient performance mark, and three labs were advised to further optimise the protocol. Interpretation of pan-TRK IHC proved to be challenging in cases with physiological TRK expression. In addition, depending on the NTRK fusion partner, the staining can vary strongly in both intensity and staining pattern. Labs using the Ventana ready-to-use system based on the EPR17341 clone and using the recommended protocol settings scored best. However, given some small optimisation, all labs scored well on the technical staining and the succeeding evaluation.

Surgical resection of colorectal liver metastases combined with systemic treatment aims to maximize patient survival. However, recurrence rates are very high postsurgery. In order to assess patient prognosis after metastasis resection, we evaluated the main patho-molecular and immune parameters of all surgical specimens. Two hundred twenty-one patients who underwent, after different preoperative treatment, curative resection of 582 metastases were analyzed. Clinicopathological parameters, RAS tumor mutation, and the consensus Immunoscore (I) were assessed for all patients. Overall survival (OS) and time to relapse (TTR) were estimated using the Kaplan-Meier method and compared by log-rank tests. Cox proportional hazard models were used for uni- and multivariate analysis. Immunoscore and clinicopathological parameters (number of metastases, surgical margin, histopathological growth pattern, and steatohepatitis) were associated with relapse in multivariate analysis. Overall, pathological score (PS) that combines relevant clinicopathological factors for relapse, and I, were prognostic for TTR (2-year TTR rate PS 0-1: 49.8.% (95% CI: 42.2-58.8) versus PS 2-4: 20.9% (95% CI: 13.4-32.8), hazard ratio (HR) = 2.54 (95% CI: 1.82-3.53), p < 0.0000; and 2-year TTR rate I 0: 25.7% (95% CI: 16.3-40.5) versus I 3-4: 60% (95% CI: 47.2-76.3), HR = 2.87 (95% CI: 1.73-4.75), p = 0.0000). Immunoscore was also prognostic for OS (HR [I 3-4 versus I 0] = 4.25, 95% CI: 1.95-9.23; p = 0.0001). Immunoscore (HR [I 3-4 versus I 0] = 0.27, 95% CI: 0.12-0.58; p = 0.0009) and RAS mutation (HR [mutated versus WT] = 1.66, 95% CI: 1.06-2.58; p = 0.0265) were significant for OS. In conclusion, PS including relevant clinicopathological parameters and Immunoscore permit stratification of stage IV colorectal cancer patient prognosis in terms of TTR and identify patients with higher risk of recurrence. Immunoscore remains the major prognostic factor for OS.

A flow-volume curve with a flattening of the inspiratory and expiratory limb suggests a proximal obstruction of the upper airways. Plasma cell neoplasms need to be considered in the differential diagnosis of an invasion of the upper respiratory tract. https://bit.ly/2K9lOXj.

Triple-negative breast cancer (TNBC) is a heterogeneous group of breast cancer and is characterized by aggressiveness and poor prognosis. MicroRNA represents a new class of biomarkers, and accumulating evidence indicates that microRNAs contribute to tumorigenesis and cancer metastasis. It has been described that miR-210 is highly expressed in TNBC, and its overexpression had been linked to poor prognosis. In a previous work, we showed that in TNBC miR-210 is expressed in tumor cells and also in the tumor microenvironment (TME), particularly in inflammatory CD45-LCA positive cells. However, the exact identity of these cells remained unknown. In this study, we performed in situ hybridization and immunohistochemistry using validated antibodies for the different specific immune cell markers on adjacent sections of 23 TNBC infiltrated with immune cells. We found that miR-210 expressing cells in the TME were stained positive with CD79a, a B-cell lineage marker. These tumor-infiltrating cells were negative for CD20 and Ki-67 but positive for MUM1 and CD38 and also expressed immunoglobulins, indicating that they are immunoglobulin-producing plasma cells (PCs). To the best of our knowledge, this is the first study demonstrating miR-210 expression in tumor-infiltrating PCs.

We hereby report a case of diffuse pelvic peritoneal involvement by immunoglobulin G4-related disease (IgG4-RD). Numerous pelvic masses and nodules showing delayed enhancement on enhanced abdominal CT were found to congregate in the pelvic organs of a 57-year-old female presenting with intestinal subocclusion. The differentiation between peritoneal IgG4-RD and pelvic peritoneal carcinomatosis was only made by histopathology and immunohistochemistry performed after surgical resection. Autoimmune pancreatitis represents the historical prototype of IgG4-RD, but the spectrum of manifestations involving various organs has expanded during the last decade. In this report, we shortly review this clinical entity.

Introduction: Colorectal cancer is the third most common cause of cancer and cancer death, and approximately 20% of all patients will present with metastatic disease. Treatment has become increasingly complex with the emergence of new chemotherapy drugs and targeted agents. To date, in metastatic colorectal cancer (mCRC), KRAS testing has been restricted to codons 12 and 13. However, phase II and III trials now suggest including both KRAS and NRAS codons 12, 13, 61, 117, and 146. Searching for these additional codons could help screen 20% more patients with mCRC for treatment with EGFR inhibitors. Other finding suggests that testing for the BRAF V600 mutation compliments KRAS and NRAS mutations analysis and may be as important for treatment decisions. Here we present a multiplexed and cost effective strategy enabling to test simultaneously these 11 hotspot mutations. Methods: A custom panel (CP-KRAS), covering exons 2, 3 and 4 of KRAS and NRAS and exon 15 of BRAF, was designed using the Ion AmpliSeq Designer module. FFPE tissue sections were macro-dissected and DNA was extracted using the Maxwell FFPE Tissue LEV purification kit. Sequencing was performed according to the AmpliSeq protocol on the Ion Torrent PGM, starting from as less as 6 ng of DNA. Data were analysed with both Torrent Suite v4 and NextGENe software v2.3 with a hotspot positions-targeting bed file. Sensitivity of the technique was assigned from 2.5% at 500 x coverage to 10% at 100 x coverage, with a forward to reverse reads ratio >0.25. Results: After validation of the CP-KRAS panel on well-characterized EQC samples, more than 250 FFPE specimens (corresponding to a 8 months of CP-KRAS testing) were sequenced. 29% of the samples were mutated for KRAS codon 12 and 6% for codon 13. Of the wild-type samples tested for additional mutations, 3% of samples were mutated for KRAS codon Q61, 4% for KRAS codon A146, 4% for NRAS G12 and 4% for NRAS Q61. 9% additional samples were mutated for BRAF V600. Each additional mutation was confirmed by pyrosequencing, mini-sequencing (SNaPshot) or Sanger sequencing. In total, 24% tumours wild type for KRAS exon 2 harboured a mutation in another of the RAS pathway genes. Conclusion: This next generation and high throughput sequencing workflow on a single-tube multiplexed amplification of those KRAS, NRAS and BRAF hotspot mutations could help to identify additional patients who possibly fail to respond to anti-EGFR treatment. Moreover, this strategy is cost-effective, less fastidious and requires minimal DNA quantity from FFPE tissue as compared to standardanalysis.

Introduction: Gastrointestinal stromal tumours (GIST) occur in the gastrointestinal track, most commonly in the stomach or small intestine. These sporadic tumours are associated with genetic changes. In GIST subsets, c-KIT or PDGFRa mutations correlate with distinct anatomical site, clinical phenotype and sensitivity to tyrosine kinase inhibitors. Here we depict a multiplexed evaluation strategy based on Next Generation Sequencing (NGS) to investigate for the most relevant regions of c-KIT and PDGFRa in a GIST context. Methods: A custom panel, covering exons 9, 11, 13 and 17 of c-KIT and exons 12, 14 and 18 of PDGFRa, was designed using the Ion AmpliSeq Designer module. FFPE tissue sections were macro-dissected and DNA was extracted using the Maxwell FFPE Tissue LEV purification kit. Sequencing was performed according to the AmpliSeq protocol on the Ion Torrent PGM, from as less as 6 ng of input DNA. Data were analysed with both Torrent Suite v4 and NextGENe software v2.3 with a whole exons-targeting bed file. Sensitivity of the technique was assigned from 2.5% at 500 x coverage to 10% at 100 x coverage, assuming a forward to reverse reads ratio >0.25. Results: DNA from samples for which c-KIT and PDGFRa mutations have been previously identified by Sanger sequencing (10 providing from EQC schemes and 30 from our regular practice), as well as from 10 wild-type samples, were tested with the designed panel. For all samples, there is a perfect correlation between our multiplexed sequencing strategy and the « gold standard » Sanger sequencing. Mutations - point mutations, duplications, deletions, insertions or both, mainly located in exons 9 and 11 of c-KIT and in exon 18 of PDGFRa – were found in all 40 mutated samples by both techniques, while no critical variant was detected in the 10 normal samples. Conclusion: This multiplexed amplification of 7 exons in GIST samples demonstrates that custom NGS panels can be designed to identify mutations in genes or regions more relevant to diagnosis, prognosis or therapeutic choices. For any tumour setting or pathway, NGS panels allow more comprehensive sequence coverage than standard techniques and are scalable in term of regions of interest and number of samples.