Molecular pathological analysis through the ages

A tribute to Jeffrey A. Kant, MD, PhD

e15050 Background: Molecular tumour boards (MTB) facilitate discussions among medical professionals, scientists, bioinformaticians and geneticists for clinical application of precision oncology, via interpretation of various genomic sequencing studies, most commonly Next Generation Sequencing (NGS). NGS test vendor recommendation may differ from the MTB recommendations. We aim to study the discordance rate between these recommendations. Methods: MTB discussions between April 1, 2021 and December 31, 2021, were retrospectively evaluated. MTBs which assessed NGS reports with or without significant genetic mutations but with drug recommendations were included. NGS testing was done with multiple vendors. Patients from all over India, Nepal, Bangladesh, Dubai and Abu Dhabi were discussed. We excluded MTBs which assessed NGS data without any drug recommendations. We compared therapeutic recommendations provided in the NGS report with the first-choice recommendation in the MTB discussion. Recommendation discordance was defined as disagreement between the NGS recommendation and the first MTB recommendation that either instructed its implementation either immediately or on disease progression. Results: 70 MTB data was studied. 49 MTBs assessing NGS reports of 49 unique patients (median age 54 years; range, 32-82) were included in the study. 13 patients had lung cancer, 10 colorectal, 6 breast, 7 hepatopancreatobiliary, 4 carcinoma of unknown primary and 9 others. 17 did not provide any treatment recommendations and hence were excluded. 4 were discussions based on NGS reports previously discussed in an MTB that was already included in the study, hence were excluded. Recommendation discordance rate was 49% (24 of 49). Conclusions: To our knowledge, this is the first study that assessed discordance rate in recommendations between NGS report and MTB from a low- and middle-income country setting. Accurate clinical translation of complex genomic data is at the forefront of personalized cancer care.

e13533 Background: The accelerated use of next generation sequencing (NGS) in oncology requires the integration of cancer genomics and precision oncology education to facilitate appropriate clinical decisions. The Molecular Tumour Board (MTB) in Ireland was established in November 2020 as a cross-institutional, educational and multi-disciplinary pilot and expanded nationally under Cancer Trials Ireland as a resource for clinicians and other oncology groups. Methods: The Irish MTB program was co-designed with Roche and local Health Care Providers (HCPs) over the pillars of co-creation, co-benefitting and co-education. Individual anonymized patient cases are discussed at each monthly meeting, supported by four multidisciplinary experts that discuss genomic findings, characterized mutations and their actionability, in addition to available therapies. The standardized end-to-end process to ensure preparation and proper handling of sensitive data is managed by an independent partner. A survey questionnaire was regularly administered to the MTB attendees to evaluate progress since the MTB initiation. Results: From November 2020 until December 2021, the program has grown to a monthly meeting where 30 patient cases have been discussed and 157 cumulative participants have been part of the sessions from 16 institutions. Survey findings revealed all of participants admitted that MTBs they attended met, exceeded, or significantly exceeded their expectations and 90% of presenters confirmed that experts communicated effectively. The majority of participants (87%) considered the discussions on clinical implications of targeted therapies or immunotherapies associated with genomic alterations as the most valuable component, 80% of presenters confirmed that the MTB discussions helped them to confirm, modify or change the treatment plan for at least one of their patients, and 65% of participants were very satisfied with the insights shared around molecular profiling and biological pathways. The pilot MTB has now been incorporated by the national trials group, Cancer Trials Ireland. Conclusions: The development of a national MTB in Ireland utilized pharmaceutical sponsorship and support to gather impetus for an independent national MTB managed by Cancer Trials Ireland. It offers a national forum for the discussion of cancers with specific molecular alterations in addition to complex clinical cases. Furthermore, it has created academic opportunities for the clinical application of precision medicine. Future directions will include leveraging the MTB infrastructure for research and the creation of a national Molecular Cancer Registry. Continued integration of experts will be key to secure the continuation of the national Irish MTB and to ultimately improving precision oncology delivery in patient care and trial enrolment through meaningful insights based on the collected data.

BackgroundMolecular tumor boards (MTBs) provide rational, genomics‐driven, patient‐tailored treatment recommendations. Worldwide, MTBs differ in terms of scope, composition, methods, and recommendations. This study aimed to assess differences in methods and agreement in treatment recommendations among MTBs from tertiary cancer referral centers in The Netherlands.Materials and MethodsMTBs from all tertiary cancer referral centers in The Netherlands were invited to participate. A survey assessing scope, value, logistics, composition, decision‐making method, reporting, and registration of the MTBs was completed through on‐site interviews with members from each MTB. Targeted therapy recommendations were compared using 10 anonymized cases. Participating MTBs were asked to provide a treatment recommendation in accordance with their own methods. Agreement was based on which molecular alteration(s) was considered actionable with the next line of targeted therapy.ResultsInterviews with 24 members of eight MTBs revealed that all participating MTBs focused on rare or complex mutational cancer profiles, operated independently of cancer type–specific multidisciplinary teams, and consisted of at least (thoracic and/or medical) oncologists, pathologists, and clinical scientists in molecular pathology. Differences were the types of cancer discussed and the methods used to achieve a recommendation. Nevertheless, agreement among MTB recommendations, based on identified actionable molecular alteration(s), was high for the 10 evaluated cases (86%).ConclusionMTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational cancer profiles. We propose a “Dutch MTB model” for an optimal, collaborative, and nationally aligned MTB workflow.Implications for PracticeInterpretation of genomic analyses for optimal choice of target therapy for patients with cancer is becoming increasingly complex. A molecular tumor board (MTB) supports oncologists in rationalizing therapy options. However, there is no consensus on the most optimal setup for an MTB, which can affect the quality of recommendations. This study reveals that the eight MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational profiles. The Dutch MTB model is based on a collaborative and nationally aligned workflow with interinstitutional collaboration and data sharing.

e23013 Background: CGP followed by MDT-based therapy recommendation plays a synergistic role in improving clinical outcomes of cancer patients. CGP in clinical practice detects broad-spectrum therapeutic, prognostic, and predictive biomarkers. MTB plays a crucial role in the assessment of molecular test results and integrating them into clinical practice. Methods: In a multicentric, IRB-approved study, 1000 biopsy-proven cancer patients were profiled by Next Generation Sequencing (NGS) using TruSight Oncology 500 gene panel . MTB recommendations were collated and followed up with an emphasis on therapeutic interventions and patient outcome. The treatment planning data before and after genetic testing were obtained from the Electronic Medical Report (EMR) to evaluate the clinical outcome. Results: Clinical outcome data were available via EMR for 618/1000 patients (62%). Actionable genetic alterations (Tier I + Tier II) were found in 458 patients (74%; 96% CI 70.6%-77.4%). Among 458 patients with ≥1 actionable genomic alteration, 200 patients (44%; 95% CI 39.1%-48.1%) were enrolled for MTB discussion. Based on inputs from MTB, treatment modifications were made in 137 patients (69%; 95% CI 62.4%-74.6%). The number of patients assigned to tumor-agnostic therapy post-MDT (n = 137) is shown in Table 1. CGP matched therapy was also recommended in 61/258 (24%) patients, not enrolled in MTB. Overall, 69% (n = 137/200) of cases discussed in MTB received a change in therapy compared to 24% (n = 61/258) of cases not discussed in MTB, thus establishing the importance of MTB in personalized genomics-driven treatment. Interim analysis of MTB-discussed cases showed 71% (n = 97/137) of cases to be alive with a median follow up of 18 months (12-24 months) with favorable clinical outcome. Conclusions: The current study revealed that the CGP results discussed in the MTB prompted a change in therapeutic decisions in > 60% of patients and established a foundation for introducing CGP and MTB in clinical practice to improve clinical outcomes and reduce healthcare costs in emerging economic countries. Clinical outcome of patients on CGP matched targeted therapy. Targeted Therapy No. of patients Indication Clinical OutcomeCR: Complete Response; PR: Partial Response; PD: Progressive Disease ICI 44 TMB>10mut/ mb and/or MSI-20% unstable sites. CR (n=5); PR (n=21); PD (n=1) Platinum/PARP 22 BRCA and/or HRR CR (n=12); PR (n=5); PD (n=1) EGFR TKIs 21 EGFR TKD mutation CR (n=12); PR (n=2); PD (n=1) Other TKIS 15 ROS1/RET/NTRK/MET/BRAF CR (n=11); PR (n=6) HER2 mAB 17 HER2 amplification CR (n=17); PR (n=4) mTOR inhibitors 5 PIK3CA/AKT/mTOR CR (n=0); PR (n=2); PD (n=1) ALK inhibitor 4 ALK Fusion CR (n=3); PR (n=1) Sotorasib 3 KRAS G12C CR (n=2); PR (n=1) FGF/FGFR inhibitor(1) 1 FGF/ FGFR PR (n=1) Others 5

Today, with the better understanding of the molecular events involved in malignancy and the mechanisms of pharmacotherapy, larger gene panels are more helpful than single biomarker detection. After the completion of the first human genome sequence in 2004, the growing need to sequence a large number of individual genomes in a fast, low-cost and accurate way has directed a shift from traditional Sanger sequencing methods towards new high-throughput genomic technologies. In 2005, the development of next generation sequencing (NGS) methods has represented one of the more significant technical advances in molecular biology. NGS, also known as massive parallel sequencing because of the ability to allow the parallel analysis of a very large number of DNA molecules, is beginning to show its full potential for diagnostic and therapeutic applications. Until recently, NGS platforms were envisioned for large-scale applications,focused on whole genome sequencing, with protocols, consumable costs and a turnaround time (TAT) unsuitable for the needs of small diagnostic laboratories. The development of miniaturised technology by benchtop NGS sequencers decreased sequencing costs, moving NGS from a few large sequencing core centers to a much larger number of individual laboratories. Currently, most pathology departments acquired and NGS benchtop sequencer, thus NGS is adopted for routine molecular diagnostics,including cytological samples. To understand the current and future application of NGS in the field of pathology,modern pathologists need to understand its basic principles. This thesis describes my research on the integration of NGS technologies in pathological diagnostics, both concerning histological and cytological specimens. Moreover, a research application of NGS on mouse xenograft cytological samples is described.

1683P Implementing next generation sequencing (NGS)/molecular tumor board (MTB)-based precision oncology practice: One-year experience at the Verona University Hospital

With the development of precision oncology, Molecular Tumor Boards (MTB) are developing in many institutions. However, the implementation of MTB in routine clinical practice has still not been thoroughly studied. Since the first drugs approved for targeted therapies, patient tumor samples were centralized to genomic testing platforms. In our institution, all tumor samples have been analyzed since 2014 by Next Generation Sequencing (NGS). In 2015, we established a regional MTB to discuss patient cases with 1 or more alterations identified by NGS, in genes different from those related to drug approval. We conducted a retrospective comparative analysis to study whether our MTB increased the prescriptions of Molecular Targeted Therapies (MTT) and the inclusions of patients in clinical trials with MTT, in comparison with patients with available NGS data but no MTB discussion. In 2014, 86 patients had UGA, but the results were not available to clinicians and not discussed in MTB. During the years 2015 and 2016, 113 patients with an UGA (unreferenced genomic alteration) were discussed in MTB. No patients with an UGA were included in 2014 in a clinical trial, versus 2 (2%) in 2015-2016. 13 patients with an UGA (12%) were treated in 2015-2016 with a MTT whereas in 2014, no patient (p = 0.001). In this retrospective analysis, we showed that the association of large-scale genomic testing and MTB was feasible, and could increase the prescription of MTT. However, in routine clinical practice, the majority of patients with UGA still do not have access to MTT.

e15068 Background: Since next generation sequencing (NGS) based profiling does not provide measures of protein abundance or activation state, clinical integration of reverse phase protein array (RPPA) with NGS could be useful for improving selection of targeted cancer therapy. We incorporated biopsy testing and proteogenomic analyses in an institutional Molecular Tumor Board (MTB) for cancer patients using CLIA-certified commercially available multi-omic platforms, including a commercially available CLIA RPPA functional protein drug target activation mapping platform. We present an interim analysis of our experience integrating functional phosphoprotein/protein based drug target activity data with NGS from 117 patients with metastatic solid tumors. Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor biopsy specimens (n = 166) were obtained prospectively from patients as part of an IRB approved MTB. A representative H&E-stained tissue section for each specimen was reviewed by a board-certified surgical pathologist. Specimens with sufficient total tumor cellularity to achieve protein lysate input requirements for RPPA were thin sectioned onto laser microdissection (LMD) membrane slides for selective harvest of tumor epithelium by LMD and RPPA analysis. In parallel, specimens from each case were also submitted for NGS analysis. The multi-omic data for each patient was reviewed by a panel of clinicians and scientists as part of the Inova Institutional MTB for clinical decision-making. Results: Patient tissue specimens (median = 1 per patient; range 1-5) were reviewed to assess feasibility of enriching tumor areas via LMD prior to RPPA analysis. Specimens from 64/117 patients were used for LMD, RPPA, and NGS. 48/117 patients were dropped due to inadequate tumor cellularity, patient death, or referral cases with inadequate specimen. At this time, 7/117 patients remained in the consenting-LMD-RPPA workflow. The median time from patient consent to RPPA analysis completion was 47.4 days. During this period, specimens were outside of the standard hospital-NGS workflow (ie, utilized for sectioning and review for LMD-RPPA) for ~10 days (median). Integrated review of the RPPA and NGS data by the MTB supported a clinical recommendation change for 34/64 patients (53%) overall, with some dependency by primary disease site. Clinical trial information: U20-11-4308.Conclusions: Incorporation of RPPA analysis of LMD enriched tumor samples with NGS was feasible in this pan-cancer cohort, with the mean time to results being less than 7 weeks. RPPA data provided additional treatment considerations for 59% of patients, the outcomes for whom continue to be monitored. [Table: see text]

5523 Background: Patients with rare gynecologic cancers often face limited systemic therapy options and participation in clinical trials is challenging due to a paucity of available options. As many as 30% of women with gynecologic cancer have rare subtypes with tumors often harbouring unique molecular aberrations that may serve as potential therapeutic targets. With accessibility of Next-Generation Sequencing (NGS), targeted drug matching in real time is an ever-increasing interest for developing personalized treatment strategies. Methods: Within the gynecologic site group at Princess Margaret (PM), we have developed a comprehensive program whereby coordination of molecular profiling, pathology review and consensus therapeutic recommendations are made for patients with rare or complex diagnoses. Patients undergo NGS predominantly through a translational study (VENUS; NCT03420118 or BioDiva; NCT03419689) and results are reviewed at ‘ComplexDiva’ rounds. These rounds gather a multidisciplinary team for review of patient demographics, treatment history, and imaging followed by literature review based on histopathology and molecular profile. This process facilitates collective analysis of genomic data and discussion of targeted treatment options to ensure recommendations are individually tailored. Results: The ComplexDiva pilot assessing feasibility of this approach has facilitated the review and discussion of 67 patients between November 2021 and December 2023. NGS was performed on at least one tissue sample for 65 patients. Whole Genome and Transcriptome Sequencing (WGTS) was performed on 14 archival tissue samples from 12 patients. Ovarian tumors comprised 63%, cervical 26%, endometrial 9%, and vulvar 2% of cases. More than 60% of patients (42/67) had rare tumor types, such as female adnexal tumor of wolffian origin (FATWO), small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), steroid cell tumor, gynandroblastoma, and mesonephric adenocarcinoma. Recommendations were made for clinical trial screening in eight patients (12%) and for Nof1 treatment (any off-label indication) in 30 patients (45%). Nof1 treatment was suggested as next line of therapy in 77% of these patients and to be considered as a future treatment line in 23%. Conclusions: Integration of molecular tumor boards, identification of targetable mutations, and real-time treatment matching at PM has allowed a functional molecular and consensus tumor board to facilitate personalized treatment strategies for patients with rare gynecologic cancers. This innovative approach holds promise for advancing precision oncology and improving therapeutic outcomes in challenging clinical contexts. This scalable initiative allows collaboration with other cancer centres. Patient outcomes are followed through a dedicated registry to systematically evaluate the efficacy of our approach.

Next Generation Sequencing (NGS) technologies have greatly impacted every field of molecular research, reducing costs and simultaneously increasing throughput of DNA sequencing. These features, together with technology’s flexibility, have open the way to a variety of applications, especially for the study of the molecular basis of human diseases. So far, several analytical approaches have been developed to selectively enrich regions of interest from the whole genome, both to identify germinal and/or somatic sequence variants. All of these have assessed their potential in research area and are now being improved also in routine molecular diagnostics. Thanks to the improvement due to NGS methods introduction, also the metagenomic field has achieved very exciting results, increasing our knowledge about the microbiome and its mutually beneficial relationships with the human host. If microbiome plays a role in the maintenance of a healthy status, it is conceivable to suppose that its quantitative and/or qualitative alterations could lead to pathological dysbiosis, as shown in an increasing number of intestinal and extra-intestinal diseases. The aim of this project was to use NGS-based strategies to study the molecular basis of human diseases. In particular, two analytic approaches were used: DNA sequence capture and metagenomics. A DNA sequence capture approach was used to analyze a large panel of genes possibly related to inherited cardiomyopathies. The obtained results indicate that this approach is useful to analyze, in a time and cost effective manner, heterogeneous diseases allowing the identification not only of the disease-causing mutation, but also of other variants involved in disease-phenotypic expression. Finally, methods reliability was higher than traditional, currently used techniques. All the above data indicate that this validated NGS-based approach can be used to improve the molecular analysis of inherited cardiomyopathies, such as of other inherited diseases, also in routine diagnostic settings. With regard to metagenomics, a 16S rRNA pyrotag analysis was carried out to deeply investigate the gut microbiome composition of Crohn and celiac diseases. Specific microbial signatures were identified in the patients. Moreover, the effects of Crohn nutritional therapy on gut microbial composition were also verified. These results suggest a role of gut microbiome in diseases pathogenesis and could, in turn, make possible to develop novel diagnostic, prognostic and, most important, therapeutic strategies. Taken together, all the above results indicate that the used NGS-based procedures can be easily applied to increase our understanding of the molecular basis of human diseases and that they can be useful also for routine diagnostic purposes.

Technical advances in the field of molecular genetics permit precise genomic characterization of malignant tumors. This has not only improved our understanding of tumor biology but also paved the way for molecularly stratified treatment strategies in routine clinical practice. A selective search of PubMed to identify literature on molecular pathology methods, their indications, the challenges associated with molecular findings, and future developments. Tumors can be characterized with the aid of immunohistochemistry, in-situ hybridization, and sequencing of DNA or RNA. The benefits of molecularly stratified tumor treatment have been demonstrated by randomized clinical trials on numerous tumor entities, e.g., non-small-cell lung cancer, colorectal cancer, and breast cancer. Therefore, initiation of specific treatment for these entities should be preceded by molecular pathology biomarker analyses, generally carried out on tumor tissue. Randomized controlled trials and non-controlled studies show that enhanced progression-free survival ensues if the pharmacological treatment is oriented on the findings of molecular pathology diagnostics. In next-generation sequencing, numerous relevant gene sequences or even whole genes can be sequenced in parallel, dispensing with complex staged diagnostics and reducing the use of biomaterials. These new methods also complement the currently relevant predictive biomarkers by permitting the investigation of genetic alterations presently of interest in the context of clinical studies. Prior to widespread routine clinical application, however, sequencing of large gene panels or whole genomes or exomes need to be even more stringently validated. Quality-assured molecular pathology assays are universally available for the determination of currently relevant predictive biomarkers. However, the integration of extensive genomic analyses into routine molecular pathology diagnostics represents a future challenge in precision oncology.

Our 2016 Cancer Cytopathology Young Investigator.

Background. Genomic screening for somatic alterations in individual tumors is informative for understanding tumor biology and identifying novel treatment approaches and research opportunities. We initiated a research study where patients with rare or poor prognosis cancers were enrolled and underwent targeted sequencing using a CLIA certified assay. Sequencing results were reviewed at a molecular tumor board and therapeutic strategies were suggested if appropriate. Preliminary results of this study are now presented. Experimental procedures. We analyzed the genomic profiles of formalin-fixed paraffin embedded tumor specimens from 72 patients using the FoundationOne™ (Foundation Medicine, Cambridge, MA) platform for targeted sequencing of the entire coding sequence of 236 genes and 47 introns of 19 genes involved in fusions in a CLIA-certified CAP accredited laboratory. A range of evaluated tumor subtypes of epithelial and mesenchymal origin were evaluated (20). The majority of cases were from rare or uncommon histologic classes, though recurrent/refractory cases of colon cancer and triple negative breast cancers were also enrolled. Both clinical history and sequencing data were presented at a multidisciplinary molecular tumor board for development of therapeutic recommendations. Results. At least one genomic alteration was found in 68 cases (95%) and none in four cases (6%). Serial specimens were obtained in six patients. Five had a gain of at least one mutation in serial specimens. The average number of mutations identified was 3.6 (range 0-10). The most common genomic alterations detected were in p53 (40%), KRAS (17%), PI3K (15%) and PTEN (10%). Alterations in the FGF pathway were surprisingly common (16%). A number of mutations occurred at low frequency but included an additional large set of potentially actionable genes: ALK, ERBB2, BRCA2, MET. The majority of cases had mutations for which our molecular tumor board had recommended action: either potential enrollment in clinical trial or off label use of approved therapy. NGS sequencing results led to implemented clinical action in ∼15% of cases. Conclusion. Use of a comprehensive clinical NGS assay in a diverse set of cancers identifies a number of actionable genomic changes associated with targeted therapies. An institutional molecular tumor board is an effective venue to systematically review sequencing data and generate clinical recommendations. Knowledge of genomic alterations has led to referral to clinical trials, off label use of targeted therapy, and planned trials of agents targeting identified pathways. Challenges to effectively implementing clinical recommendations included lack of practical access to clinical trials and limited availability of targeted agents. Expanding clinical trial portfolios, and increasing access to targeted agents is critical for successfully implementing genomic-guided precision cancer therapy. Citation Format: Kim M. Hirshfield, Siraj M. Ali, Vincent A. Miller, Philip J. Stephens, Vassiliki Karantza, Robert S. DiPaola, Lorna Rodriguez-Rodriguez, Shridar Ganesan. Prospective next generation sequencing (NGS) of rare or poor prognosis cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4261. doi:10.1158/1538-7445.AM2014-4261

Prospective Next-Generation Sequencing Molecular Profiling of Myeloid Malignancies: Assessment of Information Benefit and Impact on Patient Care