Next-generation Sequencing Findings Research Articles

Comprehensive Next Generation Sequencing Reveals that Purported Primary Squamous Cell Carcinomas of the Parotid Gland are Genetically Heterogeneous.

Squamous cell carcinoma (SCC) is one of the most common malignancies involving the parotid gland, but it has been recognized that the vast majority of parotid SCC represents metastases, especially from the ipsilateral facial skin. Bona fide primary SCC of the parotid is so rare that it is unclear whether it truly exists at all. We sought to molecularly characterize cases diagnosed as primary parotid gland SCC to see if they possess a unique genetic makeup.We identified cases in our archives which had been diagnosed as primary SCC of the parotid gland. In all cases, metastatic disease was excluded by a thorough history and physical examination. Cases with histologic evidence of a precursor neoplasm (e.g., carcinoma ex-pleomorphic adenoma) were also excluded. Targeted next-generation sequencing (NGS) was attempted on all cases.Six cases diagnosed as primary parotid SCC were identified, arising in 4 males and 2 females ranging from 8 to 73 years (mean, 51.8 years). All cases exhibited keratinization and unequivocal invasion. Four of 6 appeared to be arising from cystically dilated ducts. Five of 6 exhibited well-developed cellular atypia; the remaining case, while cytologically bland, demonstrated perineural invasion. Targeted NGS was successful in 5 of 6 cases. Two SCC harbored several mutations in a mutational profile reminiscent of SCCs seen in other organs. One case harbored YAP1::MAML2, a fusion previously reported in porocarcinoma and other neoplasms. One case harbored IRF2BP2::RUNX2, and presumably represents keratocystoma or SCC ex-keratocystoma. Finally, one case an increase of C > T mutations consistent with ultraviolet damage, suggesting that this case represented a cryptic metastasis from cutaneous SCC.Our analysis did not confirm a unifying genetic signature for purported primary parotid SCC. Indeed, our findings suggest that true primary parotid gland SCC is even rarer than already believed. In our 5 cases with results, NGS findings demonstrated that one was likely a keratocystoma, one a cryptic metastasis from a cutaneous SCC, and one a porocarcinoma, either metastatic or primary. The two remaining cases had complex genotypes reminiscent of SCCs from other sites. This may be the signature of genuine parotid primary SCC, but metastasis from an SCC from another organ cannot be excluded. Accordingly, a diagnosis of primary parotid gland SCC should be viewed with skepticism.

Utility of p53 Immunohistochemical Staining for Risk Stratification of Mantle Cell Lymphoma.

Inactivating TP53 mutations in mantle cell lymphoma (MCL) are associated with poor prognosis. While next-generation sequencing (NGS) is the gold standard for assessing TP53, p53 immunohistochemistry (IHC) is an orthogonal means of evaluating TP53 status that has not been well characterized in MCL. In this single tertiary care center laboratory study, we aimed to evaluate the concordance of p53 IHC with the TP53 status in cases of MCL in hopes of evaluating if the former could act as an accurate, timely and cost-effective way of risk stratifying these patients. A total of 47 cases of MCL that had TP53 NGS performed were included in this study. The main objective was to correlate NGS findings with p53 IHC results. Secondary objectives included assessment of possible associations between TP53 status and other variables (demographics, unique histopathological and IHC features). The turn-around time and cost for NGS and p53 IHC were also compared. Thirteen out of 47 (28%) cases were TP53-mutated by NGS. p53 IHC showed good concordance with NGS, with moderate to high sensitivity (11/13, 85%) and excellent specificity (34/34, 100%). Secondary objectives revealed increased SOX11-negative status in TP53-mutated cases (3/13, 23% vs. 1/29, 3%, P = 0.045). The cost and turn-around time of NGS were approximately of 30- and sixfold those of p53 IHC, respectively. p53 IHC shows good concordance with NGS in MCL, with high specificity and moderate sensitivity for identifying inactivating TP53 mutations. Based on our findings, p53 IHC may be an efficient and cost-effective tool in risk stratification of MCL.

Evaluating RB1 and p53 as diagnostic markers in treatment-related neuroendocrine prostate cancer through immunohistochemistry and genomic analysis of RB1 and TP53.

The diagnosis of treatment-related neuroendocrine prostate cancer (t-NEPC) often involves a pathological assessment and immunohistochemistry (IHC) for neuroendocrine markers. Genomic alterations in RB1 and TP53 are frequently observed in NEPC and are believed to play a crucial role in the transformation of adenocarcinoma to NEPC. In this study, we examined the clinicopathologic, immunohistochemical, and genetic features of patients with t-NEPC to better understand their prognosis and diagnostic utility. This retrospective study reviewed the records of patients diagnosed with t-NEPC at Kobe University Hospital between October 2018 and December 2022. Clinical data, including age, serum neuroendocrine marker levels, and treatment history, were collected. IHC was performed for conventional neuroendocrine markers (synaptophysin, chromogranin A, and CD56) and RB1 and p53 expression. Next-generation sequencing (NGS) was conducted using FoundationOne® CDx to identify mutations in RB1 and TP53. This study included 20 patients with t-NEPC. The median time from ADT initiation to development was 42.8 months. IHC revealed RB1 loss in 75% of cases and p53 abnormalities in 75% of cases. NGS identified RB1 mutations in 55% and TP53 mutations in 75% of cases. The concordance between NGS and IHC results was high, with 70% (14/20) agreement for RB1/RB1 and 80% (16/20) for p53/TP53. The immunostaining and genomic analysis of RB1/RB1 and p53/TP53 showed abnormal findings for the four negative cases for conventional neuroendocrine markers. This study indicated high concordance between IHC and NGS findings for RB1/RB1 and p53/TP53 in t-NEPC. We provide a comprehensive benchmark of NGS performance compared with IHC, and these findings may help increase the diagnostic sensitivity of t-NEPC.

Systemic ALK-negative anaplastic large cell lymphoma with NPM1::TYK2 rearrangement.

Anaplastic large cell lymphoma (ALCL) is a rare subtype of non-Hodgkin lymphoma, with most cases harboring ALK gene rearrangement (ALK + ALCL); however, 20-50% of ALCLs do not have the rearrangement (ALK- ALCL) but exhibit distinct genetic alterations. In this report, we present an unusual case of systemic ALK- ALCL with NPM1::TYK2 fusion. Diagnosis of this case was challenging prior to the NGS findings. A comprehensive panel of immunohistochemical and in-situ hybridization studies was conducted. FISH assays were utilized to target the rearrangements of DUSP22 and TP63 genes. Moreover, next-generation sequencing (NGS) assays were performed to detect clonal rearrangements of IGH and TRG genes, somatic mutations, and potential fusions. The lymphoma cells in this case are negative for all hematolymphoid markers stained, except for CD30 expression and focal and weak CD43 expression. However, NGS studies detected clonal TRG rearrangement and NPM1::TYK2 rearrangement, which aid in the diagnosis of ALK- ALCL. NPM1::TYK2 rearrangement is a rare genetic alteration that has been reported in rare cases of primary cutaneous ALCL, mycosis fungoides, and lymphomatoid papulosis. To the best of our knowledge, this is the first reported instance of such rearrangement in systemic ALK- ALCL.

Integrated diagnosis of grade 3-4 gliomas using targeted next-generation sequencing and traditional diagnostic data, and its overall implications on patient management

Adult gliomas vary significantly in terms of their genetics, epigenetics, phenotypes as well as clinical outcomes [1]. Optimal tumor classification that leads to more precise diagnoses, and which influences therapy selection is hence critically important. Targeted next-generation sequencing (NGS) is a highly reliable and efficient novel tool for the identification of biomarkers for the diagnosis of CNS tumors [2]. In this study, we aimed to determine the implications of integrating NGS findings with the traditional diagnostic data on clinical diagnostics and patient management. All patients who were diagnosed with glioma at our institution between 2019 and 2020 and whose tumor biopsy materials were analyzed by both traditional histomorphological and immunohistochemical diagnostic techniques as well as NGS were included in our study. Patients’ information was retrieved from the electronic medical record. Data analyzed included the histopathologic diagnosis, tumor grade, ki67, and the mutation status of TP53 and /or IDH, as well as other genes such as BRAF and HER2 for some patients. The overall concordance between NGS and the traditional diagnostic tools for IDH and TP53 mutations was also evaluated. Further, we derived the overall survival of patients using the Kaplan–Meier estimate. We identified 9 patients with grade 3-4 gliomas whose biopsy materials were analyzed by NGS as well as traditional diagnostic tools. Our patients were 33.3 % females and had a median age of 43 years old at diagnosis. In terms of the identification of gene mutations by NGS in comparison to immunohistochemistry (IHC), it was found that the concordance for mutant identification was 77.8 % (7/9) for IDH mutations and 55.6 % (5/9) for TP53 mutations. NGS further led to a change in patient management in which evidence-based targeted therapy (dabrafenib plus trametinib) was used for one patient (11.1 %) with BRAF V600E mutated astroblastoma. Integrating NGS with traditional diagnostic tools for the diagnosis of gliomas can have significant implications on patients. Such integrated diagnosis can lead to more precise diagnosis, enhanced identification of molecular markers, and offers the potential of using targeted therapies for patients.

Real-World Experiences of Next-Generation Sequencing in Oncology: From an Indian Multicenter Registry and Collaborative Centers

Abstract Background The integration of next-generation sequencing (NGS) in guiding personalized therapy for oncology faces the challenges, primarily, of cost and drug accessibility. Limited data from Indian academic centers accentuate the need for comprehensive insights into the real-world applications of NGS in oncology. Methods The Network of Oncology Clinical Trials in India (NOCI), accessible at www.noci-india.com, compiled data on patients who underwent NGS for solid organ cancers from January 1, 2018, to December 31, 2021. This study aimed to elucidate the testing indications, sample types analyzed, and the resultant impact on patient care. Results Analysis of data from six centers included 278 subjects, with 24 specimens (9%) excluded due to quality test failure. Tissue constituted 59.7% of specimens, blood 38.5%, and both 1.8%. Predominantly, NGS was employed for identifying BRCA1/2 mutations (56%) and for targeted therapy in non-small-cell lung cancer (NSCLC; 28%). Only 41 (16%) patients with other cancers underwent multigene NGS panels in pursuit of targetable mutations. Among them, 13 exhibited targetable mutations, and 3 received treatment based on NGS findings. Conclusion This study underscores that the majority of NGS applications focused on screening for BRCA1/2 mutations and identifying targetable mutations in NSCLC. However, among those undergoing NGS for advanced cancers, only a limited number received personalized therapy. The findings underscore the challenges of utilizing NGS in off-label indications within resource-constrained settings.

Osteoarthritis and the Microbiome: Knock, Knock, Anybody Home?

Commentary The human microbiome—the ecosystem of bacteria living and interacting on (and in) human tissues—is enormously complex. Our understanding of its impact is evolving. Next-generation sequencing (NGS) techniques allow for the identification of microbial species and can be used to elaborately interrogate the composition of the bacterial community in specific anatomic sites. This has been extensively described in relation to the gut microbiome, the largest concentration of bacteria in the human body, where complex ecological interactions alter immunology1, pathogenesis2 and metabolic functions3. Far less is known about the microbiomes of anatomic sites previously considered sterile, such as the placenta, the central nervous system, and now the synovium. No one disputes that bacteria and bacterial DNA may transiently penetrate these protected spaces, but whether bacteria truly live there remains to be demonstrated. This preliminary study by Goswami et al. represents a relevant attempt to identify the microbiome of the human osteoarthritic hip and knee. The investigators obtained synovial fluid, tissue, and swabs from patients undergoing primary total hip or knee arthroplasty for osteoarthritis—patients with no overt signs of infection. Concordance in bacterial community composition among samples from the same patient was observed. Variations in diversity were seen depending on the hospital of enrollment and on whether patients had received a corticosteroid injection; these differences may be proxies for broader differences in patient-specific microbial composition. Surprisingly, comorbidities and demographic variables had no significant impact on the microbes found within a joint in this study. The detected microbes ranged from common to obscure—including some that are traditionally considered pathogenic and many others that are not. Some of the common sequences obtained included those of Staphylococcus, Streptococcus, and Pseudomonas. However, even though these are recognized orthopaedic pathogens, it would be surprising to confirm that they persist in clinically uninfected joints. Other bacteria found with frequency included some that are typically found in the environment but only occasionally cause opportunistic infections, such as Paenarthrobacter and Brevundimonas. The limitations of NGS techniques are important to consider. The presence of nucleic acids does not mean the presence of living microbes: remnants of dead bacteria, contamination introduced during sample collection and processing, and even miscategorized mammalian DNA in the vast NGS sequence libraries can all lead to positive findings even when no living bacteria are present. NGS findings are also difficult to confirm by alternate means (such as microscopy or culture), as the microbial burden is so low. A healthy degree of skepticism is warranted, given recent studies of low-biomass microbiomes in other tissues not known to harbor bacteria, such as the human fetus4,5. To their credit, the authors acknowledge these important limitations of their study. So, what can we learn from this study? First, the study serves as a reminder that the presence of microbial DNA in joints, in the absence of other findings of infection, is not diagnostic of infection. This is an important point particularly as it pertains to the molecular diagnosis of periprosthetic joint infection, which is gaining faster in popularity than in evidence. Optimizing molecular tests to diagnose infections is complicated by the implication that all joints may have subclinical quantities of bacterial DNA. Second, it is plausible that microbial nucleic acids are present in osteoarthritic joints. If the findings of this study are confirmed, further work should be done to establish whether these products are being produced intra-articularly by living bacteria, whether they are unique to diseased joints, and whether they represent a disease state that might benefit from treatment. The burden of proof should be high. Further confirmation of these findings with optimal controls, using alternate methodologies, and with replication across other populations (for instance, comparing osteoarthritic and healthy joints) will be needed to convince us that a stable microbiome exists within the joint capsule. Lastly, the techniques used in this study are less broadly available and much more complex than other existing diagnostic tools. The expertise to critically evaluate this type of methodology is beyond the scope of many clinicians. Further education and elaboration on clinically relevant findings are critical to realize the full clinical benefit of NGS in orthopaedics. In summary, this provocative paper offers evidence that microbial DNA is present in the osteoarthritic joint. The next steps that are needed will be to confirm these findings with appropriate controls at every juncture, and to evaluate whether these nucleic acids derive from living intra-articular bacterial communities or from transient circulation of dead microbes.

Mutations in Classical Signaling Pathways and Their Functional Impact in Microsatellite Instability High Colorectal Cancer.

Aims: Colorectal carcinomas with microsatellite instability high (MSI-H) are a distinctive group among colorectal cancers (CRCs). This study investigated the mutations of genes in the common signaling pathways and their potential clinical implications in MSI-H CRC. Materials and Methods: Twenty-five MSI-H tumors were selected from 384 primary CRCs, and the related clinical and pathological information were also collected from medical records. A commercial kit was used to detect the mutational status of crucial oncogenes within these tumors using next generation sequencing (NGS). Fluorescence in situ hybridization and immunohistochemistry were used to validate the NGS findings. Result: In the present study, MSI-H cases accounted for 6.51% of primary CRCs, with special clinicopathological features. NGS showed that the average number of mutations per tumor in the target genes evaluated was 3.36 and ranged from 1 to 9. In total, there were 17 cases (68%) with mutations in the RAS-RAF pathway and 18 cases (72%) with mutations in the PI3K pathway among the MSI-H CRCs. The remaining two cases included an EMAP Like 4-ALK Receptor Tyrosine Kinase (EML4-ALK) fusion and one with a Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2) missense mutation. Conclusion: This study found multiple variants within different signaling pathways that were mutually present in MSI-H CRCs, suggesting that such a heterogeneous group of tumors requires complex treatment responses. Thus, additional clinical molecular testing is recommended for such patients, such as NGS, to inform the appropriate treatment strategies.

Genomic characterization of Lumpy Skin Disease virus (LSDV) from India: Circulation of Kenyan-like LSDV strains with unique kelch-like proteins

Lumpy skin disease (LSD) is an economically important poxviral disease endemic to Asia, Europe, and Africa. Recently, LSD has spread to naïve countries, including India, China, Bangladesh, Pakistan, Myanmar, Vietnam, and Thailand. Here, we describe the complete genomic characterization of LSDV from India, LSDV-WB/IND/19 isolated from an LSD affected calf in 2019 determined by Illumina next-generation sequencing (NGS). The LSDV-WB/IND/19 has a genome size of 150,969 bp encoding 156 putative ORFs. Phylogenetic analysis based on complete genome sequence suggested that LSDV-WB/IND/19 is closely related to Kenyan LSDV strains with 10–12 variants with non-synonymous changes confined to LSD_019, LSD_049, LSD_089, LSD_094, LSD_096, LSD_140, and LSD_144 genes. In contrast to complete kelch-like proteins in Kenyan LSDV strains, LSDV-WB/IND/19 LSD_019 and LSD_144 genes were found to encode truncated versions (019a, 019b, and 144a, 144b). LSD_019a and LSD_019b proteins of LSDV-WB/IND/19 resemble that of wild-type LSDV strains based on SNPs and the C-terminal part of LSD_019b except for deletion at K229, whereas the LSD_144a and LSD_144b proteins resemble that of Kenyan LSDV strains based on SNPs, however, C-terminal part of LSD_144a resembles that of vaccine-associated LSDV strains due to premature truncation. The NGS findings were confirmed by Sanger sequencing of these genes in Vero cell isolate as well as in the original skin scab along with similar findings in another Indian LSDV from scab specimen. LSD_019 and LSD_144 genes are thought to modulate virulence and host range in capripoxviruses. This study demonstrates the circulation of unique LSDV strains in India and highlights the importance of constant monitoring of the molecular evolution of LSDV and associated factors in the region in light of the emergence of recombinant LSDV strains.

Combined Reverse-Transcriptase Multiplex Ligation-Dependent Probe Amplification and Next-Generation Sequencing Analyses to Assign Unclassified BCL2−/BCL6− Nonrearranged Small B-Cell Lymphoid Neoplasms as Follicular or Nodal Marginal Zone Lymphoma

Distinguishing between follicular lymphoma (FL) and nodal marginal zone lymphoma (NMZL) can be difficult when morphologic and phenotypic features are unusual and characteristic cytogenetic rearrangements are absent. We evaluated the diagnostic contribution of ancillary techniques—including fluorescence in situ hybridization (FISH)-detected 1p36 deletion; reverse-transcriptase, multiplex, ligation-dependent probe amplification (RT-MLPA); and next-generation sequencing (NGS)—for tumors that remain unclassified according to standard criteria. After review, 50 CD5-negative small B-cell lymphoid neoplasms without BCL2 and BCL6 FISH rearrangements were diagnosed as FLs (n = 27), NMZLs (n = 5), or unclassified (n = 18) based on the 2016 World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. FISH helped identify the 1p36 deletion in 3 FLs and 1 unclassified tumor. Most classified FLs had an RT-MLPA germinal center B-cell (GCB) signature (93%) or were noncontributive (7%). Classified NMZLs had an RT-MLPA activated B-cell signature (20%), had an unassigned signature (40%), or were noncontributive (40%). Among unclassified tumors, the RT-MLPA GCB signature was associated with mutations most commonly found in FLs (CREBBP, EZH2, STAT6, and/or TNFRSF14) (90%). An RT-MLPA-detected GCB signature and/or NGS-detected gene mutations were considered as FL identifiers for 13 tumors. An activated B-cell signature or NOTCH2 mutation supported NMZL diagnosis in 3 tumors. Combining the RT-MLPA and NGS findings successfully discriminated 89% of unclassified tumors in favor of one or the other diagnosis. NGS-detected mutations may be of therapeutic interest. Herein, we detected 3 EZH2 and 8 CREBBP mutations that might be eligible for targeted therapies.

Abstract A056: Landscape of the molecular changes in the most frequent tumors in a safety net hospital

Abstract INTRODUCTION The advent of deep molecular analysis using next generation sequencing (NGS) leads to a better understanding of carcinogenesis and the ability to identify and target specific mutations. METHOD After obtaining IRB approval, patients were approached in several oncology clinics in Grady Health System and explained the risk and benefit of being tested for molecular testing to better guide clinical management of their disease. After informed consent, we carried out Clinical Laboratory Improvement Amendments of 1988 (CLIA) NGS for a large number of targetable genes, tumor mutation burden (TMB), microsatellite instability (MSI), and the somatic transcriptome. We identified germline mutations, actionable genes, biologically significant genes, pertinent negative, variants of unknown significance, tumor profile-matched clinical trials and the recommended therapies based on CLIA NGS findings. All data and medical reports were made available to treating physicians. RESULTS We tested 280 patients, of which 206 were women and 74 men. 95% are represented by African American population. The majority of our patients had endometrial cancer (33%), breast cancer (21%), lung (9%), colon (8&), ovary (7.5%), prostate (3.5%) and head and neck (3.5%). Fewer carcinomas were of thyroid, other gastrointestinal and genitourinary origin. Of the most frequent cancers in our pilot study, endometrial carcinomas, 26% displayed significant tumor burden with 53% demonstrating > 50 mutations. In additional, endometrial cancers were found to be MSI stable in 84% of cases, 11% had a germline mutation, 63% had actionable mutations (range of 1-9), 68% with biologically relevant mutations (range 2-5) and 100% had VUS (range 1 to 21). Importantly, 18% of the cases studied had novel treatment indications. Of the breast carcinomas, 44% of cases displayed a significant tumor burden with 29% demonstrating > 50 mutations. In additional, 76% of breast cancer case were found to be MSI stable in, 10% had a germline mutations, 71% had actionable mutations, 90% with biologically relevant mutations (range 1-7), and 86% had VUS (range 1 to 14). Clinical trials were available in 66% and 76% cases had unique treatment indications. Of the lung carcinomas, 89% had tumor mutation burden with > 50 mutations. 100% had VUS (range 2-15) and 44% had treatment indication. CONCLUSION Providing access to CLIA NGS to safety net hospital cancer patients revealed unique somatic changes and treatment options. Taken together, this approach is an important step to democratize precision medicine and improve health outcomes to advance health equity. Citation Format: Gabriella M. Oprea-Ilies, LaTrisha L. Horne, Eddie R. Stanley, Regina Lee, Joel Okoli, Giuseppe Del Priore, Pooja Mishra, Roland Matthew, Eric L. Flenaugh, Brian Rivers, James W. Lillard, Sam Sirotnikov, Mallik Matthews. Landscape of the molecular changes in the most frequent tumors in a safety net hospital [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A056.

Circulating miRNAs Respond to Denosumab Treatment After 2 Years in Postmenopausal Women With Osteoporosis-the MiDeTe study.

MicroRNAs (miRNAs)-short, single-stranded, noncoding RNAs-regulate several biological processes, including bone metabolism. We investigated circulating miRNAs as promising biomarkers for treatment monitoring in women with postmenopausal osteoporosis on denosumab (DMAB) therapy. In this prospective, observational, single-center study, 21 postmenopausal women treated with DMAB were included for a longitudinal follow-up of 2 years. Next-generation sequencing (NGS) was performed to screen for serological miRNAs at baseline, month 6, and month 24. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to confirm NGS findings in the entire cohort. Bone turnover markers (BTM) P1NP and CTX, and bone mineral density (BMD) by dual x-ray absorptiometry were assessed and correlated to miRNAs. BMD at the hip (5.5%, P = 0.0006) and lumbar spine significantly increased (11.4%, P = 0.017), and CTX (64.1%, P < 0.0001) and P1NP (69.3%, P < 0.0001) significantly decreased during treatment. NGS analysis revealed significant changes in miRNAs after 2 years of DMAB treatment but not after 6 months. Seven miRNAs were confirmed by RT-qPCR to be significantly changed during a 2-year course of DMAB treatment compared to baseline. Four of these were mainly transcribed in blood cells, including monocytes. Correlation analysis identified significant correlation between change in miRNA and change in BTMs as well as BMD. Based on effect size and correlation strength, miR-454-3p, miR-26b-5p, and miR-584-5p were defined as top biomarker candidates, with the strongest association to the sustained effect of denosumab on bone in osteoporotic patients. Two years of DMAB treatment resulted in upregulation of 7 miRNAs, 4 of which are mainly transcribed in monocytes, indicating a potential impact of DMAB on circulating osteoclast precursor cells. These changes were associated to BMD gain and BTM suppression and could therefore be useful for monitoring DMAB treatment response.

Defining Primary Vs Secondary AML: Correlation between Pathology and NGS/FISH

Introduction: The treatment of acute myelogenous leukemia (AML) is often determined by factors that include an antecedent dysplasia. At presentation it is often unknown whether a patient has had dysplastic bone marrow features or not. We opted to look back at our leukemia patients to determine the frequency of antecedent dysplasia and to determine if next generation sequencing (NGS) or Florescent in situ hybridization (FISH) findings correlate with the pathologic review. Methods: We chart reviewed 43 charts from a period of 9/21/2021 to current from Orlando Health records. We focused on 29 charts that we could gather complete information from. We reviewed initial bone marrow biopsies and NGS/ FISH findings. We determined if a patient had a known history of myelodysplasia from previous records. If there was no known history of myelodysplastic syndrome (MDS) but dysplasia was seen on the bone marrow biopsy, we looked at NGS/FISH. While there can be overlap with AML, findings that suggest MDS are as follows: NGS: TET2, NRAS, U2AF1, RUNX1, TP53, SF3B1 FISH: 5q-, -5 (5p15, 5q31, 5q33), 7q-, -7 (Cen 7, 7q22, 7q31), Trisomy 8 (Cen 8), MLL (11q23), 20q- (20q12, 20qter) We then examined what treatments were used based on these findings. Results: Overall: 11/29 patients had dysplasia mentioned in their initial pathology report. 4/11 had a known preexistent dysplasia. Of the 7 who didn't have known dysplasia, 6/7 had an NGS or FISH suggesting MDS. Age: Of the patient who had dysplastic findings as well as NGS/FISH to support those findings, the patients had an average age of 57. The one patient without MDS/FISH findings has an age of 69. The average age of all patients with dysplastic findings on their bone marrow was 60. Of the patients who did not have any dysplastic findings on the bone marrow, the average age was 58. Treatment: Vyexos (liposomal daunorubicin and cytarabine), a therapy generally used for secondary leukemias was only used in two patients. Age and performance status was cited as a reason to use alternative therapy. Although one patient did receive 7+3 (idarubicin and cytarabine) instead of Vyxeos. One patient also had severe COVID pneumonia at diagnosis as the reason for not getting induction with Vyexos and instead getting venetoclax/azacitidine. Four patients were either treated with hypomethylating agent of either azacitidine or decitabine, again this was normally chosen due to performance status, age, or active COVID pneumonia as above. Conclusion: These results show that pathology findings of underlying dysplastic changes seem to correlate with NGS or FISH findings. Of the 7 patients with these bone marrow changes but no known MDS, 6 of them had NGS or FISH to support an underlying disease. Factors such as age did not help to predict antecedent MDS. Based on this information it may be appropriate to start with induction therapies such as Vyxeos when dysplastic changes are seen on the bone marrow while FISH and NGS are still pending, if their performance status and age allow to do so. Further larger retrospective studies to compare underlying dysplastic findings versus FISH/NGS findings should be done to further support this concept.

Clinical actionability and utilization of next-generation sequencing for prostate cancer in a changing treatment landscape

BackgroundUntil recently there were no genome-directed therapies (GDTs) requiring next-generation sequencing (NGS) in prostate cancer. We examined whether the US approval of poly-(ADP-ribose) polymerase (PARP) inhibitors in May 2020 influenced the actionability and utilization of NGS in patients with prostate cancer.MethodsThis was a single-center, retrospective study including men with prostate cancer who received NGS testing from a single lab between 7/11/2018-7/6/2021. Clinical and testing data were derived from the electronic medical record.ResultsThere were 346 patients with prostate cancer and qualifying NGS testing during the study period. Overall, 55 patients (15.9%) had qualifying homologous recombination repair (HRR) alterations for PARP inhibitor treatment. A greater proportion of alterations were actionable post-approval compared to pre-approval (22.7% vs 0%, Chi-squared p&amp;lt;0.001). 9 patients received olaparib during the study period. Patients receiving NGS testing after the PARP inhibitor approval were more likely to have metastatic disease than patients sequenced before the approval (74.2% vs. 41.1% Chi-squared p&amp;lt;0.001). Only 10.4% of patients with metastatic prostate cancer received NGS testing within 30 days of diagnosis. NGS testing was performed after a median of 1 prior line of systemic therapy. The median number of days between metastatic diagnosis and NGS testing was 196 (Q1-Q3: 54-832). The median time from NGS testing to the next treatment was 255 days (95% CI 151-300). These characteristics were not significantly different before or after the PARP inhibitor approval.ConclusionIn this single-center cohort, the approval of PARP inhibitors for later-line treatment of metastatic prostate cancer increased the actionability of NGS findings but did not lead to earlier use of NGS testing.

Glioma 2021 WHO Classification: The Superiority of NGS Over IHC in Routine Diagnostics.

The accurate detection of genetic variants such as single substitutions (IDH1/2, TERT), chromosomal abnormalities (CDKN2A, 1p/19q deletions, and EGFR amplifications), or promoter methylations (MGMT) is critical for glioma patient management, as emphasized in the World Health Organization's (WHO's) most recent classification in 2021 (WHO CNS5). The purpose of this study was to evaluate novel innovative methods for determining IDH1/2 status in the context of WHO CNS5. Multiple biomarkers were simultaneously screened using next-generation sequencing (NGS) on 34 glioma samples. In cases where the IDH1/2 status determined by immunohistochemistry (IHC) or multiplex ligation-dependent probe amplification (MLPA) was inconsistent with the NGS results, quantitative polymerase chain reaction (qPCR) and Sanger sequencing were performed to resolve the adjudicated discrepancy. IDH1/2 NGS results differ from IHC (7/13 samples) as well as MLPA reports (1/4 samples). All NGS findings were confirmed by qPCR and Sanger sequencing. WHO CNS5 requires assessment of multiple mutations for glioma classification. We demonstrated that qPCR or NGS performed in reference genetic laboratories, rather than IHC, is the most reliable method for IDH1/2 analysis. Clinicians should be aware of discrepancies in MLPA or IHC results and seek reconsultation in facilities with extensive access to advanced molecular technologies. Moreover, we proposed a new algorithm for the molecular diagnostic procedures in glioma patients based on the WHO CNS5.

Performance of Idylla KRAS assay on extracted DNA and de-stained cytology smears: Can we rescue small sample?

ObjectiveKRAS is a frequently mutated gene in cancers, and with recent FDA-approved targeted therapy for the G12C mutation, testing for KRAS variants is essential. We evaluated the performance of the Idylla KRAS assay on extracted DNA and cytology smears in order to expand the utility of the assay. MethodsIn total, fifty-seven human samples were analyzed. Idylla results from sixteen DNA extracted from formalin-fixed, paraffin-embedded tissues (FFPE DNA) and thirty cytology smears were compared to the reference method. We evaluated the performance of the Idylla assay using corresponding cytology smears to rescue cellblocks or surgical blocks that were quantity not sufficient (QNS) for next generation sequencing (NGS). ResultIn the FFPE DNA cohort, 10 ng DNA input yielded valid results in all 16 samples, with 15 of 16 (93 %) concordant with NGS findings. In the cytology smear cohort, the Idylla KRAS assay demonstrated 100 % concordance with previous NGS results in 30 cases. In the QNS cohort, the assay was valid in all cases and KRAS mutations were identified in 3 of 11 cytology smears, including one G12C mutation. ConclusionThe Idylla KRAS assay is a high-performing, feasible, and convenient option for testing extracted DNA and cytology smears. It rescues QNS samples allowing it to be integrated into the molecular workflow as an initial screening test with remarkably quick turnaround times.

Comprehensive genomic profiling: Insights into diagnostic value and treatment strategy.

e15028 Background: The clinical application of next-generation sequencing (NGS) has continued to advance precision oncology by enabling the identification of biomarkers that may predict potential response to targeted therapy, yet the benefit of tissue-based NGS to patient care is not solely confined to this narrow application. Not uncommonly, NGS can reveal genomic characteristics of the tumor that may be inconsistent with initial pathology review and clinical presentation. When this occurs, these genomic insights along with other clinical and diagnostic findings may be utilized to reconsider the initial diagnosis and seek to explore alternative diagnostic explanations that may be more consistent with the genomic findings. Methods: Patient diagnoses and clinical data were reviewed and compared against NGS results to determine if the genomic profile was consistent with and supports the initial diagnosis. If the genomic profile of the tumor was found to be inconsistent with the initial diagnosis, a secondary review of the case was initiated. Out of a cohort of &gt;2,500 patient cases, notable examples of cases were identified that highlight the impact of NGS findings on the diagnostic classification of the tumor. Genomic profiling was conducted utilizing PathGroup’s molecular pathology-directed tumor profiling solution, Endeavor, which is powered by the FDA-cleared PGDx elio tissue complete test. Results: Among the identified patient cases where initial pathological diagnosis was altered following review of NGS results, several examples include 1) a sarcoma case being re-classified as melanoma, 2) a lung lesion initially diagnosed as NSCLC that was metastatic basal cell carcinoma and 3) a cancer of unknown primary that was determined to be a cholangiocarcinoma. Treating oncologists were kept apprised of all diagnostic findings and modifications to either the initial diagnosis or to the tumor classification which led to an altered treatment strategy in each case. Conclusions: NGS can be broadly applied throughout the cancer care continuum, with a new perspective on its potential value diagnostically. In these cases, re-evaluation and subsequent re-classification led to treatment strategy shifts that are more specific to the patient’s tumor type. These findings provide critical real-world evidence that supports the value of broad genomic profiling in complex cancer cases, especially when the genomic findings are inconsistent with the initial diagnosis, or the diagnosis is unclear.

Comparison of comprehensive genomic profiles between young-onset and average-onset colorectal cancer.

167 Retraction The abstract by Torrejon et al entitled, “Comparison of comprehensive genomic profiles between young-onset and average-onset colorectal cancer.,” ( Journal of Clinical Oncology 40, no. 4, suppl 167) published on January 19, 2022, has been retracted due to calculation errors. Current age was used for the analysis instead of age at diagnosis. This error significantly changed the results and conclusion of the abstract. Background: The incidence of young-onset colorectal cancer (yoCRC) is rising for unknown reasons. This study assessed for differences in comprehensive genomic profiles between yoCRC and average-onset colorectal cancer (aoCRC). Methods: All patients with CRC seen at Cleveland Clinic that had tumor-based next generation sequencing (NGS) performed as part of their care with a clinically available assay between January 2017 and September 2020 were included. The cohort was divided based on age of diagnosis, with yoCRC defined as age of CRC diagnosis &lt;50 years old. All clinical data and genomic alterations were included for analysis. We assessed for differences in clinical data and NGS findings between yoCRC and aoCRC using Fisher’s exact test, adjusted for primary tumor sidedness. Overall survival (OS) by genomic alteration was estimated by Kaplan-Meier methods and compared using log rank test. Results: The study population comprised of 51 yoCRC patients and 211 aoCRC patients. There were no significant differences in sex, race or ethnicity between yoCRC and aoCRC patients. Compared to aoCRC patients, yoCRC patients were more likely to present at diagnosis with stage IV disease (81% vs. 56%, p = 0.02) and have left-sided primary tumors (69% vs. 60%, p = 0.26). YoCRC tumors were more likely to have mutations in APC, KRAS, TP53 and FLT3 compared to aoCRC tumors, independent of tumor sidedness. These data are summarized in Table. Compared to left-sided tumors, right-sided tumors had significantly higher frequency of KRAS (67.7% vs. 48.8%, p = 0.003), BRAF (15.2% vs. 3.1%, p = 0.0006) and PTEN (16.2% vs. 3.1%, p value = 0.0003) alterations. AoCRC patients had significantly longer OS compared to yoCRC patients (median OS: 70.0 months vs. 36.3 months, p=0.004). Black (37.2 months, n=35) and Asian (37.8 months, n=7) patients had significantly worse median OS compared to White patients (67.8 months, n=199), p-value 0.005. In the overall population, patients with APC mutations had significantly better OS compared to those with APC wildtype tumors (median OS: 92.6 months vs. 54.4 months, p=0.001). Patients with FLT3 mutations had worse median OS compared to those with FLT3 wildtype tumors (median OS: 42.0 months vs. 64.8 months, p=0.007). There were no survival differences based on MSI status. Conclusions: In this series, yoCRC patients were more likely to present with stage IV disease and experienced worse OS compared to aoCRC patients. YoCRC patients were more likely to have mutations in APC, KRAS, TP53 and FLT3, independent of tumor sidedness. Mutations in FLT3 correlated with worse OS.[Table: see text]

Rapid point of care NGS in colorectal cancer.

172 Background: Next generation sequencing (NGS) is the laboratory cornerstone of precision oncology treatment. In advanced colorectal cancer (CRC), current guidelines recommend testing RAS, BRAF and MMR biomarkers as standard of care. The added value of comprehensive genomic profiling is so far unclear. Traditional NGS operations are complicated, requiring specialized equipment and personnel. In many jurisdictions, cancer patients are treated in publicly-funded community hospitals, where NGS is not typically utilized and access to testing via send-out services is associated with lengthy turnaround times. Here, we have validated and implemented one of the world's first "point of care" NGS services. Our early experience on NGS implementation and impact in CRC patients is described. Methods: All NGS studies were performed using the Oncomine Precision Assay (OPA) on the genexus integrated sequencer. NGS was performed at the request of the treating physician. All NGS was performed in a local community pathology lab by histotechnologists, simultaneously responsible for IHC testing (such as MMR) and interpreted by anatomic pathologists in conjunction with routine diagnostic pathology services. Retrospective chart review was performed for all patients undergoing sequencing studies and key data, including turnaround time and NGS findings were extracted from the electronic medical record for analysis. Results: A total of 51 cases with CRC were tested using point of care NGS from November 2020-August 2021, initiated by treating physicians. The median turnaround time for results was 3 days. Oncogenic driver events were identified in 46 (90%) cases, including canonical mutations in KRAS, NRAS and BRAF (Table). Actionable mutations were identified in 13 (25%) samples that would not have been identified with single-gene testing. Conclusions: Here, we show that comprehensive NGS can reveal occult resistance mechanisms to standard therapy and identify actionable biomarkers in a substantial proportion of patients with CRC. NGS added valuable information compared to guideline-recommended testing standards. Our study demonstrates that local testing can have rapid turnaround times. To our knowledge, this is the first report of “point of care” NGS in CRC. Further follow up is needed to explore the utility of these expanded roles for NGS testing. [Table: see text]

A geno-clinical decision model for the diagnosis of myelodysplastic syndromes

AbstractThe differential diagnosis of myeloid malignancies is challenging and subject to interobserver variability. We used clinical and next-generation sequencing (NGS) data to develop a machine learning model for the diagnosis of myeloid malignancies independent of bone marrow biopsy data based on a 3-institution, international cohort of patients. The model achieves high performance, with model interpretations indicating that it relies on factors similar to those used by clinicians. In addition, we describe associations between NGS findings and clinically important phenotypes and introduce the use of machine learning algorithms to elucidate clinicogenomic relationships.