Case Report: Significant Clinical Benefit From Pemetrexed-Based Therapy in ROS1- and ALK-rearranged Lung Cancer With Adenosquamous Histology

Pemetrexed (used as a platinum doublet or as a maintenance regimen) is an established therapy for patients with advanced non-squamous non-small-cell lung cancer (NSCLC). In addition, certain gene rearrangements (e.g., ALK, ROS1, RET) appear to especially benefit from the use of pemetrexed. Inferior outcomes with pemetrexed compared to other chemotherapies in patients with NSCLC demonstrating squamous histology removed these patients from the labeled indication for the drug. While most squamous cases do not harbor driver oncogenes, rare exceptions exist. Whether the poor outcomes with pemetrexed extend to NSCLC with squamous component harboring driver oncogenes remains unexplored. In this case series, we describe two patients with adenosquamous histology harboring an ROS1 and ALK gene arrangement, respectively, who derived significant benefit from pemetrexed-based therapy. These cases suggest that the value of pemetrexed may need to be re-explored in adenosquamous NSCLC harboring such alterations.

Cranial Fasciitis in Children: Expanding the Spectrum of USP6-Associated Clonal Transient Neoplasms

Background: Cranial fasciitis (CF) is a benign (myo)fibroblastic proliferation of children. Typical presentation consists of a rapidly growing solitary mass on the temporal or parietal cranium in the first 2 years of age. CF is characterized by a rapid growth followed by a relative slowdown and even growth arrest. The finding of somatic USP6 gene rearrangements demonstrating clonality in CF together with its clinical behavior places it in the category of diseases recently termed “transient neoplasia.” Methods: Histological, immunohistochemical, and molecular findings of 18 patients with CF were retrospectively studied. Results: The tumor typically presented as a painless rapidly enlarging mass in the temporal region. Sixty-six percent of the cases harbored USP6 gene rearrangement. Nine patients were treated with gross total resection (GTR) and 9 with subtotal tumor resection (STR). Two patients treated with GTR had recurrence. Five patients treated with STR had progression-free disease for at least 10 months after surgery and in four patients the tumor regressed spontaneously a median 16 months after surgery. Conclusions: In this largest series to date, we reported the clinicopathological, immunohistochemical, and molecular findings of 18 pediatric cases of CF with emphasis on the clinical growth pattern of these tumors.

Clonality assessment and detection of clonal diversity in classic Hodgkin lymphoma by next-generation sequencing of immunoglobulin gene rearrangements

Clonality analysis in classic Hodgkin lymphoma (cHL) is of added value for correctly diagnosing patients with atypical presentation or histology reminiscent of T cell lymphoma, and for establishing the clonal relationship in patients with recurrent disease. However, such analysis has been hampered by the sparsity of malignant Hodgkin and Reed-Sternberg (HRS) cells in a background of reactive immune cells. Recently, the EuroClonality-NGS Working Group developed a novel next-generation sequencing (NGS)-based assay and bioinformatics platform (ARResT/Interrogate) to detect immunoglobulin (IG) gene rearrangements for clonality testing in B-cell lymphoproliferations. Here, we demonstrate the improved performance of IG-NGS compared to conventional BIOMED-2/EuroClonality analysis to detect clonal gene rearrangements in 16 well-characterized primary cHL cases within the IG heavy chain (IGH) and kappa light chain (IGK) loci. This was most obvious in formalin-fixed paraffin-embedded (FFPE) tissue specimens, where three times more clonal cases were detected with IG-NGS (9 cases) compared to BIOMED-2 (3 cases). In total, almost four times more clonal rearrangements were detected in FFPE with IG-NGS (N = 23) as compared to BIOMED-2/EuroClonality (N = 6) as judged on identical IGH and IGK targets. The same clonal rearrangements were also identified in paired fresh frozen cHL samples. To validate the neoplastic origin of the detected clonotypes, IG-NGS clonality analysis was performed on isolated HRS cells, demonstrating identical clonotypes as detected in cHL whole-tissue specimens. Interestingly, IG-NGS and HRS single-cell analysis after DEPArray™ digital sorting revealed rearrangement patterns and copy number variation profiles indicating clonal diversity and intratumoral heterogeneity in cHL. Our data demonstrate improved performance of NGS-based detection of IG gene rearrangements in cHL whole-tissue specimens, providing a sensitive molecular diagnostic assay for clonality assessment in Hodgkin lymphoma.

NGS-Based MRD Quantitation: An Alternative to qPCR Validated on a Large Consecutive Cohort of Children with ALL

Together with multicolor flow cytometry, quantitation of clonal immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements represents the current standard for the detection of minimal / measurable residual disease (MRD) in treatment protocols for pediatric acute lymphoblastic leukemia (ALL) patients. Despite the adoption of next generation sequencing (NGS) in the routine identification of clonal IG/TR gene rearrangements as markers for MRD detection, real-time quantitative (q)PCR is still the standard for MRD quantitation in follow-up samples. So far, no large-scale direct comparison of qPCR- and NGS-based MRD quantitation has been performed. We compared qPCR- and NGS-MRD evaluation in a cohort of children with B-cell precursor (BCP) ALL treated on the AIEOP-BFM ALL 2009 protocol and assessed the feasibility and relevance of this method for the stratification at day 33 (EOI). In total, 459 patients were diagnosed with BCP-ALL from 2010 to 2018, and 437 of them were included in our study based on the availability of residual DNA material isolated from day 33 bone marrow aspirates and having at least one IG/TR MRD marker detectable by standard qPCR with protocol-required sensitivity of 10 -4. Sequencing libraries were prepared according to the EuroClonality-NGS group SOP (Brüggemann et al, Leukemia 2019) with the total DNA input normalized to the equivalent of 150,000 nucleated cells to reach MRD sensitivity of 10 -5 and sequenced on Illumina NovaSeq and MiSeq instruments. In total of 780 IG/TR markers evaluated by both NGS and qPCR. Sequencing data were analyzed using the ARResT/Interrogate (Bystry et at, Bioinformatics 2017) pipeline and a custom bioinformatic analysis process and the NGS-MRD results were normalized to the EuroClonality-NGS central in-tube quality/quantification control (cIT-QC; Knecht et al, Leukemia 2019). From the total 780 IG/TR MRD markers evaluated by both methods, 629 (80.6%) were concordant with 242 markers being MRD positive and 387 negative. From 82 markers that were only positive by qPCR and not by NGS, 76 were positive below the quantitative range (positive non-quantifiable). Specificity analysis was performed for each marker by searching for the junction sequence across the dataset of all patients' NGS results. Based on these results, 22 out of 82 markers positive only by qPCR were classified as potentially unspecific (false positive) and similarly 32 unspecific markers were identified among the 69 positive only by NGS. This was also supported by unspecific amplification of the polyclonal control in 27 out of these 32 corresponding qPCR systems, in some cases leading to qPCR negative classification determined by the EuroMRD guidelines. Overall stratification of patients based only on day 33 MRD by qPCR or NGS was concordant in 76% of patients by both methods, while in 19% of patients, NGS-MRD quantitation led to the assignment to a lower-risk group, mainly due to the elimination of false-positive results. Furthermore, analysis of all positive markers across all patients' NGS libraries showed, that one out of 10 markers (mainly in the IGK, TRG and TRD loci) used for qPCR-MRD stratification did not provide satisfactory specificity, although they fully met EuroMRD criteria during the optimization of qPCR patient-specific assays. Our results show that NGS-MRD is highly concordant with traditional qPCR-based strategy and has comparable sensitivity and clinical value in the setting of a BFM-based clinical protocol, while being less laborious and providing significantly more specific results and additional information on the IG/TR repertoire (Kotrova et al, Blood 2016). Our study also emphasizes the importance of selecting MRD markers of adequate specificity at diagnosis. Currently, this selection can be assisted by these broad sequencing data on IG/TR repertoire of large number of patients. Based on these results, we propose that frontline NGS-MRD evaluation developed by the EuroClonality-NGS working group can be used as an alternative to traditional qPCR-based MRD quantitation in future MRD-based treatment protocols. Supported by grants NU20-03-00284 and NU20-07-00322 from the Czech Health Research Council and 534120 from Charles University. All methods were established through collaboration within the EuroClonality-NGS and EuroMRD groups. Disclosures van der Velden: Agilent: Research Funding; Navigate: Other: Service Level Agreement; Janssen: Other: Service Level Agreement; EuroFlow: Other: Service Level Agreement, Patents & Royalties: for network, not personally; BD Biosciences: Other: Service Level Agreement. Brüggemann: Incyte: Other: Advisory Board; Janssen: Speakers Bureau; Amgen: Other: Advisory Board, Travel support, Research Funding, Speakers Bureau. Langerak: Erasmus MS, University Medical Center: Current Employment; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Research Funding; Gilead: Research Funding; Janssen: Speakers Bureau.

Pan-cancer landscape of CD274 (PD-L1) rearrangements in 283,050 patient samples, its correlation with PD-L1 protein expression, and immunotherapy response

BackgroundImmune checkpoint inhibitors (ICIs) benefit patients with multiple cancer types, however, additional predictive biomarkers of response are needed. CD274 (programmed cell death ligand-1, PD-L1) gene rearrangements are positively associated with PD-L1 expression and may confer benefit to ICI, thus a pan-cancer characterization of these alterations is needed.MethodsWe analyzed 283,050 patient samples across multiple tumor types that underwent comprehensive genomic profiling for activating CD274 rearrangements and other alterations. The DAKO 22C3 Tumor Proportion Scoring (TPS) method was used for PD-L1 immunohistochemistry (IHC) testing in a small subset with available data (n=55,423). A retrospective deidentified real-world clinico-genomic database (CGDB) was examined for ICI treatment outcomes. We also report a detailed case of CD274-rearranged metastatic rectal adenocarcinoma.ResultsWe identified 145 samples with functional rearrangements in CD274. There were significant enrichments for PIK3CA, JAK2, PDCD1LG2, CREBBP, and PBRM1 co-mutations (ORs=2.1, 16.7, 17.8, 3.6, and 3.4, respectively, p<0.01). Genomic human papillomavirus (HPV)-16, Epstein-Barr virus, and mismatch repair genes also co-occurred (OR=6.2, 8.4, and 4.3, respectively, p<0.05). Median tumor mutational burden (TMB) was higher compared with CD274 wild-type samples (7.0 vs 3.5 mutations/Mb, p=1.7e-11), with disease-specific TMB enrichment in non-small cell lung, colorectal, unknown primary, and stomach cancers. PD-L1 IHC skewed toward positivity (N=39/43 samples with ≥1% positivity). Of eight patients from the CGDB, three remained on ICI treatment after 6 months. Separately, one patient with metastatic rectal adenocarcinoma experienced a pathologic complete response on chemoimmunotherapy.ConclusionsCD274 gene rearrangements are associated with increased PD-L1 IHC scores, higher TMB, and potential clinical benefit in ICI-treated patients with cancer.

Molecular Diagnosis of T-cell Lymphoma: A correlative study of PCR-based T-cell clonality assessment and targeted NGS

Immunomorphological diagnosis of T-cell lymphomas (TCL) may be challenging, especially on needle biopsies. Multiplex polymerase chain reaction (PCR) assays to assess T-cell receptor (TCR) gene rearrangements are now widely used to detect T-cell clones and provide diagnostic support. However, PCR assays only detect 80% of TCL, and clonal lymphocyte populations may also appear in non-neoplastic conditions. More recently, targeted next-generation sequencing (t-NGS) technologies have been deployed to improve lymphoma classification. To the best of our knowledge, the comparison of these techniques' performance in TCL diagnosis has not been reported yet. In this study, 82 TCL samples and 25 non-neoplastic T-cell infiltrates were divided into two cohorts (test and validation) and analyzed with both multiplex PCR and t-NGS to investigate TCR gene rearrangements and somatic mutations, respectively. The detection of mutations appeared to be more specific (100.0%) than T-cell clonality assessment (41.7% to 45.5%), whereas no differences were observed in terms of sensitivity (95.1% to 97.4%). Furthermore, t-NGS provided a reliable basis for TCL diagnosis in samples with partially degraded DNA that was impossible to assess with PCR. Finally, although multiplex PCR assays appeared to be less specific than t-NGS, both techniques remain complementary, as PCR recovered some t-NGS negative cases.