Analysis of immunoglobulin/T-cell receptor repertoires by high-throughput RNA sequencing reveals a continuous dynamic of positive clonal selection in follicular lymphoma.

Follicular lymphoma (FL) course is highly variable, making its clinical management challenging. In this incurable and recurring pathology, the interval between relapses tends to decrease while aggressiveness increases, sometimes resulting in the transformation to higher-grade lymphoma. These evolutions are particularly difficult to anticipate, resulting from complex clonal evolutions where multiple subclones compete and thrive due to their capacity to proliferate and resist therapies. Here, to apprehend further these processes, we used a high-throughput RNA sequencing approach to address simultaneously the B-cell immunoglobulin repertoires and T-cell immunoglobulin repertoires repertoires of lymphoma cells and their lymphoid microenvironment in a large cohort of 131 FL1/2-3A patients. Our data confirm the existence of a high degree of intra-clonal heterogeneity in this pathology, resulting from ongoing somatic hyper-mutation and class switch recombination. Through the evaluation of the Simpson ecological-diversity index, we show that the contribution of the cancerous cells increases during the course of the disease to the detriment of the reactive compartment, a phenomenon accompanied by a concomitant decrease in the diversity of the tumoral population. Clonal evolution in FL thus contrasts with many tumors, where clonal heterogeneity steadily increases over time and participates in treatment evasion. In this pathology, the selection of lymphoma subclones with proliferative advantages progressively outweighs clonal diversification, ultimately leading in extreme cases to transformation to high-grade lymphoma resulting from the rapid emergence of homogeneous subpopulations.

Diagnosis of chronic B-cell lymphoproliferative disease in peripheral blood = how machine learning may help to the interpretation of flow cytometry data.

Flow cytometry (FCM) has become a method of choice for immunologic characterization of chronic lymphoproliferative disease (CLPD). To reduce the potential subjectivities of FCM data interpretation, we developed a machine learning random forest algorithm (RF) allowing unsupervised analysis. This assay relies on 16 parameters obtained from our FCM screening panel, routinely used in the exploration of peripheral blood (PB) samples (mean fluorescence intensity values (MFI) of CD19, CD45, CD5, CD20, CD200, CD23, HLA-DR, CD10 in CD19-gated B cells, ratio of kappa/Lambda, and different ratios of MFI B-cells/T-cells [CD20, CD200, CD23]). The RF algorithm was trained and validated on a large cohort of more than 300 annotated different CLPD cases (chronic B-cell leukemia, mantle cell lymphoma, marginal zone lymphoma, follicular lymphoma, splenic red pulp lymphoma, hairy cell leukemia) and non-tumoral selected from PB samples. The RF algorithm was able to differentiate tumoral from non-tumoral B-cells in all cases and to propose a correct CLPD classification in more than 90% of cases. In conclusion the RF algorithm could be proposed as an interesting help to FCM data interpretation allowing a first B-cells CLPD diagnostic hypothesis and/or to guide the management of complementary analysis (additional immunologic markers and genetic).

IGH 3'RR recombination uncovers a non-germinal center imprint and c-MYC-dependent IGH rearrangement in unmutated chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B cells that weakly express a B-cell receptor. The mutational status of the variable region (IGHV) within the immunoglobulin heavy chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLL are genetically imprinted by activation-induced cytidine deaminase (AID). AID is also required for IGH rearrangements: class switch recombination and recombination between switch Mu (Sµ) and the 3' regulatory region (3'RR) (Sµ-3'RRrec). The great majority of CLL B cells being unswitched led us to examine IGH rearrangement blockade in CLL. Our results separated CLL into two groups on the basis of Sµ-3'RRrec counts per sample: Sµ-3'RRrecHigh cases (mostly unmutated CLL) and Sµ-3'RRrecLow cases (mostly mutated CLL), but not based on the class switch recombination junction counts. Sµ-3'RRrec appeared to be ongoing in Sµ-3'RRrecHigh CLL cells and comparison of Sµ-3'RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sµ-3'RRrecHigh CLL harbor a non-germinal center experienced B-cell imprint while Sµ-3'RRrecLow CLL are from AID-experienced B cells from a secondary lymphoid organ. In addition to the proposals already made concerning the CLL cell of origin, our study highlights that analysis of IGH recombinatory activity can identify CLL cases from different origins. Finally, on-going Sµ-3'RRrec in Sµ-3'RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sµ-3'RRrec, even in the absence of AID for the latter.

High PDL1/PDL2 gene expression correlates with worse outcome in primary mediastinal large B-cell lymphoma.

Primary mediastinal B-cell lymphoma (PMBL) is an uncommon entity of aggressive B-cell lymphoma with an unusually good prognosis, except for 10-15% of chemotherapy-refractory cases. To identify earlier these higher risk patients, we performed molecular characterization of a retrospective multicenter cohort of patients treated with firstline immunochemotherapy. The traits of the patients with gene-expression profiling data (n = 120) were as follows: median age of 34 years (range, 18-67 years); female sex, 58.3%; elevated lactate dehydrogenase, 82.5%; Eastern Cooperative Oncology Group performance status score of 0 to 1, 85.7%; Ann Arbor stage I/II, 55%; International Prognostic Index score of 1 to 2, 64.4%; and median metabolic tumor volume, 290.4 cm3 (range, 15.7-1147.5 cm3). Among all 137 markers tested for correlation with survival data, only programmed death-ligand (PDL) 1 and PDL2 expression showed a prognostic impact. Overall, both PDL1 and PDL2 genes were highly expressed in 37 patients (30.8%; PDL1high/PDL2high). The baseline clinical characteristics of patients with PDL1high/PDL2high were similar to those of other patients. In univariate analysis, PDL1high/PDL2high status was associated with poor progression-free survival (PFS) (hazard ratio [HR], 4.292) and overall survival (OS; HR, 8.24). In multivariate analysis, PDL1high/PDL2high status was an independent prognostic factor of adverse outcomes (PFS: HR, 5.22; OS: HR, 10.368). We validated these results in an independent cohort of 40 patients and confirmed the significant association between PDL1high/PDL2high status and inferior PFS (HR, 6.11). High PDL1/PDL2 gene expression defines a population with strong immune privilege and poorer outcomes from standard chemotherapy who might benefit from firstline checkpoint inhibitor therapy.

Detection of salivary gland and sinonasal fusions by a next-generation sequencing based, ligation-dependent, multiplex RT-PCR assay.

AIMS: The discovery of tumour type-specific gene fusion oncogenes in benign and malignant salivary gland and sinonasal (SGSN) tumours has significantly increased our knowledge about their molecular pathology and classification. METHODS AND RESULTS: We developed a new targeted multiplexed next-generation sequencing (NGS)-based method that utilizes ligation dependent reverse-transcriptase polymerase chain reaction (LD-RT-PCR) to detect oncogenic fusion transcripts involving 116 genes, leading to 96 gene fusions known to be recurrently rearranged in these tumours. In all, 180 SGSN tumours (formalin-fixed, paraffin-embedded samples, 141 specimens and 39 core needle biopsies) from the REFCORpath (French network for rare head and neck cancers) with previously identified fusion genes by fluorescent in situ hybridisation (FISH), RT-PCR, or molecular immunohistochemistry were selected to test its specificity and sensitivity and validate its diagnostic use. Tested tumours encompassed 14 major tumours types, including secretory carcinoma, mucoepidermoid carcinoma, adenoid cystic carcinoma, salivary gland intraductal carcinoma, clear cell carcinoma, pleomorphic adenoma, adamantinoma-like Ewing Sarcoma, EWSR1::COLCA2 sinonasal sarcoma, DEK::AFF2 sinonasal carcinoma, and biphenotypic sinonasal sarcoma. In-frame fusion transcripts were detected in 97.8% of cases (176/180). Gene fusion assay results correlated with conventional techniques (immunohistochemistry [IHC], FISH, and RT-PCR) in 176/180 tumours (97.8%). CONCLUSION: This targeted multiplexed NGS-based LD-RT-PCR method is a robust, highly sensitive method for the detection of recurrent gene fusions from routine clinical SGSN tumours. It can be easily customized to cover new fusions. These results are promising for implementing an integrated NGS system to rapidly detect genetic aberrations, facilitating accurate, genomics-based diagnoses, and accelerate time to precision therapies in SGSN tumours.

Complete hematologic response after belinostat treatment and allogeneic stem cell transplantation for multiple relapsed/refractory angioimmunoblastic T-cell lymphoma: A case report.

Key Clinical Message: This case report highlights the potential of belinostat for the treatment of relapsed/refractory peripheral T-cell lymphomas, for which effective therapies are still scarce. Abstract: Peripheral T-cell lymphomas have an aggressive disease course associated with poor outcomes. We report a young patient with highly pretreated relapsed/refractory nodal follicular helper T-cell lymphoma (angioimmunoblastic-type [nTFHL-AI]), who successfully received an allogeneic stem cell transplantation following belinostat therapy. The complete hematologic response achieved has lasted more than 2 years.

Disequilibrium between BRCA1 and BRCA2 Circular and Messenger RNAs Plays a Role in Breast Cancer.

Breast cancer is a frequent disease for which the discovery of markers that enable early detection or prognostic assessment remains challenging. Circular RNAs (circRNAs) are single-stranded structures in closed loops that are produced by backsplicing. CircRNA and messenger RNA (mRNA) are generated co-transcriptionally, and backsplicing and linear splicing compete against each other. As mRNAs are key players in tumorigenesis, we hypothesize that a disruption of the balance between circRNAs and mRNAs could promote breast cancer. Hence, we developed an assay for a simultaneous study of circRNAs and mRNAs, which we have called splice and expression analyses by exon ligation and high-throughput sequencing (SEALigHTS). Following SEALigHTS validation for BRCA1 and BRCA2, our hypothesis was tested using an independent research set of 95 pairs from tumor and adjacent normal breast tissues. In this research set, ratios of BRCA1 and BRCA2 circRNAs/mRNAs were significantly lower in the tumor breast tissue compared to normal tissue (p = 1.6 × 10-9 and p = 4.4 × 10-5 for BRCA1 and BRCA2, respectively). Overall, we developed an innovative method to study linear splicing and backsplicing, described the repertoire of BRCA1 and BRCA2 circRNAs, including 15 novel ones, and showed for the first time that a disequilibrium between BRCA1 and BRCA2 circRNAs and mRNAs plays a role in breast cancer.

A Simple Molecular Tool for the Assessment of Kidney Transplant Biopsies.

BACKGROUND: The Banff Classification for Allograft Pathology recommendations for the diagnosis of kidney transplant rejection includes molecular assessment of the transplant biopsy. However, implementation of molecular tools in clinical practice is still limited, partly due to the required expertise and financial investment. The reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) assay is a simple, rapid, and inexpensive assay that permits simultaneous evaluation of a restricted gene panel using paraffin-embedded tissue blocks. The aim of this study was to develop and validate a RT-MLPA assay for diagnosis and classification of rejection. METHODS: A retrospective cohort of 220 kidney transplant biopsies from two centers, which included 52 antibody-mediated rejection, 51 T-cell-mediated rejection, and 117 no-rejection controls, was assessed. A 17-gene panel was identified on the basis of relevant pathophysiological pathways. A support vector machine classifier was developed. A subset of 109 biopsies was also assessed using the Nanostring Banff Human Organ Transplant panel to compare the two assays. RESULTS: The support vector machine classifier train and test accuracy scores were 0.84 and 0.83, respectively. In the test cohort, the F1 score for antibody-mediated rejection, T-cell-mediated rejection, and control were 0.88, 0.86, and 0.69, respectively. Using receiver-operating characteristic curves, the area under the curve for class predictions was 0.96, 0.89, and 0.91, respectively, with a weighted average at 0.94. Classifiers' performances were highest for antibody-mediated rejection diagnosis with 94% correct predictions, compared with 88% correct predictions for control biopsies and 60% for T-cell-mediated rejection biopsies. Gene expression levels assessed by RT-MLPA and Nanostring were correlated: r = 0.68, P < 0.001. Equivalent gene expression profiles were obtained with both assays in 81% of the samples. CONCLUSIONS: The 17-gene panel RT-MLPA assay, developed here for formalin-fixed paraffin-embedded kidney transplant biopsies, classified kidney transplant rejection with an overall accurate prediction ratio of 0.83. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/CJASN/2023_04_10_CJN10100822.mp3.

A novel SMARCA2-CREM fusion expending the molecular spectrum of salivary gland hyalinazing clear cell carcinoma beyond the FET genes.

Hyalinizing clear cell carcinoma (HCCC) is a rare salivary gland carcinoma with a generally indolent behavior, characterized by recurrent chromosomal translocation involving EWSR1 (22q12.2) leading to two fusion genes EWSR1::ATF1 or EWSR1::CREM. We report one case of HCCC with a novel SMARCA2::CREM fusion, identified by targeted RNA next generation sequencing by LD-RT-PCR, which has until now never been described in salivary glands. The exon 4 of SMARCA2 is fused to exon 5 of CREM. This fusion has been described previously in only one tumor, a central nervous system tumor (intracranial mesenchymal tumor) but not in other FET::CREB fused tumors. This fusion was confirmed by CREM break-apart FISH and reverse transcriptase polymerase chain reaction (RT-PCR). The tumor cells showed retained expression of INI1, SMARCA2, and SMARCA4 by immunohistochemistry. We compare its clinical, histopathological, immunophenotypic, genetic features with those previously described in HCCC, FET::CREB fusion-positive. Our results added data suggesting that different histomolecular tumor subtypes seem to be included within the terminology "HCCC, FET::CREB fusion-positive," and that further series of cases are needed to better characterize them.

5' Rapid amplification of cDNA ends (5'RACE): A simpler method to analyze immunoglobulin genes and discover the value of the light chain in chronic lymphocytic leukemia.

The mutational status of the variable region of the immunoglobulin heavy chain gene (IGHV) is a very important biomarker for chronic lymphocytic leukemia (CLL) patients. However, the routine detection of IGHV mutational status is time-consuming and costly. Therefore, we performed 5' Rapid amplification of cDNA ends (5' RACE) in 81 CLL patients who previously underwent detection using Biomed-2. The agreement rate of these two methods was 93.8 %. Regarding the discordant cases, 5' RACE was more sensitive to identify unproductive and multiple rearrangements. Furthermore, 5' RACE can also be used to simultaneously sequence light chains. In most CLL cases, the mutational statuses of heavy and light chains are concordant, except in IGLV3-21. Most IGLV3-21 (24/25) rearrangement shared a similar LCDR3 (QVWDSSSDHPWV) and harbored a single point mutation, namely, IGLV3-21R110. Compared to mutated-CLL non IGLV3-2R110, IGLV3-21R110-CLL exhibited a shorter overall survival (OS) and time to first treatment (TTFT) (p = 0.05, p < 0.0001, respectively) even though 75 % (18/24) of these patients expressed mutated heavy chains. Altogether, IGLV3-21R110 defines a CLL subgroup with specific biological features and an unfavorable prognosis independent of the IGHV mutational status and emphasizes the important value of the light chain. This study is the first to use 5' RACE to detect the mutational status of IGH in CLL. Here, 5' RACE was a reliable and effective method to test the mutational status of heavy and light chains. In addition, 5' RACE can be combined with other assays in the NGS workflow to obtain more detailed insight into subclonal architecture and intraclonal diversity.

UMI-Varcal: A Low-Frequency Variant Caller for UMI-Tagged Paired-End Sequencing Data.

The rapid transition from traditional sequencing methods to Next-Generation Sequencing (NGS) has allowed for a faster and more accurate detection of somatic variants (Single-Nucleotide Variant (SNV) and Copy Number Variation (CNV)) in tumor cells. NGS technologies require a succession of steps during which false variants can be silently added at low frequencies. Filtering these artifacts can be a rather difficult task especially when the experiments are designed to look for very low frequency variants. Recently, adding unique molecular barcodes called UMI (Unique Molecular Identifier) to the DNA fragments appears to be a very effective strategy to specifically filter out false variants from the variant calling results (Kukita et al. DNA Res 22(4):269-277, 2015; Newman et al. Nat Biotechnol 34(5):547-555, 2016; Schmitt et al. Proc Natl Acad Sci U S A 109(36):14508-14513). Here, we describe UMI-VarCal (Sater et al. Bioinformatics 36:2718-2724, 2020), which can use the UMI information from UMI-tagged reads to offer a faster and more accurate variant calling analysis.

Integrative diagnosis of primary cutaneous large B-cell lymphomas supports the relevance of cell of origin profiling.

Primary cutaneous large B-cell lymphomas (PCLBCL) represent a diagnostic challenge because they are classified as PCLBCL, leg type (PCLBCL, LT) or primary cutaneous follicle centre lymphoma, large cell (PCFCL, LC), which differ by prognosis and therapeutic requirement. Unclassified cases with discordant clinical presentations, morphologies, and immunophenotypes may be classified into the not otherwise specified (PCLBCL, NOS) category based on ancillary molecular analyses. Cell-of-origin profiling as germinal centre (GC) type or non-GC type by immunohistochemistry is not considered reproducible because of variable CD10 expression. In a series of 55 PCLBCL cases with > 80% large cells, we reported 21 PCFCL, LC cases as GC-type and 27 PCLBCL, LT as non-GC-type; 7 cases were considered PCLBCL, NOS. Here, we demonstrate the accuracy of molecular profiling of PCLBCL as GC or non-GC type using a reverse transcriptase multiplex ligation assay (RT-MLPA). RT-MLPA classified the seven PCLBCL, NOS cases in accordance with their mutational profile. An integrative principal component analysis confirmed the main criteria and the relevance of genomic profiling of PCFCL, LC as GC-derived, and PCLBCL, LT as non-GC-derived. Both the cell-of-origin classification of PCLBCL and the integrative analysis identified two clinically relevant subgroups according to overall survival, which may help to standardize PCLBCL diagnosis and patient management.

Detection of sarcoma fusions by a next-generation sequencing based-ligation-dependent multiplex RT-PCR assay.

Morphological, immunohistochemical, and molecular methods often need to be combined for accurate diagnosis and optimal clinical management of sarcomas. Here, we have developed, a new molecular diagnostic assay, for the detection of gene fusions in sarcomas. This targeted multiplexed next-generation sequencing (NGS)-based method utilizes ligation dependent reverse-transcriptase polymerase chain reaction (LD-RT-PCR-NGS) to detect oncogenic fusion transcripts involving 137 genes, leading to 139 gene fusions known to be recurrently rearranged in soft-tissue and bone tumors. 158 bone and soft-tissue tumors with previously identified fusion genes by fluorescent in situ hybridization (FISH) or RT-PCR were selected to test the specificity and the sensitivity of this assay. RNA were extracted from formalin-fixed paraffin-embedded (n = 143) or frozen (n = 15) material (specimen; n = 42 or core needle biopsies; n = 116). Tested tumors encompassed 23 major translocation-related sarcomas types, including Ewing and Ewing-like sarcomas, rhabdomyosarcomas, desmoplastic small round-cell tumors, clear-cell sarcomas, infantile fibrosarcomas, endometrial stromal sarcomas, epithelioid hemangioendotheliomas, alveolar soft-part sarcomas, biphenotypic sinonasal sarcomas, extraskeletal myxoid chondrosarcomas, myxoid/round-cell liposarcomas, dermatofibrosarcomas protuberans and solitary fibrous tumors. In-frame fusion transcripts were detected in 98.1% of cases (155/158). Gene fusion assay results correlated with conventional techniques (FISH and RT-PCR) in 155/158 tumors (98.1%). These data demonstrate that this assay is a rapid, robust, highly sensitive, and multiplexed targeted RNA sequencing assay for the detection of recurrent gene fusions on RNA extracted from routine clinical specimens of sarcomas (formalin-fixed paraffin-embedded or frozen). It facilitates the precise diagnosis and identification of tumors with potential targetable fusions. In addition, this assay can be easily customized to cover new fusions.

Clinico-biological features of T-cell acute lymphoblastic leukemia with fusion proteins.

T-cell acute lymphoblastic leukemias (T-ALL) represent 15% of pediatric and 25% of adult ALL. Since they have a particularly poor outcome in relapsed/refractory cases, identifying prognosis factors at diagnosis is crucial to adapting treatment for high-risk patients. Unlike acute myeloid leukemia and BCP ALL, chromosomal rearrangements leading to chimeric fusion-proteins with strong prognosis impact are sparsely reported in T-ALL. To address this issue an RT-MPLA assay was applied to a consecutive series of 522 adult and pediatric T-ALLs and identified a fusion transcript in 20% of cases. PICALM-MLLT10 (4%, n = 23), NUP214-ABL1 (3%, n = 19) and SET-NUP214 (3%, n = 18) were the most frequent. The clinico-biological characteristics linked to fusion transcripts in a subset of 235 patients (138 adults in the GRAALL2003/05 trials and 97 children from the FRALLE2000 trial) were analyzed to identify their prognosis impact. Patients with HOXA trans-deregulated T-ALLs with MLLT10, KMT2A and SET fusion transcripts (17%, 39/235) had a worse prognosis with a 5-year EFS of 35.7% vs 63.7% (HR = 1.63; p = 0.04) and a trend for a higher cumulative incidence of relapse (5-year CIR = 45.7% vs 25.2%, HR = 1.6; p = 0.11). Fusion transcripts status in T-ALL can be robustly identified by RT-MLPA, facilitating risk adapted treatment strategies for high-risk patients.

Concomitant occurrence of genetically distinct Hodgkin lymphoma and primary mediastinal lymphoma.

Synchronous Hodgkin Lymphoma and Primary Mediastinal B-cell Lymphoma is possible, with molecular analyses proving the absence of clonal filiation between both entities. This suggests a common etiology but the existence of two divergent clones.

Detection of Gene Fusion Transcripts in Peripheral T-Cell Lymphoma Using a Multiplexed Targeted Sequencing Assay.

The genetic basis of peripheral T-cell lymphoma (PTCL) is complex and encompasses several recurrent fusion transcripts discovered over the past years by means of massive parallel sequencing. However, there is currently no affordable and rapid technology for their simultaneous detection in clinical samples. Herein, we developed a multiplex ligation-dependent RT-PCR-based assay, followed by high-throughput sequencing, to detect 33 known PTCL-associated fusion transcripts. Anaplastic lymphoma kinase (ALK) fusion transcripts were detected in 15 of 16 ALK-positive anaplastic large-cell lymphomas. The latter case was further characterized by a novel SATB1_ALK fusion transcript. Among 239 other PTCLs, representative of nine entities, non-ALK fusion transcripts were detected in 24 samples, mostly of follicular helper T-cell (TFH) derivation. The most frequent non-ALK fusion transcript was ICOS_CD28 in nine TFH-PTCLs, one PTCL not otherwise specified, and one adult T-cell leukemia/lymphoma, followed by VAV1 rearrangements with multiple partners (STAP2, THAP4, MYO1F, and CD28) in five samples (three PTCL not otherwise specified and two TFH-PTCLs). The other rearrangements were CTLA4_CD28 (one TFH-PTCL), ITK_SYK (two TFH-PTCLs), ITK_FER (one TFH-PTCL), IKZF2_ERBB4 (one TFH-PTCL and one adult T-cell leukemia/lymphoma), and TP63_TBL1XR1 (one ALK-negative anaplastic large-cell lymphoma). All fusions detected by our assay were validated by conventional RT-PCR and Sanger sequencing in 30 samples with adequate material. The simplicity and robustness of this targeted multiplex assay make it an attractive tool for the characterization of these heterogeneous diseases.

Pan-TRK Immunohistochemistry Is Highly Correlated With NTRK3 Gene Rearrangements in Salivary Gland Tumors.

AIMS: Secretory carcinoma (SC) is characterized by ETV6 rearrangements, most often ETV6-NTRK3 fusion. Given its histologic overlap with other salivary gland tumors (SGTs), SCs can be difficult to diagnose without genetic confirmation. A recently developed pan-TRK (tropomyosin receptor kinase) antibody shows promise for identifying tumors with NTRK (neurotrophic tyrosine kinase receptor 3) fusions. The aim of this study was to evaluate the utility of pan-TRK immunohistochemistry in distinguishing SCs from mimics and selecting patients eligible for TRK inhibitor clinical trials. We examined whole-tissue sections from 111 SGTs with molecular characterization, including 26 SCs (23 with ETV6-NTRK3 fusion and 3 with ETV6-RET fusion detected by ligation-dependent reverse transcription-polymerase chain reaction, next-generation sequencing and 85 non-SC SGTs (no ETV6-NTRK3 fusion). Immunohistochemistry was performed with a pan-TRK rabbit monoclonal antibody. When any pan-TRK staining (nuclear or cytoplasmic with any staining intensity) was considered to indicate positivity, 22 of 23 SCs with ETV6-NTRK3 fusion (95.7%) and 33 of 85 non-SC (38.8%) salivary neoplasms were positive, mainly basal cell adenoma, pleomorphic adenomas, adenoid cystic carcinomas, and epithelial-myoepithelial carcinomas. All SCs with ETV6-RET fusion were entirely negative. When only nuclear pan-TRK staining with any staining intensity was considered positive, 18 of 23 SCs with ETV6-NTRK3 fusion (78.3%) were positive, 11 among them with diffuse staining (>30% of cells). All non-SCs and SCs with ETV6-RET fusion were entirely negative. In comparison to molecular analysis (ligation-dependent reverse transcription-polymerase chain reaction, next-generation sequencing), nuclear pan-TRK IHC has a sensitivity of 78.3% and a specificity of 100% for diagnosing SCs with ETV6-NTRK3 fusion, 69% and 100% for SCs (all fusions). Pan-TRK is a reasonable screening test for diagnosing SCs among SGTs when taking only nuclear staining into account. Although pan-TRK expression is not entirely sensitive for SCs, nuclear staining is highly specific for SCs with ETV6-NTRK3 fusion. The lack of pan-TRK immunoreactivity in a subset of SCs is suggestive of atypical exons 4 to 14 or exons 5 to 14 ETV6-NTRK3 fusion or non-NTRK alternative fusion partners such as ETV6-RET. Pan-TRK staining can serve as a strong diagnostic marker to distinguish SC from it mimics and to select patients eligible for TRK inhibitor clinical trials.

Improving high-resolution copy number variation analysis from next generation sequencing using unique molecular identifiers.

BACKGROUND: Recently, copy number variations (CNV) impacting genes involved in oncogenic pathways have attracted an increasing attention to manage disease susceptibility. CNV is one of the most important somatic aberrations in the genome of tumor cells. Oncogene activation and tumor suppressor gene inactivation are often attributed to copy number gain/amplification or deletion, respectively, in many cancer types and stages. Recent advances in next generation sequencing protocols allow for the addition of unique molecular identifiers (UMI) to each read. Each targeted DNA fragment is labeled with a unique random nucleotide sequence added to sequencing primers. UMI are especially useful for CNV detection by making each DNA molecule in a population of reads distinct. RESULTS: Here, we present molecular Copy Number Alteration (mCNA), a new methodology allowing the detection of copy number changes using UMI. The algorithm is composed of four main steps: the construction of UMI count matrices, the use of control samples to construct a pseudo-reference, the computation of log-ratios, the segmentation and finally the statistical inference of abnormal segmented breaks. We demonstrate the success of mCNA on a dataset of patients suffering from Diffuse Large B-cell Lymphoma and we highlight that mCNA results have a strong correlation with comparative genomic hybridization. CONCLUSION: We provide mCNA, a new approach for CNV detection, freely available at https://gitlab.com/pierrejulien.viailly/mcna/ under MIT license. mCNA can significantly improve detection accuracy of CNV changes by using UMI.

Challenging conventional karyotyping by next-generation karyotyping in 281 intensively treated patients with AML.

Although copy number alterations (CNAs) and translocations constitute the backbone of the diagnosis and prognostication of acute myeloid leukemia (AML), techniques used for their assessment in routine diagnostics have not been reconsidered for decades. We used a combination of 2 next-generation sequencing-based techniques to challenge the currently recommended conventional cytogenetic analysis (CCA), comparing the approaches in a series of 281 intensively treated patients with AML. Shallow whole-genome sequencing (sWGS) outperformed CCA in detecting European Leukemia Net (ELN)-defining CNAs and showed that CCA overestimated monosomies and suboptimally reported karyotype complexity. Still, the concordance between CCA and sWGS for all ELN CNA-related criteria was 94%. Moreover, using in silico dilution, we showed that 1 million reads per patient would be enough to accurately assess ELN-defining CNAs. Total genomic loss, defined as a total loss ≥200 Mb by sWGS, was found to be a better marker for genetic complexity and poor prognosis compared with the CCA-based definition of complex karyotype. For fusion detection, the concordance between CCA and whole-transcriptome sequencing (WTS) was 99%. WTS had better sensitivity in identifying inv(16) and KMT2A rearrangements while showing limitations in detecting lowly expressed PML-RARA fusions. Ligation-dependent reverse transcription polymerase chain reaction was used for validation and was shown to be a fast and reliable method for fusion detection. We conclude that a next-generation sequencing-based approach can replace conventional CCA for karyotyping, provided that efforts are made to cover lowly expressed fusion transcripts.