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.

Single Capture High Throughput Sequencing Assay for Combined V(D)J Clonality Analysis and Oncogene Mutations in the Diagnosis of T and B Lymphoid Malignancies

Introduction The diagnosis of B and T cell malignancies relies on the demonstration of B-cell (BCR) or T-cell (TCR) antigen receptor clonality. This can be studied through the analysis of V(D)J rearrangements of BCR and TCR genes by PCR (van Dongen Leukemia 2003) or, more recently, by high-throughput sequencing (HTS). Amplification of a clonal population with a "primers approach" could fail in case of hybridization problems due to too fragmented DNA, somatic mutations or polymorphic variations. Here we evaluated the performance of a HTS capture system for the analysis of B and T-cell clonality in clinical samples from mature T or B malignancies. We further combined this technology to concomitant sequencing of oncogenes of interest. Patients and Methods DNA was extracted from 58 tumoral samples from fresh/frozen (FF) cells or tissues or formalin-fixed paraffin-embedded tissue (FFPE) (n=19). These samples comprised various T-cell [i.e. 1T-cell prolymphocytic leukemia, 1 T large granular lymphocytic leukemia, 2 Sézary syndrome, 4 peripheral T-cell lymphoma not otherwise specified, 14 angioimmunoblastic T-cell lymphoma] or B-cell [i.e. 14 chronic lymphocytic leukemia, 1 mantle cell lymphoma, 5 diffuse large B-cell lymphoma, 1 grey-zone lymphoma, 13 Hodgkin lymphoma, 1 Poppema, 2 Waldenström and 1 multiple myeloma] malignancies. The Biomed-2 PCR technique was used as standard for assessing the performance of TRG, IGH and IGK clonality analysis. An extensive panel of capture probes was designed (SureSelect XT HS2 DNA system, Agilent Technologies) that covered the variable (V), + diversity (D) and junction (J) segments of the IGH, IGK, TRG, TRB loci and diagnostic/theranostic genes of interest i.e. B2M, BTK, CARD11, CD28, DNMT3A, IDH2, JAK3, PLCG1, PLCG2, ROHA, SOCS1, STAT3, STAT5B, STAT6, TET2, TNFAIP3, TP53. Paired-End sequencing was performed on a MiSeq system (Illumina) in 300, 500 and 600 cycles. Analysis of clonality profiles was performed using Vidjil software and SeqOne. Results HTS runs resulted in a median total read count of 1,6M (0.7-2.9) per sample. V(D)J rearrangements were identified with a median of 1503 reads (189-6824) per sample. Five samples were excluded because less than 300 rearranged reads were obtained. The number of rearranged reads and of clonotypes identified are influenced by the number of sequencing cycles (300<500 or 600) but not by the quality of DNA (FFPE vs FF). Analyses of tumoral samples with HTS versus PCR were compared. For the IGH locus (n=47), comparable PCR/HTS clonal (n=22) and polyclonal (PCL, n=20) profiles were identified. One discordant case showed a clonal PCR profile and a PCL HTS profile but the IGK was clonal. For the IGK locus (n=23), 10 clonal and 12 PCL cases were similar with both techniques. One case appeared discordant with a PCL PCR profile but a clonal HTS profile. For the TRG locus (n=31), PCR and HTS profiles were similar in 14 clonal, 5 oligoclonal and 9 PCL cases respectively. Three cases were discordant with oligoclonal PCR profiles but a clonal HTS profile. Overall in the 38 cases of B-cell malignancies, 27 and 11 cases had a concordant B-cell clonal or PCL profile with PCR and HTS. Among PCL cases, only one was discordant with a clonal HTS profile. This case and 3 other PCL cases were Hodgkin lymphomas which all disclosed another mutation (i.e. TP53, TNFAIP3, SOCS1). Of the 20 cases of T-cell malignancies, 14 displayed a clonal TRG profile with PCR and HTS. Among them, 13 showed oncogene mutations that confirmed the oncogenic nature of the clonal proliferation. Among 6 patients with a non-clonal PCR TRG profile, two cases of AITL and T-LGL had a discordant clonal TRG HTS profile and both also had specific mutations (SOCS1, RHOA and STAT3 respectively). Two other AITL samples showed a T-PCL profile with PCR and HTS but also had a mutation/CNV (RHOA, SOCS1). Conclusion A very good performance of B and T cell clonality assessment was obtained here with capture-HTS compared to Biomed-2 PCR. The combined identification of mutation/CNV allowed to confirm the malignant character in cases of clonal or PCL lymphoproliferations, while concomitantly specifying the type of lymphoproliferative disorder. The combined capture-HTS of B and T repertoires and oncogenes of diagnostic or theranostic interest thus appears as an efficient, accurate and useful approach for the diagnosis of mature B and T lymphoid malignancies in clinical practice. Disclosures No relevant conflicts of interest to declare.

805. Outbreak of Ralstonia pickettii Bacteremia Caused by Contaminated Hydromorphone in a Universitary Hospital in Bogota, Colombia

Background Ralstonia pickettii are aerobic non fermenter gram negative bacilli isolated in water and soil. It is related to nosocomial infection outbreaks and considered an opportunistic pathogen. There have been outbreaks reports due to contaminated water systems and sterile drug solutions which mainly occurs during manufacturing. We present the report of an outbreak of R. pickettii bacteremia secondary to a contamination of hydromorphone vials. Methods In February 2021 an outbreak of R. pickettii bacteremia was identified. All isolates were from blood cultures with slow growth, thus indicating the culturing of liquid inputs, intravenous administration solutions and commonly used drugs among patients including hydromorphone. Mass spectrometry (MALDI-TOF) was used for the identification and automated microdilution to determine sensitivity to antimicrobials of the isolates and clonality analysis of genetic relationships was carried out using the DICE coefficient, UPGMA algorithm Results During the outbreak, 19 patients with R. pickettii bacteremia were identified The global attack rate was 1,9%. 11/19 (58%) were women and 13/19 (68%) of the isolations were from inward patients and 6/19 (32%) were from intensive care unit. Factors that could contribute to the appearance of the outbreak were underlying pathology, 2 patients with a diagnosis of diabetes mellitus, 10 patients with a diagnosis of arterial hypertension, 5 patients with obesity, 6 patients with heart disease, additionally 7 patients with a diagnosis of SARS COV 2 and 6 patients with the use of corticosteroids. The global attack rate was 1,9% and mortality was 31.5% (6 patients). R. pickettii was identified from two batches of hydromorphone by MALDI-TOF and the clonality study concluded that the isolates analyzed, were clonal with a 100% similarity. The associated mortality rate was 5/29 (26.3%). Conclusion We confirmed an outbreak of R. pickettii due to the contamination of two hydromorphone badges in Colombia. It is crucial to acknowledge the importance of infection control and surveillance during the COVID-19 pandemic as well as maintaining adequate quality control of medication production in order to avoid presenting this kind of outbreaks. Disclosures Sandra Liliana. Valderrama-Beltrán, MD, MSc, Biotoscana (Speaker’s Bureau)MSD (Grant/Research Support, Scientific Research Study Investigator, Research Grant or Support, Speaker’s Bureau)Pfiezer (Speaker’s Bureau)

Expansion of tumor-infiltrating lymphocytes with substantial stem cell properties from vulvar cancer

Tumor-infiltrating lymphocyte (TIL) therapy has been clinically proved as a promising therapeutic approach for patients with solid tumor. TIL therapy could effectively control tumor growth in cervical cancer as indicated by a phase 2 pivotal trial with an objective response rate of 44.4%. Vulvar cancer is believed to share a similar biological and immunological phenotype with cervical cancer. However, the therapeutic potential of TIL in vulvar cancer remains to be explored. In this study, we described a manufacturing procedure that can expand clinical-scale TILs from both vulvar cancer and cervical cancer with a high success rate. Characterization of the phenotype of TIL populations showed that TILs from vulvar cancer are prone to maintaining a higher percentage of progenitor-like phenotype and have stronger tumor-killing capacity compared to TILs from cervical cancer. TCR clonality analysis indicated that all TIL samples have more enriched TCR clones than PBMC, which might be expanded during anti-tumor responses and tend to be patient specific. Thus, our study provides a feasible method of TIL preparation from and a potential new therapeutic strategy for vulvar cancer patients.

Evaluation of a worldwide EQA scheme for complex clonality analysis of clinical lymphoproliferative cases demonstrates a learning effect

Clonality analysis of immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements is routine practice to assist diagnosis of lymphoid malignancies. Participation in external quality assessment (EQA) aids laboratories in identifying systematic shortcomings. The aim of this study was to evaluate laboratories’ improvement in IG/TR analysis and interpretation during five EQA rounds between 2014 and 2018. Each year, participants received a total of five cases for IG and five cases for TR testing. Paper-based cases were included for analysis of the final molecular conclusion that should be interpreted based on the integration of the individual PCR results. Wet cases were distributed for analysis of their routine protocol as well as evaluation of the final molecular conclusion. In total, 94.9% (506/533) of wet tests and 97.9% (829/847) of paper tests were correctly analyzed for IG, and 96.8% (507/524) wet tests and 93.2% (765/821) paper tests were correctly analyzed for TR. Analysis scores significantly improved when laboratories participated to more EQA rounds (p=0.001). Overall performance was significantly lower (p=0.008) for non-EuroClonality laboratories (95% for IG and 93% for TR) compared to EuroClonality laboratories (99% for IG and 97% for TR). The difference was not related to the EQA scheme year, anatomic origin of the sample, or final clinical diagnosis. This evaluation showed that repeated EQA participation helps to reduce performance differences between laboratories (EuroClonality versus non-EuroClonality) and between sample types (paper versus wet). The difficulties in interpreting oligoclonal cases highlighted the need for continued education by meetings and EQA schemes.

Detection of Oxazolidinone Resistance Genes and Characterization of Genetic Environments in Enterococci of Swine Origin, Italy

One hundred forty-five florfenicol-resistant enterococci, isolated from swine fecal samples collected from 76 pig farms, were investigated for the presence of optrA, cfr, and poxtA genes by PCR. Thirty florfenicol-resistant Enterococcus isolates had at least one linezolid resistance gene. optrA was found to be the most widespread linezolid resistance gene (23/30), while cfr and poxtA were detected in 6/30 and 7/30 enterococcal isolates, respectively. WGS analysis also showed the presence of the cfr(D) gene in Enterococcus faecalis (n = 2 isolates) and in Enterococcus avium (n = 1 isolate). The linezolid resistance genes hybridized both on chromosome and plasmids ranging from ~25 to ~240 kb. Twelve isolates were able to transfer linezolid resistance genes to enterococci recipient. WGS analysis displayed a great variability of optrA genetic contexts identical or related to transposons (Tn6628 and Tn6674), plasmids (pE035 and pWo27-9), and chromosomal regions. cfr environments showed identities with Tn6644-like transposon and a region from p12-2300 plasmid; cfr(D) genetic contexts were related to the corresponding region of the plasmid 4 of Enterococcus faecium E8014; poxtA was always found on Tn6657. Circular forms were obtained only for optrA- and poxtA-carrying genetic contexts. Clonality analysis revealed the presence of E. faecalis (ST16, ST27, ST476, and ST585) and E. faecium (ST21) clones previously isolated from humans. These results demonstrate a dissemination of linezolid resistance genes in enterococci of swine origin in Central Italy and confirm the spread of linezolid resistance in animal settings.