Institutional surgical pathology archives were queried from 2016 to 2022. Cases selected for inclusion were required to have available formalin-fixed-paraffin-embedded (FFPE) tissue and evidence of CLL/SLL by compelling clinical, histologic, and/or flow cytometry evidence, as determined by a pathologist (MM) upon review of pathology reports and the electronic medical record. Select cases with what was previously described as low-grade or “classic” morphology were chosen (cases 1–5), with available whole-tissue sections (as opposed to core biopsies). All cases with previously documented atypical/accelerated/”high-grade” features or known Richter transformation were included. In cases with a predominance of large cells, diffuse large B-cell-like morphology, or “Richter transformation” noted in the pathology report, patients were required to have a well-documented history of CLL/SLL (by prior documented flow cytometry or histology) to be included. Cases of transformation with non-DLBCL morphology (Hodgkin lymphoma, plasmablastic lymphoma, etc.) were not included.

Clinical information was extracted from the medical record, as available. Rai/Binet/Ann Arbor staging was not consistently documented at the time of biopsy and therefore was not included for consideration. Results (prior and concurrent) of TP53 mutation testing, IgHV hypermutation testing, karyotype analysis, and fluorescence in situ hybridization (FISH) performed on peripheral blood were noted when available.

For all cases, an H&E slide and IHC stains for CD3 (Leica Biosystems, Clone LN10), CD20 (Leica Biosystems, L26), CD5 (Leica Biosystems, 4C7), CD23 (Leica Biosystems, 1B12), LEF1 (Abcam, EPR2029Y), LAG3 (Abcam, EPR4392), C-MYC (Epitomics, EP121), PD1 (Abcam, NAT105), MUM1 (Leica Biosystems, eau32), Cyclin D1 (Leica Biosystems, P2D11F11), BCL-6 (Leica Biosystems, LN22), p53 (Dako, M7001 or Leica Biosystems, D0-7), and Ki-67 (Leica Biosystems, MM1 or Abcam, Ab16667) were available for review. Three board-certified hematopathologists (EC, IO, and NA) with daily practice experience in lymphoma diagnosis independently reviewed the H&E and IHC stains for each case. The reviewing pathologists were blinded to the clinical information related to the case, the prior rendered diagnosis (i.e., the diagnosis on record rendered for clinical care), and the interpretation of the other pathologists. In cases in which the reviewing pathologist was also the historic pathologist, a wash-out period of at least one month was employed.

Evaluations were performed on BX51 and BX53 Olympus microscopes (all with 20 × objective of CN20x/0.50 and an ocular WHN 10x/22, Evident/Olympus Scientific Solutions). Reviewers were required to score cases using a standardized worksheet with a checklist of H&E and IHC features. Reviewers documented the quality of the H&E preparation, the presence/absence of identifiable proliferation centers, and the presence/absence of expanded (broader than 20 × field) or confluent proliferation centers, according to the criteria outlined by Giné et al. [5]. An average mitotic rate (by H&E examination) across 10 proliferation centers was calculated, if proliferation centers were deemed identifiable. If proliferation centers were not readily identifiable, an average mitotic index was calculated across 10, 40x (0.238 mm2) high-power fields. DLBCL/RT was defined as diffuse sheets or nodules of large cells, in keeping with DLBCL morphology outlined in the World Health Organization Classification of Haematolymphoid Tumours, 5th edition [1].

IHC stains for CD20, CD5, CD23, LEF1, LAG3, C-MYC, PD-1, MUM1, BCL-6, and Cyclin D1 were scored as diffusely positive, weakly/partially positive, or negative in lesional B cells. In addition, reviewers documented whether LEF1, LAG3, C-MYC, PD-1, MUM1, BCL-6, or Cyclin D1 had differential staining in proliferation centers compared to background lesional cells. p53 staining was scored as the percentage of positive nuclei in lesional B-cells, as averaged across 10, 40 × high-power fields. Ki-67 was scored in intervals (< 5%, 5–20%, 21–40%, 41–60%, 61–80%, and 81–100%) inside and outside of proliferation centers, when proliferation centers were identifiable. For cases in which the pathologist did not feel they could confidently delineate proliferation centers, an overall estimate of the Ki-67 proliferation index was assigned (< 5%, 5–20%, 21–40%, 41–60%, 61–80%, and 81–100%). Reviewers were provided a free-text field to provide additional comments about each case. Finally, they were required to select the diagnosis that they felt best aligned with the H&E and IHC findings from the following options: (1) CLL/SLL, no evidence of aggressive features, (2) CLL/SLL with expanded and/or confluent proliferation centers or increased Ki-67, or (3) large cell (Richter) transformation/DLBCL.

DNA extraction was performed on FFPE tissue from select cases using a QIAamp DNA FFPE tissue kit, per manufacturer specifications. OncoScan microarray (OncoScan, ThermoFisher) and PGDx elio™ tissue complete next-generation sequencing (NGS, 505 gene panel) analyses were performed on FFPE samples from select cases using manufacturer’s recommended protocols. Analysis of OncoScan and PGDx data was performed by molecular pathology faculty according to professional guidelines [19, 20].