Background: The diagnosis of lymphoid malignancies primarily relies on histopathologic evaluation, immunophenotypic profiling, and targeted fluorescence in situ hybridization (FISH) in select entities. Structural alterations such as IGH-MYC, IGH-CCND1, and IGH-BCL2 are hallmark events in Burkitt lymphoma, mantle cell lymphoma, and follicular lymphoma, respectively, and their detection by FISH serves as a defining diagnostic criterion. While conventional cytogenetics and FISH have improved diagnostic accuracy, some rearrangements remain cryptic due to structural complexity or non-canonical breakpoints. In clinical practice, this can lead to diagnostic uncertainty when the expected rearrangement is not identified by standard methods. High-throughput chromosome conformation capture (Hi-C) is a genome-wide approach, that interrogates 3D chromatin architecture and can reveal structural variants by capturing and sequencing DNA segments in physical proximity. Here, we present three cases with different outcomes to illustrate the clinical utility of Arima Hi-C technology in uncovering cryptic rearrangements in lymphoid neoplasms and its potential role as a complementary diagnostic tool.

Methods: Unstained formalin-fixed-paraffin-embedded (FFPE) tissue sections from three lymphoma cases were selected for Arima Hi-C analysis. Nucleic acid was extracted, and spatially adjacent DNA segments were ligated, resulting in a genome-wide mapping of interacting genetic loci. Sequencing of those proximity-ligated fragments followed by a bioinformatics pipeline allowed for detection of structural rearrangements and other genomic aberrations. All patients provided informed consent.

Results: Three morphologically challenging cases of newly diagnosed lymphomas with suspected but unconfirmed rearrangements were evaluated.

Case 1. An 81-year-old man presented with PET-avid lymphadenopathy. Axillary lymph node biopsy showed histopathologic findings highly suggestive of Burkitt lymphoma, but break-apart FISH for MYC rearrangement was negative. Hi-C sequencing revealed a cryptic IGH-MYC rearrangement, confirming the diagnosis of Burkitt lymphoma. CNS prophylaxis was added to the treatment regimen based on these results. Despite initial improvement, the patient's course was complicated by multiple comorbidities, and he ultimately succumbed to the disease.

Case 2. A 27-year-old man with a large sacral mass underwent biopsy revealing infiltrating neoplastic cells with high proliferative index and morphology concerning for high-grade B-cell lymphoma or Burkitt lymphoma. FISH for MYC, BCL2, and BCL6 was negative. Arima Hi-C testing identified a structural rearrangement involving BCL3 gene. Although this rearrangement is not immediately actionable, the Hi-C findings confirmed the FISH results and effectively ruled out Burkitt lymphoma, and high-grade B-cell lymphoma with MYC and BCL2 rearrangement or 11q aberration. While BCL3 rearrangements are most often described in chronic lymphocytic leukemia, recent data suggest that lymphomas harboring these rearrangements may represent a distinct clinicopathologic entity. Characterization of additional cases is needed to determine the frequency of BCL3 rearrangements in large B-cell lymphomas withhigh-grade morphology.

Case 3. A 74-year-old female with a history of invasive breast carcinoma presented with a large skull base lesion involving the left posterior sinonasal region. Biopsy revealed involvement by diffuse large B-cell lymphoma (DLBCL), which was negative for BCL2, BCL6, and MYC rearrangement by FISH studies. These findings were confirmed by Hi-C testing, which additionally revealed a deletion on chr. 10 involving PTEN gene, a deletion on chr. 12 involving ETV6, and a rearrangement near the AFAP1L1 and ARHGAP26 genes (chr. 5). Fusions involving ARHGAP26 have been reported in gastric cancer and linked to tumor invasiveness; however, the relevance of these genes in lymphoid malignancies remains unclear. The patient is currently receiving polatuzumab vedotin in combination with CHP-R and shows clinical response to therapy.

Conclusions: Strong histopathologic and clinical suspicion for a rearrangement-defined lymphoma that is not detected by conventional FISH studies should prompt additional testing, particularly when precise subclassification impacts clinical management. Hi-C is a powerful tool to uncover cryptic structural variants in hematologic malignancies and can complement existing diagnostic methods.

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2025
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