Fluorescence in situ hybridization (FISH) with DNA probes allows the visualization of gene copy number and localization of specific DNA targets with fluorescence microscopy. Cells in culture, metaphase chromosomes, and tissue sections are fixed and prepared on glass slides. Both the DNA in the cells and fluorescently labeled probe are denatured, and the labeled probe is allowed to hybridize to the cellular DNA. The slides are washed, counterstained, and viewed via fluorescence microscopy. We describe the basic method for preparing slides and probes for studies involving DNA copy number changes and structural chromosome rearrangements in formalin-fixed paraffin-embedded (FFPE) tissue sections and cell culture preparations.

Background: Double-protein-expression lymphomas (DEL) is a unique sub-group of diffuse large B-cell lymphoma (DLBCL) with co-expression of MYC and BCL-2 on immunohistochemistry. Compared with classic DLBCL, DEL has a lower response to standard induction chemoimmunotherapy with rituximab added to cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and poorer prognosis. Autologous stem cell transplant (ASCT) is a potentially curative treatment of high-risk of relapsed/refractory lymphomas. However, the role of ASCT in patients with DEL is controversy. Our pre-clinic and clinic studies showed the combination of chidamide, cladribine, gemcitabine and busulfan (ChiCGB) worked synergistically against lymphomas. Aims: So, in this study, we used ASCT conditioning with ChiCGB as consolidation in patients with DEL in first or second complete remission. Methods: This study was a phase 2 trial done at the West China Hospital of Sichuan University from May. 2016 to Dec. 2018. Eligible patients were aged 16-65 years and had received R-CHOP or chidamide-R-CHOP as first-line or any second-line therapies. They achieve first or second complete remission (CR) before transplant. The DEL was diagnosed as MYC expression ≥ 40% and BCL-2 ≥ 50% on immunohistochemistry. Additional eligibility criteria were an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2, adequate major organs function, no active hepatitis bacteria, fungal or virus infection. Patients provided written informed consent to participate in the trial. Patients with DLBCL without MYC and BCL-2 over-expression were assigned to the control group (non-DEL). Patients received ChiCGB conditioning regimen consisting of oral chidamide 30 mg on days −7, −4, 0, +3, intravenous cladribine at a dose of 6 mg/m 2 from days −6 to −2, intravenous gemcitabine 4 hours after cladribine at a dose of 2500 mg/m 2 on days −6 and −2, and intravenous busulfan at a fixed dose of 3.2 mg/kg from days −6 to −2. Dexamethasone was prophylactically given for adverse effect of high dose gemcitabine from the day −6 to −2. Peripheral-blood stem-cells were infused on day 0.Results: A total of 44 patients with DLBCL were enrolled, with 22 DEL and 22 non-DEL. The median age was 46 (range, 23 to 61) and 34.5 (range, 16 to 61) years in DEL and non-DEL groups. There were 8 (36.3%) and 12 (54.5%) patients in second CR in DEL and non-DEL group respectively. In DEL group, 5 patients receive chidamide-R-CHOP as first-line therapy. Stem cells source was peripheral blood. Median infused CD34+cells count was 2.2 × 10 6 /Kg (range, 1.0 to 11.6 × 10 6 /Kg). Neutrophils and platelets were engrafted on a median day +10 (+8 to +14) and day +12 (range, days +8 to +38), respectively. The median follow-up was 17.8 (range, 4.1 to 34.9) and 20 (range, 3 to 46.3) months in DEL and non-DEL group. The 2-year progression free survival of patients with DEL or non-DEL was 94.4% and 84.2% (P = 0.3731), while the 2-year overall survival of tow groups was 94.1% and 100% (P = 0.2904) (Figure -1). Summary/C...