iNKT Cells After Allogeneic Stem Cell Transplant | Tomáš Kříž
Reconstitution of Invariant Natural Killer T Cells Post-Allogeneic stem cell transplant: Pilot data for phase I Clinical trial
Authors: Tomáš Kříž (1,2,3), Hana Gmucová (1), Tereza Dekojová (1,2,3), Robin Klieber (1,2,3), Daniel
Lysák (1), Pavel Jindra (1)
Supervisor: Monika Holubová (1,2,3)
(1) Department of Hematology and Oncology, Faculty of Medicine in Pilsen, Charles University and University Hospital, Pilsen (2) Laboratory of Tumor Biology and Immunotherapy, Faculty of Medicine in Pilsen, Charles University (3) Consortium for iNKT Research and Therapy (CiRT)
State-of-the-Art: Invariant natural killer T cells (iNKTs) constitute a distinct subset of T cells,
with attributes of T cells and natural killer (NK) cells, bridging innate and adaptive immunity, with profound immunomodulatory potential. Haematopoietic stem cell transplant (alloSCT) remains the only potentially curative option for several hematological malignancies. Despite advances in optimal donor selection, tailored conditioning regimes, and supportive care, alloSCT is still burdened by high mortality and morbidity. Graft-versus-host disease (GvHD) and relapse are the most frequent and serious post-transplant complications. Based on studies monitoring reconstitution of immune subsets after alloSCT, higher levels of iNKTs positively correlate with lower incidence of GvHD and relapse.
Objective: The study aimed to characterize the kinetics of iNKT cells reconstitution in patients
post alloSCT. Results will serve as a comparator for an ensuing phase I clinical trial, investigating
the administration of allogeneic, donor-derived, ex-vivo expanded iNKTs to prevent GvHD.
Material and Methods: Peripheral blood (PB) iNKTs were monitored in 59 consecutive patients transplanted at our department. INKTs were enumerated 1, 2 and 3 months after alloSCT. PB samples were labeled with anti-CD45, anti-CD3, anti-TCR Vα24-Jα18, anti-TCR Vβ11 antibodies and analyzed using a BD ARIA Fusion flow cytometer. To determine the absolute number of iNKTs per mililiter of blood, the percentages of iNKTs from total leukocyte counts were integrated with the absolute leukocyte count quantified in our standard clinical laboratory. Mann–Whitney test was used for statistical analyses (p≤0.05). The association of GvHD occurrence or relapse with iNKTs levels was tested. The effect of different GvHD prophylaxis drugs on post-transplant iNKT levels reconstitution was evaluated.
Results & Discussion: Patients who developed aGvHD had lower median (m) iNKT cell counts at all 3 time points compared to those without aGvHD. At 1 month, m iNKT counts were 67.16 versus 216.94 (p=0.17), at 2 months 80.44 versus 164.70 (p=0.87), and at 3 months 103.54 versus 228.80 (p=0.85). Similar trend was observed when comparing relapsed and non-relapsed patients. Median iNKT cell counts were 55.4 vs. 467.48 at 1 month (p=0.25), 36.8 vs. 600.7 at 2 months (p=0.09), and 19.85 vs. 320.32 (p=0.01) at 3 months, where the difference reached statistical signifficance. Patients receiving PTCy as GvHD prophylaxis had significantly decreased iNKT cells compared to patients treated with other prophylaxis regimens. This difference was observed at all time points.
Conclusion: Consistent with previous findings, we observed an association between higher
post-transplant iNKT cell counts and a lower incidence of aGvHD or relapse. We also determined
a statistically significant decrease in iNKT cells post-transplant in patients receiving PTCy as GvHD prophylaxis.
Funding: Supported by Ministry of Health of the Czech Republic Conceptual Development of Research Organization (Faculty Hospital in Pilsen – FNPl, 00669806) and the grant of Bone Marrow Foundation.
Study program: Doctoral study – Anatomy, Histology and Embryology | Year of study: 2
ID: 1103
Exome Sequencing in Rare Diseases | Lukáš Strych
Clinical exome sequencing in the diagnostics of rare diseases
Authors: Lukáš Strych (1), Tomáš Zavoral (1)
Supervisor: Ivan Šubrt (1)
(1) Department of Medical Genetics, Faculty of Medicine in Pilsen, Charles University and University
Hospital, Pilsen
State-of-the-Art: Rare diseases comprise a heterogeneous group of over 8000 diseases, predominantly
affecting children. The majority of rare diseases have a genetic origin, and thus, accurate diagnosis is confirmed/ established by genetic testing. Although molecular diagnosis is still challenging, clinical exome sequencing (CES) offers a powerful tool for identifying rare diseases. In some cases, only a candidate variant of unknown significance is identified, which requires further validation of its pathogenicity through additional methods.
Objective: The aim of this study was to identify the genetic cause of rare diseases in pediatric patients and their families with rare diseases of unknown etiology using CES and other methods (if required).
Material and Methods: We performed CES with phenotype-driven analysis of 29 patients and
their families with a rare disease of unknown etiology. Twenty-eight pediatric patients underwent
singleton CES and one fetus trio CES. Causal and candidate sequence variants were subsequently
verified by Sanger sequencing, which was also used for segregation analysis in parents when samples were available. Potential splice site variants were further characterized by RNA analysis (Sanger sequencing and quantitative real-time PCR). Detailed in silico protein analysis was used for candidate missense variants. We also used methylation-sensitive restriction analysis to study the skewed X chromosome inactivation in X-linked diseases.
Results & Discussion: Causal pathogenic variants were found in 7 patients (24%), consisting of
7 sequence variants in UBR1, L1CAM, KDM5C, SPTBN2, POLG, and two structural variants. Two
of the seven sequence variants were splicing variants (novel and previously identified) and five
missense variants (three novel and two previously identified). In other two patients (7%), we
found two candidate sequence variants in VRK1 and SCN3A genes. These are novel missense
variants that cannot be classified as pathogenic (causal) at present.
Conclusion: By combining CES and other methods, we found a genetic cause in 7 patients (24 %). In another two patients (7%), we identified two candidate sequence variants. Identifying a genetic cause enables improvement in genetic counseling for patients and their family members.
Funding: LM2023067, SVV 260773
Study program: Doctoral study – Medical Biology and Genetics | Year of study: 4
ID: 1086
Senescent Cells in Colorectal Cancer | Safaa Andarawi
Senescent cells gene expression in Colorectal Ca ncer in relation to DN A repair capacity and chemotherapy resistance
Authors: Safaa Andarawi (1,2), Zdeněk Hodný (3), Ludmila Vodičková (1,4), Pavel Vodička (1,4)
Supervisor: Pavel Vodicka (1,4)
(1) Biomedical Center, Faculty of Medicine in Pilsen, Charles University (2) Laboratory of Haematooncology, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague (3) Laboratory of Genome Integrity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague (4) Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University
State-of-the-Art: Chemotherapy resistance in colorectal cancer (CRC) is driven by factors like genetic mutations and tumor microenvironment. Recently, the role of senescent cells (SnCs) in chemotherapy resistance has been suggested. These cells exhibit traits such as increased SA-β-gal activity, impaired DNA repaired mechanisms, and resistance to apoptosis, impacting treatment effectiveness. Research indicates SnCs could predict CRC outcomes, aligning with the National Cancer Institute‘s proposed strategy of inducing and removing senescent cells to improve therapy. Our study hypothesizes that the characteristics of SnCs and their relationship to DNA repair capacity may serve as predictive biomarkers for CRC prognosis and chemotherapy responses.
Objective: (1) To investigate how senescent cells affect CRC progression by analyzing SnCs gene expression in tumor and non-cancerous samples from varied responders. (2) To monitor senescent cells in PBMCs for non-invasive diagnostics. (3) To evaluate the influence of dABT-263 (Navitoclax) on 5-FU sensitivity.
Material and Methods: For the first aim of our project, we will measure SASP genes in patients‘ Paired tissues, using RT-PCR. For the second aim, base excision repair DNA damage will be assessed in 100 paired samples using comet assay. For the third aim, the buffy coat will be isolated from patients‘ blood using Ficoll, after which the cells will be fixed and incubated with SA-β-gal. Senescence will then be confirmed using flow cytometry. For the in vitro experiments, we will utilize two cell lines: HT29, which is negative for replication error (RER-)colorectal cancer without mismatch repair deficiency (RER-), and HCT116 (Lynch syndrome), which is positive for replication error (RER+). Both cell lines will be employed to assess the impact of (Navitoclax) drug on the sensitivity to 5-FU.
Results & Discussion: Results from previous CRC NGS project shows that several SnCs genes was differentially expressed in tumors vs adjacent healthy mucosa. In addition, we identified two antiapoptotic genes (BCL9 and BCL2L12) differentially expressed between tumor tissue and control. That might indicate, senescent tumor cells inhibition of apoptosis by upregulating anti-apoptotic BCL2 family members. In our objective to monitor senescent cells in PBMCs, patient exhibited 85% SA-β-GAL activity, compared to 41% in a healthy control. Interestingly, while some patients showed decreased SA-β-GAL activity after tumor removal, others showed increased activity, indicating variability in responses. BER comet assay results shows significant increase in DNA damage in Tumor in comparison to healthy mucosa.
Conclusion: Our preliminary results suggest that chemotherapy resistance stems from therapy-induced senescence (TIS), which upregulates anti-apoptotic BCL2 pathways that protect cancer cells. Inhibiting these proteins with Navitoclax, a senolytic, may reduce resistance and induce apoptosis in cancer cells.
Funding: The authors acknowledge support by the project National Institute for Cancer Research
(Programme EXCELES, ID Project No. LX22NPO5102) – Funded by the European Union – Next Generation EU, and support by AZV NU-21-03-00145 and NU21-03-00506.
Study program: Doctoral study – Experimetnal Surgery | Year of study: 2
ID: 1102
AMACR Stains up to 77.8 % of CCRCCs | Josef Skopal
ALPHA-METHYL COA RACEMASE (AMACR) REACTIVITY ACROSS THE SPECTRUM OF CLEAR CELL RENAL CELL NEOPLASMS
Authors: Josef Skopal (1,3), Pavla Rotterová (3), Reza Alaghehbandan (9), Joanna Rogala (4), Maryna Slisarenko (1,5), Andrea Straková Peteříková (1,3), Květoslava Michalová (1,3), Delia Perez Montiel (6), Mihaela Farcas (1,7), Monika Ulamec (8), Petr Stránský Jr (2), Ondřej Fiala (10,11), Tomáš Pitra (2), Milan Hora (2), Michal Michal (1,3), Kristýna Pivovarčíková (1,3), Ondřej Hes (1,3)
Supervisor: Kristýna Pivovarčíková (1,3)
(1) Department of Pathology, Faculty of Medicine in Pilsen, Charles University and University Hospital, Pilsen (2) Department of Urology, Faculty of Medicine in Pilsen, Charles University and University Hospital, Pilsen (3) Biopticka laborator s.r.o., Pilsen (4) Department of Pathology, University Hospital Wroclaw, Poland (5) Department of Pathology, CSD LAB, Kyiv, Ukraine (6) Department of Pathology, Institute Nacional de Cancerologia, Mexico City, Mexico (7) Onco Team Diagnostic, București, Romania (8) Department of Pathology and Cytology, University Hospital Centre Zagreb, Zagreb, Croatia (9) Robert J. Tomsich Pathology and Laboratory Medicine Institute, Department of Anatomic Pathology, Cleveland Clinic, Cleveland, OH, USA (10) Department of Oncology and Radiotherapeutics, Faculty of Medicine and University Hospital in Pilsen. (11) Biomedical Center, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Clear cell morphology can be seen in a wide range of renal epithelial neoplasms including clear cell renal cell carcinoma (CCRCC), clear cell papillary renal cell tumor (CCPRCT), papillary renal cell carcinoma (PRCC), multilocular cystic renal neoplasm of low malignant potential (MCRNLMP), “MiT family” translocation renal cell carcinomas, renal cell carcinomas with fibromyomatous stroma. Except for “MiT family” translocation renal cell carcinoma and renal cell carcinomas with fibromyomatous stroma, most of the above-mentioned tumors can be frequently diagnosed on pure histologic basis. In challenging cases, particularly in limited samples and metastatic settings, a panel of immunohistochemical stains (i.e., PAX-8, CA-IX) can provide adjunctive diagnostic support.
Objective: The aim of this study was to investigate AMACR immunoreactivity within the spectrum of clear cell renal cell neoplasms including low- and high-grade CCRCC, CCRCC with cystic changes, and MCRNLMP.
Material and Methods: The Plzen tumor registry was searched for clear cell renal tumors to aid in assembling the following four cohorts: low grade (LG) CCRCC, high grade (HG) CCRCC, CCRCC with cystic changes, and MCRNLMP. The diagnostic inclusion criteria were: LG CCRCC with predominately (>90 %) solid growth pattern and WHO/ISUP histologic grade 1–2, HG CCRCC with predominately solid growth (>90 %) pattern and WHO/ISUP histologic grade ≥ 3, CCRCC with cystic changes (>50 % of the tumor volume), and MCRNLMP as per the WHO definition . All tumors were reviewed and the diagnosis was confirmed by two urologic pathologists. For each tumor, two representative blocks were stained for AMACR using two different clones. Immunohistochemical staining evaluation was performed independently by three pathologists.
Results & Discussion: Clear cell renal cell carcinoma is a morphologically and immunohistochemically heterogeneous neoplasm. Majority of CCRCCs can solely be diagnosed on H&E basis (or with a limited immunohistochemical panel). However, in diagnostically difficult cases, particularly in limited samples and metastatic settings, immunohistochemistry plays more important role. Data on AMACR reactivity in CCRCC is limited, with variable and conflicting findings. There were at least some AMACR immunoreactivity in 77.8 % CCRCCs . Moderate to strong positivity, or positivity in more than one third of the tumor was detected in 48.9 % of CCRCC using OV-TL12/30 clone. Strong and diffuse AMACR positivity was detected in 8.9 % of all CCRCCs. AMACR immunoreactivity in MCRNLMP was up to 37.5 %.
Conclusion: We demonstrated relatively high expression rate of AMACR in CCRCC, while very variable in intensity and distribution. This finding may have diagnostic implications especially in limited samples (i.e., core biopsies), as AMACR positivity does not exclude the diagnosis of CCRCC.
Funding: This work was supported by the Cooperatio Program, Research Area SURG, the Institutional
Research Fund FN 00669806, and SVV 260652 from the Ministry of Education, Czech Republic.
Study program: Doctoral study – Pathology | Year of study: 3
ID: 1085