Bulky Lesions Research Articles

Synthetic Lesions with a Fluorescein Carbamoyl Group As Analogs of Bulky Lesions Removable by Nucleotide Excision Repair: A Comparative Study on Properties.

Mammalian nucleotide excision repair (NER), known for its broad substrate specificity, is responsible for removal of bulky lesions from DNA. Over 30 proteins are involved in NER, which includes two distinct pathways: global genome NER and transcription-coupled repair. The complexity of these processes, the use of extended DNA substrates, and the presence of bulky DNA lesions induced by chemotherapy have driven researchers to seek more effective methods by which to assess NER activity, as well as to develop model DNAs that serve as efficient substrates for studying lesion removal. In this work, we conducted a comparative analysis of model DNAs containing bulky lesions. One of these lesions, N-[6-{5(6)-fluoresceinylcarbamoyl}hexanoyl]-3-amino-1,2-propanediol (nFluL), is known to be efficiently recognized and excised by NER. The second lesion, N-[6-{5(6)-fluoresceinylcarbamoyl}]-3-amino-1,2-propanediol (nFluS), has not previously been tested as a substrate for NER. To evaluate the efficiency of lesion excision, a 3'-terminal labeling method was employed to analyze the excision products. The results showed that nFluS is removed approximately twice as efficiently as nFluL. Comparative analyses of the effects of nFluL and nFluS on the geometry and thermal stability of DNA duplexes - combined with spectrophotometric and spectrofluorimetric titrations of these DNAs with complementary strands - were performed next. They revealed that the absence of an extended flexible linker in nFluS alters the interaction of the bulky fluorescein moiety with neighboring nitrogenous bases in double-stranded DNA. This absence is associated with the enhanced efficiency of excision of nFluS, making it a more effective synthetic analog for studying bulky-lesion removal in model DNA substrates.

STK19 facilitates the clearance of lesion-stalled RNAPII during transcription-coupled DNA repair.

Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and transcription factor IIH (TFIIH) around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryoelectron microscopy (cryo-EM) and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.

A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function.

Cockayne syndrome (CS) is a premature ageing condition characterized by microcephaly, growth failure, and neurodegeneration. It is caused by mutations in ERCC6 or ERCC8 encoding for Cockayne syndrome B (CSB) and A (CSA) proteins, respectively. CSA and CSB have well-characterized roles in transcription-coupled nucleotide excision repair, responsible for removing bulky DNA lesions, including those caused by UV irradiation. Here, we report that CSA dysfunction causes defects in the nuclear envelope (NE) integrity. NE dysfunction is characteristic of progeroid disorders caused by a mutation in NE proteins, such as Hutchinson-Gilford progeria syndrome. However, it has never been reported in Cockayne syndrome. We observed CSA dysfunction affected LEMD2 incorporation at the NE and increased actin stress fibers that contributed to enhanced mechanical stress to the NE. Altogether, these led to NE abnormalities associated with the activation of the cGAS/STING pathway. Targeting the linker of the nucleoskeleton and cytoskeleton complex was sufficient to rescue these phenotypes. This work reveals NE dysfunction in a progeroid syndrome caused by mutations in a DNA damage repair protein, reinforcing the connection between NE deregulation and ageing.

Liver Transplantation for Intrahepatic Cholangiocarcinoma After Chemotherapy and Radioembolization: An Intention-To-Treat Study.

Liver transplantation (LT) is a potentially curative experimental treatment for unresectable intrahepatic cholangiocarcinoma (iCC). Pre-transplant downstaging may help defining tumor aggressiveness and drive patient selection. We report the preliminary results of LT for liver-limited unresectable iCC after sequential downstaging with systemic chemotherapy and radioembolization (SYS-TARE). In case of sustained disease stability after SYS-TARE, patients underwent surgical nodal sampling and, if negative, were listed for LT. In this study, 13 patients with unresectable iCC underwent downstaging with SYS-TARE. The median age was 70years and 77% were female. All had single bulky lesions at diagnosis. After SYS-TARE, 9 (69%) dropped out: 3 due to progressive disease after TARE with no response to second-line, 4 due to extrahepatic disease development and 2 due to positive nodal disease at pre-listing abdominal exploration. The median OS after dropout was 11.5 months. Four (31%) were successfully listed and transplanted. At pathology, viable tumor ranged from 30% to less than 5%. All four patients are alive and disease-free at 73, 40, 12, and 8months from LT. LT for unresectable iCC after downstaging with SYS-TARE appears to select suitable patients for LT, achieving optimal oncological outcomes in case of response to therapy and no lymphnodal spread.

Strand dependent bypass of DNA lesions during fork reversal by ATP-dependent translocases SMARCAL1, ZRANB3, and HLTF.

During DNA replication, the replisome encounters obstacles including DNA lesions, transcription-replication conflicts, and other sources of replication stress. These obstacles must be efficiently overcome to complete DNA synthesis and minimize genome instability. One pathway to tolerate replication stress is replication fork reversal, in which parental template DNA strands are reannealed and a nascent-nascent DNA duplex is formed. Several enzymes promote replication fork reversal, including the ATP-dependent translocases SMARCAL1, ZRANB3, and HLTF. How these enzymes translocate on DNA that contains fork-stalling lesions is unknown. Here, we examined the abilities of SMARCAL1, ZRANB3, and HLTF to tolerate various lesions on leading or lagging template strands. We demonstrate that SMARCAL1 and ZRANB3 are selectively inhibited by lesions on the leading template strand, whereas HLTF is insensitive to bulky lesions on either strand. These results suggest that SMARCAL1 and ZRANB3 contact the leading strand during fork reversal and therefore are more sensitive to inhibition by bulky lesions on this strand. In contrast, HLTF DNA translocation is inherently insensitive to DNA lesions. These biochemical differences between the fork reversal enzymes provide insights into their mechanism of DNA remodeling and suggest they may act in lesion-specific contexts.

Histone variant H2A.Z is needed for efficient transcription-coupled NER and genome integrity in UV challenged yeast cells.

The genome of living cells is constantly challenged by DNA lesions that interfere with cellular processes such as transcription and replication. A manifold of mechanisms act in concert to ensure adequate DNA repair, gene expression, and genome stability. Bulky DNA lesions, such as those induced by UV light or the DNA-damaging agent 4-nitroquinoline oxide, act as transcriptional and replicational roadblocks and thus represent a major threat to cell metabolism. When located on the transcribed strand of active genes, these lesions are handled by transcription-coupled nucleotide excision repair (TC-NER), a yet incompletely understood NER sub-pathway. Here, using a genetic screen in the yeast Saccharomyces cerevisiae, we identified histone variant H2A.Z as an important component to safeguard transcription and DNA integrity following UV irradiation. In the absence of H2A.Z, repair by TC-NER is severely impaired and RNA polymerase II clearance reduced, leading to an increase in double-strand breaks. Thus, H2A.Z is needed for proficient TC-NER and plays a major role in the maintenance of genome stability upon UV irradiation.

478. OVERVIEW ON USE OF THE TRANSCERVICAL MEDIASTINOSCOPIC APPROACH FOR MINIMALLY INVASIVE TREATMENT OF ESOPHAGEAL CANCER: ESTABLISHMENT OF INTERNATIONAL COLLABORATIVE GROUP

Abstract Background Minimally invasive esophagectomy has emerged as a promising alternative to open surgery, demonstrating reduced postoperative complications and comparable oncological outcomes. Despite advancements, pulmonary complications and anastomotic leakages remain concerns. Since 2015, a novel technique combining single-port transcervical and trans-hiatal laparoscopic mediastinal dissection for esophageal resection was introduced. This procedure exhibits lower pulmonary complication rates compared to traditional approaches. However, its global adoption is limited, necessitating collaborative efforts to standardize techniques and outcomes. Following the IDEAL framework stage 2B (exploration), an international collaborative group for transcervical esophagectomy has been established, aiming to exchange insights, experiences, and lessons learned. Methods A cross-sectional survey involving 13 surgeons currently practicing transcervical esophagectomy was conducted, revealing variations in surgical approaches, indications, and outcomes. Results Surgeons from China, Germany, Japan, and The Netherlands participated. Indications for surgery varied, with less selectivity in higher-volume centers. Large tumors near the carina, bulky primary lesions or T4 tumors were identified as contraindications. Different surgical approaches were reported, with the left transcervical and abdominal laparoscopic/robotic dissection being the most common. The mean operative time was about 382.2±117.9 minutes. The mean blood loss was of 100.2±71.1 milliliters. Surgeons reported high rates of R0 resection and lymph node harvesting, as well as low rates of pulmonary complications (8.6±3.7) and anastomotic leaks (10.1±5.2); however, concerns persist regarding recurrent laryngeal nerve injuries (27.2±18.2). Conclusion Transcervical esophagectomy is gradually gaining recognition, with varying adoption rates. The collaborative group aims to align techniques, share experiences, and standardize the procedure. Challenges include differences in indications, surgical approaches, and outcomes, highlighting the need for ongoing discussion and research. Despite concerns about recurrent laryngeal nerve injuries, the procedure shows promise in reducing pulmonary complications. The establishment this collaborative group signifies a crucial step toward global acceptance. By fostering collaboration, the group aims to enhance patient care, minimize learning-associated morbidity, and contribute to the continuous advancement of this surgical technique. Future efforts will focus on standardization, comparative studies, and training protocols.

Evaluation of the incidence, predictors, risk assessment scores and outcomes of thromboembolism in a cohort of Egyptian NHL patients - Real World Experience

Non-Hodgkin’s Lymphoma (NHL) is the most common subtype of lymphoma. The incidence of venous thromboembolism (VTE) in aggressive NHL was estimated recently to be 11%. Several risk assessment scores and factors are available to help identify cancer patients at risk for developing VTE. Patients with a pathologically confirmed diagnosis of NHL were identified at the Oncology Center of Mansoura University. The study included 777 patients: 719 with DLBCL-NOS, 26 with Anaplastic-B-cell, and 32 with T-cell-rich-NHL. Data were retrospectively collected from electronic medical records, including clinical, radiological, and laboratory information related to VTE and NHL. The median age at NHL diagnosis was 53 years, (range: 18–98). There was a male predominance, 51.4% of the cases. At initial lymphoma diagnosis, VTE was identified in 46 (5.9%) patients, and 61 (7.9%) patients experienced VTE while undergoing chemotherapy. According to logistic regression analysis, a PS (performance status) ≥ 2, bulky lesions, and mediastinal masses were significant predictors of VTE at presentation, with P-values of 0.022, 0.002, and < 0.001, respectively. Meanwhile, NHL patients who developed VTE during chemotherapy had significantly poorer PS, higher absolute neutrophilic counts (ANC), neutrophil/lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR) and lactate dehydrogenase (LDH) levels than lymphoma patients without VTE, with P-values of 0.003, 0.034, 0.049, 0.01 and 0.007, respectively, as determined by multivariate analysis. The ROC curve identified the cut-off values of 4.875 × 109/L for ANC, 2.985 for NLR, 144.85 for PLR, and 417.5 U/L for LDH as potential markers for predicting VTE in NHL patients. Patients with a PS ≥ 2 and values exceeding these cut-offs for ANC, NLR, and PLR experienced significantly higher incidences of VTE than other groups, with P-values of 0.003, < 0.001, < 0.001, and < 0.001, respectively. At the end of the follow-up, the overall survival was significantly shortened by VTE occurring during chemotherapy, hypoalbuminemia, intermediate-high and high international prognostic index (IPI) scores (intermediate-high and high), responses other than CR and relapse, all with P-values < 0.05. ECOG PS and Inflammatory markers such as NLR, PLR, and neutrophilic count could serve as predictors of the development of thrombotic events in patients with NHL-DLBCL. Additionally, the occurrence of VTE during chemotherapy is an independent poor prognostic marker for overall survival (OS).

STK19 facilitates the clearance of lesion-stalled RNAPII during transcription-coupled DNA repair.

Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and TFIIH around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a new TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryo-EM and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.

A study of a new mechanism: Intracellular retention allo-CART safety and efficacy for extranodal bulky B-NHL.

e19007 Background: Extranodal bulky lesions, a significant adverse prognostic factor in lymphoma, commonly denote a more aggressive disease course, mandating prompt and intensified treatment strategies. In response, we developed ThisCART19A, a non-genetic editing and off-the-shelf anti-CD19 CAR T-cell therapy incorporating intracellular retention of membrane proteins, representing a novel approach that downregulates surface expression of TCRαβ/CD3 complexes. ThisCART19 has exhibited a favorable safety profile and promising efficacy in patients with relapsed/refractory bulky B-cell Non-Hodgkin Lymphoma. Methods: This is an investigator-initiated trial employing an open-label, dose escalation, and expansion design to evaluate the safety and efficacy of ThisCART19A in patients with relapsed or refractory bulky B-cell non-Hodgkin lymphoma (B-NHL). Bulky disease was defined as the longest diameter of the mass &gt;5 cm at baseline. Results: Between June, 2021, and June, 2023, 13 extranodal bulky disease were enrolled in the studies and successfully received ThisCART19A. The median age was 56 (range, 37~67) years. All 13 patients had 3 median prior lines of therapy (range, 2-5). All patients received anti-CD20 and multi- agent chemotherapy. 8 (61.54%) patients had prior auto CART. mSPD was 48 (range, 16~162) cm². mTMTV was 114.9 (range, 14~483) ml. As of February 2024, The most common adverse events were CRS (100%) , infections (30.77%), ICANS (23.08%) and cytopenias(100%); This is no grade 3 or higher CRS occurred;only 1(7.69%)patient experienced grade 4 ICANS and relieved after intravenous dexamethasone treatment. This is no treatment-related deaths occurred. Among the 13 patients, 10 were evaluable, with an ORR and CR of 90% and 80%, respectively, at 28 days post-infusion. mPFS was 62 days (range, 28~227). The patient who achieved a PR received chemotherapy and radiotherapy as consolidation, As of February 5th, 2024, the patient has survived for 263 days and is still under follow-up. Additionally, two patients progressed after CR received sequential chemotherapy and BCL-2 Inhibitor, with an average OS of 326.5 days, still being under follow-up. In 13 evaluable patients, the mean C-max was 1043.13 cells/μL(range, 0.59~10045.4), and the mean AUC0-28d was 1542.23 cells/μL × day(range, 0.91~11724).In this cohort, two patients received low-dose infusions at 1×106 cells/kg and still achieved excellent expansion, the respective C-max were 726.432 cells/μL and 336.67 cells/μL. Conclusions: This CART19A exhibited favorable safety, remarkable expansion potential, and efficacy profiles in the treatment of r/r bulky lymphoma. Notably, it achieved a high ORR of 90%, providing a favorable window of opportunity for subsequent consolidation and intensification treatments. Moreover, this transition from a bulky to a non-bulky disease state reduces the intricacy of follow-up treatments. Clinical trial information: NCT04384393 .

UV damage induces production of mitochondrial DNA fragments with specific length profiles.

UV light is a potent mutagen that induces bulky DNA damage in the form of cyclobutane pyrimidine dimers (CPDs). Photodamage and other bulky lesions occurring in nuclear genomes can be repaired through nucleotide excision repair (NER), where incisions on both sides of a damaged site precede the removal of a single-stranded oligonucleotide containing the damage. Mitochondrial genomes (mtDNAs) are also susceptible to damage from UV light, but current evidence suggests that the only way to eliminate bulky mtDNA damage is through mtDNA degradation. Damage-containing oligonucleotides excised during NER can be captured with antidamage antibodies and sequenced (XR-seq) to produce high-resolution maps of active repair locations following UV exposure. We analyzed previously published datasets from Arabidopsis thaliana, Saccharomyces cerevisiae, and Drosophila melanogaster to identify reads originating from the mtDNA (and plastid genome in A. thaliana). In A. thaliana and S. cerevisiae, the mtDNA-mapping reads have unique length distributions compared to the nuclear-mapping reads. The dominant fragment size was 26 nt in S. cerevisiae and 28 nt in A. thaliana with distinct secondary peaks occurring in regular intervals. These reads also show a nonrandom distribution of di-pyrimidines (the substrate for CPD formation) with TT enrichment at positions 7-8 of the reads. Therefore, UV damage to mtDNA appears to result in production of DNA fragments of characteristic lengths and positions relative to the damaged location. The mechanisms producing these fragments are unclear, but we hypothesize that they result from a previously uncharacterized DNA degradation pathway or repair mechanism in mitochondria.

Оптимизация терапии у пациентов с впервые диагностированной диффузной В-крупноклеточной лимфомой: эффективность и безопасность программы R-SD-EPOCH ± HDMTX в рамках одноцентрового нерандомизированного проспективного клинического исследования (предварительные результаты)

Оптимизация терапии у пациентов с впервые диагностированной диффузной В-крупноклеточной лимфомой: эффективность и безопасность программы R-SD-EPOCH ± HDMTX в рамках одноцентрового нерандомизированного проспективного клинического исследования (предварительные результаты)

Comparing Mfd- and UvrD-dependent models of transcription coupled DNA repair in live Escherichia coli using single-molecule tracking

During transcription-coupled DNA repair (TCR) the detection of DNA damage and initiation of nucleotide excision repair (NER) is performed by translocating RNA polymerases (RNAP), which are arrested upon encountering bulky DNA lesions. Two opposing models of the subsequent steps of TCR in bacteria exist. In the first model, stalled RNAPs are removed from the damage site by recruitment of Mfd which dislodges RNAP by pushing it forwards before recruitment of UvrA and UvrB. In the second model, UvrD helicase backtracks RNAP from the lesion site. Recent studies have proposed that both UvrD and UvrA continuously associate with RNAP before damage occurs, which forms the primary damage sensor for NER. To test these two models of TCR in living E. coli, we applied super-resolution microscopy (PALM) combined with single particle tracking to directly measure the mobility and recruitment of Mfd, UvrD, UvrA, and UvrB to DNA during ultraviolet-induced DNA damage. The intracellular mobilities of NER proteins in the absence of DNA damage showed that most UvrA molecules could in principle be complexed with RNAP, however, this was not the case for UvrD. Upon DNA damage, Mfd recruitment to DNA was independent of the presence of UvrA, in agreement with its role upstream of this protein in the TCR pathway. In contrast, UvrD recruitment to DNA was strongly dependent on the presence of UvrA. Inhibiting transcription with rifampicin abolished Mfd DNA-recruitment following DNA damage, whereas significant UvrD, UvrA, and UvrB recruitment remained, consistent with a UvrD and UvrA performing their NER functions independently of transcribing RNAP. Together, although we find that up to ∼8 UvrD-RNAP-UvrA complexes per cell could potentially form in the absence of DNA damage, our live-cell data is not consistent with this complex being the primary DNA damage sensor for NER.

The hemiclamshell approach to bulky cervico-mediastinal lesions: how to do it.

The hemiclamshell incision is a combination of partial median sternotomy and anterolateral thoracotomy, allowing excellent exposure of mediastinum, one pleural cavity and the neck. It can be used for superior sulcus tumors with mediastinal involvement or to resect bulky mediastinal lesions or lesions requiring cervical, mediastinal and pleural exposition. Although the vast majority of mediastinal lesions can now be approached by minimally invasive techniques, the hemiclamshell incision still plays a pivotal role in the case of bulky lesions involving cervical, mediastinal and pleural cavities. In the case of cardiac or great vessel involvement, the procedure should be performed in experienced high-volume centers with the availability of cardio-pulmonary bypass, extra-corporeal membrane oxygenator or venous shunts. Although the vast majority of mediastinal lesions can now be approached by minimally invasive techniques, the hemiclamshell incision still plays a pivotal role in the case of bulky lesions involving cervical, mediastinal and pleural cavities. It consists of a combination of subtotal median vertical sternotomy and antero-lateral thoracotomy in the 4th intercostal space, providing excellent exposure of the neck, mediastinum and one pleural cavity. The objective of this paper is to describe the standard steps for performing a hemiclamshell incision by using modern devices and technology which contribute to make this procedure easier, faster and safer.

Определение соматических мутаций в гене EZH2 и оценка их прогностической значимости при фолликулярной лимфоме 1–3А цитологического типа

Aim. To determine the incidence and prognostic value of mutations in exon 16 of EZH2 as well as those of polymorphism с.1582-21А&gt;G (rs2072407) in EZH2 in patients with follicular lymphoma (FL) grades 1–3А in relation to morphologic and cytogenetic tumor characteristics.&#x0D; Materials &amp; Methods. The prospective cohort study conducted by the National Research Center for Hematology from January 2017 to April 2021 enrolled 80 patients with newly diagnosed FL grades 1/2 and 3А. The median follow-up was 53 months. Molecular and cytogenetic analyses were based on biopsy samples of lymph nodes obtained before chemotherapy. The mutation status of exon 16 in EZH2 and the presence of intronic polymorphism rs2072407 in EZH2 were examined by Sanger sequencing method. Translocation t(14;18)(q32;q21) was detected by karyotyping or FISH.&#x0D; Results. Mutations in exon 16 of EZH2 (mutEZH2) were identified in 10/80 (13 %) patients. All patients showed missense mutation in codon 646 of EZH2. Translocation t(14;18) was detected in 45/80 (56 %) cases. Poor outcome in the cohort with no t(14;18) was observed 3 times more often than in the group of patients with t(14;18) (p = 0.0001). The presence of t(14;18) was associated with favorable prognosis irrespective of either the mutation status of exon 16 in EZH2 or the FL grade. The analysis of the polymorphism rs2072407 status yielded the following genotypes: AA in 24 % (n = 19), AG in 42 % (n = 34), and GG in 34 % (n = 27) of cases. The variants АА and AG were associated with higher risk of death (hazard ratio 2.9; 95% confidence interval 1.2–10.6; p = 0.01), whereas the genotype GG was associated with wtEZH2 (10 % vs. 37 %) and favorable prognosis (p = 0.065).&#x0D; Conclusion. Significant biological markers for favorable prognosis in FL appeared to be the presence of t(14;18)(q32;q21) and GG genotype of polymorphism rs2072407 in EZH2. The previously identified prognostic factors (grade 3А, bulky tumor lesions &gt; 6 cm, Ki-67 &gt; 35 %, and a short interval between symptom onset and chemotherapy start) were incorporated into a new unified personalized predictive (index PPI) FL model by supplementing it with two additional biological markers: the presence of t(14;18)(q32;q21) and GG genotype of polymorphism rs2072407. This approach may increase the prognostic value of the new personalized design which will provide the basis for risk-adapted algorithms for FL treatment.

Elf1 promotes Rad26's interaction with lesion-arrested Pol II for transcription-coupled repair.

Transcription-coupled nucleotide excision repair (TC-NER) is a highly conserved DNA repair pathway that removes bulky lesions in the transcribed genome. Cockayne syndrome B protein (CSB), or its yeast ortholog Rad26, has been known for decades to play important roles in the lesion-recognition steps of TC-NER. Another conserved protein ELOF1, or its yeast ortholog Elf1, was recently identified as a core transcription-coupled repair factor. How Rad26 distinguishes between RNA polymerase II (Pol II) stalled at a DNA lesion or other obstacles and what role Elf1 plays in this process remains unknown. Here, we present cryo-EM structures of Pol II-Rad26 complexes stalled at different obstacles that show that Rad26 uses a common mechanism to recognize a stalled Pol II, with additional interactions when Pol II is arrested at a lesion. A cryo-EM structure of lesion-arrested Pol II-Rad26 bound to Elf1 revealed that Elf1 induces further interactions between Rad26 and a lesion-arrested Pol II. Biochemical and genetic data support the importance of the interplay between Elf1 and Rad26 in TC-NER initiation. Together, our results provide important mechanistic insights into how two conserved transcription-coupled repair factors, Rad26/CSB and Elf1/ELOF1, work together at the initial lesion recognition steps of transcription-coupled repair.

Outcomes of Targeted Therapy Plus Immunotherapy and High-Dose Stereotactic Ablative Body Radiotherapy (SABR) for Metastatic Renal Cell Cancer Patients.

Stereotactic ablative body radiotherapy (SABR) is one of the treatment options for metastatic renal cell carcinoma (mRCC) but is limited by a lack of data to evaluate targeted therapy plus immunotherapy concurrently with high-dose SABR to multiple sites. We evaluated the safety and disease control for mRCC patients who concurrently received the above tri-modality treatment. Patients were treated with SABR (40-70 Gy/5-10 fractions) for small lesions or partial-SABR (tumor center boosted with 6-8 Gy/3-5 fractions with 50-60 Gy/20-25 fractions to the whole tumor volume) for bulky tumors or tumors adjacent to critical organs. When SABR/partial-SABR was not feasible, a moderate fractionated radiotherapy plan, usually 60Gy/20 fractions were applied. of Targeted therapy plus immunotherapy (PD-1 inhibitor) was not interrupted during or after radiotherapy (RT). Adverse events (AEs) were evaluated. Disease control rate (DCR), objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were calculated. The PFS1 was defined as the first progression since the start of RT. The PFS2 was defined as the second progression after the second RT course, if new metastases occurred after first RT were all re-irradiated, and the systemic therapy was not changed. The Kaplan-Meier method was used for time-to-event endpoints. A total of 51 patients, with a median age of 57 yr, were enrolled. The median follow-up was 12 months. There were 75% of patients with intermediate-risk and 18% with favorable-risk disease. 61% of the patients were oligometastatic. 71% had clear cell renal cancer. There were 241 metastases while 161 (67%) were irradiated. 80% of the lesions received SABRP/partial SABR. 1 patient with 14 lesions irradiated received proton therapy. All the surviving patients are continuing using targeted therapy while 81% patients complete at least 1-year PD-1 therapy. 10 patients (20%) had grade 3 drug-related AEs: pneumonitis (n = 2), elevated alanine transaminase (n = 4), myositis (n = 1), hand-foot syndrome myositis (n = 1), enteritis (n = 1), fatigue (n = 1). There were 1 grade 4 AEs of upper gastrointestinal bleeding. No grade 3-5 RT-related AEs was found. ORR and DCR for irradiated lesion were 51% and 98%. Median OS and PFS2 was not reached. Median PFS1 was 14(6-22) months. Estimated 1- and 2-yr OS, PFS1 and PFS2 were 90% and 90%, 56% and 38%, 74% and 51% respectively. Univariate analysis showed that an PFS1 benefit was found in patients who received radiation before systemic therapy failure (p = 0.038). We investigated the high-dose RT in combination of concurrent targeted and immunotherapy in patients with metastatic RCC. We found that this treatment regimen was well tolerated, with good cancer control. Early use of high-dose RT to multi-lesions may improve PFS. Partial-SABR for bulky lesions close to critical organs could be safely and effectively applied under certain circumstances. These encouraging findings warrant further investigation.

Hybrid Immuno-RT for Bulky Tumors: Standard Fractionation with Partial Tumor SBRT

Bulky tumors remain challenging to be treated. Stereotactic body radiation therapy (SBRT) is effective against radioresistant tumor cells and can induce immunogenic cell death (ICD) that leads to T-cell-mediated antitumor effects. Low-dose radiation (LDRT) can inflame the tumor microenvironment (TME) by recruiting T cells. We designed a novel radiotherapy technique (RT, ERT) whose dose distribution map resembles the "eclipse" by concurrently delivering LDRT to the whole tumor, meanwhile SBRT to only a part of the same tumor. This study examined the safety and efficacy of ERT to bulky lesions with PD-1 inhibitors in mice and patients. In mice with CT26 colon or LLC1 lung bulky tumors (400 - 500 cm3), the whole tumor was irradiated by LDRT (2 Gy x 3), meanwhile the tumor center was irradiated by SBRT (10 Gy x 3); αPD-1 was given weekly. The dependence of therapeutic effects on CD8+ T cells was determined using depleting antibodies. Frequencies of CD8+ T cells and M1 macrophages (Mφ) were determined by flow cytometry. Multiplex Immunohistochemistry (mIHC) was applied to analyze the number and the location of CD8+ T cells and their subpopulations, as well as the phospho-eIF2α level (the ICD marker) of tumor cells in TME. Patients with advanced lung or liver bulky tumors who failed standard treatment or with oncologic emergencies were treated. Kaplan-Meier method was applied to estimate patients' progression-free survival (PFS) and overall survival (OS). ERT/αPD-1 is superior to SBRT/αPD-1 or LDRT/αPD-1 in controlling bulky tumors in both mouse models in a CD8+ T-cell dependent manner. In the CT26 model, ERT/αPD-1 resulted in complete tumor regression in 3/11 mice and induced more CD8+ T cells and M1 Mφ in TME compared to other groups. mIHC analysis showed that ERT/αPD-1 induced higher bulk, stem-like (TCF1+ TIM3- PD-1+), and more differentiated (TCF1- TIM3+ PD-1+) CD8+ T cells infiltration into the tumor center and periphery compared to other groups. Compared to untreated or LDRT-treated tumor centers, tumor centers irradiated with ERT or SBRT showed elevated phospho-eIF2α accompanied by higher dendritic cell infiltration. In total, 39 advanced cancer patients were treated with ERT/αPD-1 or plus chemotherapy. Radiation-induced pneumonitis occurred in 1 of 26 patients receiving thoracic ERT. There were two cases of grade III toxicity associated with PD-1 inhibitors. No toxicity above grade III was observed. The objective response rate was 38.5%. The median PFS was 5.6 months and median OS was not reached at a median follow-up of 11.7 months. ERT/αPD-1 showed superior efficacy in controlling bulky tumor in two mouse models. The hybrid immuno-RT (ERT) combing PD-1 inhibitors was safe and effective in patients with bulky tumors. Further clinical trials in combination with bioimaging to identify the optimal SBRT target region for the bulky tumor are warranted.

Laryngeal Papillomatosis: A Non-traditional Flexible Endoscopic Approach

To evaluate the efficacy of flexible endoscopic laryngeal surgery (FELS) using unconventional tools in the treatment of laryngeal papillomatosis (LP). We applied FELS using forceps, diathermy snare, and Nd:YAG laser for 47 patients with LP. In 38 cases (81%), surgeries were performed under topical anesthesia with spontaneous respiration and in seven cases we used general anesthesia with superimposed high-frequency jet ventilation. In two cases, both types of anesthesia were used in different sessions. In 22 cases (46.8%), the interventions were performed as outpatient procedures. The Nd:YAG laser was used in the majority (89.4%) of cases. The expected result (complete eradication of the visible lesion) was obtained in all treated patients without procedure complications. Disease recurrence was established in 16 cases (34%) that led to repeated surgeries. FELS, using Nd:YAG laser as a main tool, is a safe and effective approach that can be a feasible alternative to the conventional approach in the treatment of LP due to possibility to avoid repeated general anesthesia with its associated risks; applicability for the patients, who have contraindications for general anesthesia and transoral microsurgery; possibility to perform the intervention in an outpatient setting that leads to essential reduction of medical costs; economy of time. Nd:YAG laser ablation, preceded by diathermy snare resection, allows reducing the time of the intervention and thermal injury of the adjacent tissues, compared to using laser alone, and can be used in bulky and obstructive lesions that present a limitation for nonablative techniques.

The role of Transcription Factor IIH complex in nucleotide excision repair.

DNA damage occurs throughout life from a variety of sources, and it is imperative to repair damage in a timely manner to maintain genome stability. Thus, DNA repair mechanisms are a fundamental part of life. Nucleotide excision repair (NER) plays an important role in the removal of bulky DNA adducts, such as cyclobutane pyrimidine dimers from ultraviolet light or DNA crosslinking damage from platinum-based chemotherapeutics, such as cisplatin. A main component for the NER pathway is transcription factor IIH (TFIIH), a multifunctional, 10-subunit protein complex with crucial roles in both transcription and NER. In transcription, TFIIH is a component of the pre-initiation complex and is important for promoter opening and the phosphorylation of RNA Polymerase II (RNA Pol II). During repair, TFIIH is important for DNA unwinding, recruitment of downstream repair factors, and verification of the bulky lesion. Several different disease states can arise from mutations within subunits of the TFIIH complex. Most strikingly are xeroderma pigmentosum (XP), XP combined with Cockayne syndrome (CS), and trichothiodystrophy (TTD). Here, we summarize the recruitment and functions of TFIIH in the two NER subpathways, global genomic (GG-NER) and transcription-coupled NER (TC-NER). We will also discuss how TFIIH's roles in the two subpathways lead to different genetic disorders.