The DNA damage response network in the treatment of head and neck squamous cell carcinoma.

BACKGROUND: We sought to determine whether DNA damage response (DDR)-related aberrations predict therapeutic benefit in cisplatin-treated head and neck squamous cell carcinoma (HNSCC) patients and how DDR pathways are modulated after treatment with olaparib alone or in combination with cisplatin or durvalumab. PATIENTS AND METHODS: Oxidative stress, abasic sites and DDR-related parameters, including endogenous DNA damage, DNA repair mechanisms and apoptosis rates, were evaluated in HNSCC cell lines and peripheral blood mononuclear cells from 46 healthy controls (HC) and 70 HNSCC patients at baseline and following treatment with cisplatin-containing chemoradiation or nivolumab or enrolled in the OPHELIA phase II trial (NCT02882308; olaparib alone, olaparib plus cisplatin, olaparib plus durvalumab). RESULTS: HNSCC patients at diagnosis exhibited deregulated DDR-related parameters and higher levels of oxidative stress and abasic sites compared with HC (all P < 0.05). Accordingly, nucleotide excision repair (NER; ERCC1, ERCC2/XPD, XPA, XPC) and base excision repair (APEX1, XRCC1) genes were downregulated in patients versus HC whereas double-strand breaks repair (MRE11A, RAD50, RAD51, XRCC2) and mismatch repair (MLH1, MSH2, MSH3) genes were overexpressed. Corresponding results were obtained in cell lines (all P < 0.001). Excellent correlations were observed between individual ex vivo and in vivo/therapeutic results, with cisplatin non-responders showing higher levels of endogenous DNA damage, augmented oxidative stress and abasic sites, increased NER capacities and reduced apoptosis than responders (all P < 0.05). Also, longer progression-free survival correlated with lower NER capacity (P = 0.037) and increased apoptosis (P = 0.029). Interestingly, treatment with olaparib-containing regimens results in the accumulation of cytotoxic DNA damage and exerts an extra antitumor effect by elevating oxidative stress (all P < 0.05). Nivolumab induced no significant changes in the DDR parameters examined. CONCLUSIONS: Aberrations in DDR signals are implicated in the response to HNSCC chemotherapy and can be exploited as novel therapeutic targets, sensitive/effective non-invasive biomarkers as well as for the design of novel clinical trials.

Correction: Chromatin structure, transcriptional activity and DNA repair efficiency affect the outcome of chemotherapy in multiple myeloma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chromatin structure, transcriptional activity and DNA repair efficiency affect the outcome of chemotherapy in multiple myeloma.

BACKGROUND: Melphalan is one of the most active chemotherapeutic agents in the treatment of multiple myeloma (MM). However, the mechanism underlying differential patient responses to melphalan therapy is unknown. METHODS: Chromatin structure, transcriptional activity and DNA damage response signals were examined following ex vivo treatment with melphalan of both malignant bone marrow plasma cells (BMPCs) and peripheral blood mononuclear cells (PBMCs) of MM patients, responders (n=57) or non-responders (n=28) to melphalan therapy. PBMCs from healthy controls (n=25) were also included in the study. RESULTS: In both BMPCs and PBMCs, the local chromatin looseness, transcriptional activity and repair efficiency of the transcribed strand (TS) were significantly higher in non-responders than in responders and lowest in healthy controls (all P<0.05). Moreover, we found that melphalan-induced apoptosis inversely correlated with the repair efficiency of the TS, with the duration of the inhibition of mRNA synthesis, phosphorylation of p53 at serine 15 and apoptosis rates being higher in responders than in non-responders (all P<0.001). CONCLUSIONS: Our findings provide a mechanistic basis for the link between DNA repair efficiency and response to melphalan therapy. Interestingly, the observation of these phenomena in PBMCs provides a novel approach for the prediction of response to anti-myeloma therapy.

Progressive changes in chromatin structure and DNA damage response signals in bone marrow and peripheral blood during myelomagenesis.

The molecular pathways implicated in multiple myeloma (MM) development are rather unknown. We studied epigenetic and DNA damage response (DDR) signals at selected model loci (N-ras, p53, d-globin) in bone marrow plasma cells and peripheral blood mononuclear cells (PBMCs) from patients with monoclonal gammopathy of undetermined significance (MGUS; n=20), smoldering/asymptomatic MM (SMM; n=29) and MM (n=18), as well as in healthy control-derived PBMCs (n=20). In both tissues analyzed, a progressive, significant increase in the looseness of local chromatin structure, gene expression levels and DNA repair efficiency from MGUS to SMM and finally to MM was observed (all P<0.002). Following ex vivo treatment with melphalan, a gradual suppression of the apoptotic pathway occurred in samples collected at different stages of myelomagenesis, with the severity and duration of the inhibition of RNA synthesis, p53 phosphorylation at serine15 and induction of apoptosis being higher in MGUS than SMM and lowest in MM patients (all P<0.0103). Interestingly, for all endpoints analyzed, a strong correlation between plasma cells and corresponding PBMCs was observed (all P<0.0003). We conclude that progressive changes in chromatin structure, transcriptional activity and DDR pathways during myelomagenesis occur in malignant plasma cells and that these changes are also reflected in PBMCs.

Transcription factor-pathway coexpression analysis reveals cooperation between SP1 and ESR1 on dysregulating cell cycle arrest in non-hyperdiploid multiple myeloma.

Multiple myeloma is a hematological cancer of plasma B cells and remains incurable. Two major subtypes of myeloma, hyperdiploid MM (HMM) and non-hyperdiploid MM (NHMM), have distinct chromosomal alterations and different survival outcomes. Transcription factors (TrFs) have been implicated in myeloma oncogenesis, but their dysregulation in myeloma subtypes are less studied. Here, we developed a TrF-pathway coexpression analysis to identify altered coexpression between two sample types. We apply the method to the two myeloma subtypes and the cell cycle arrest pathway, which is significantly differentially expressed between the two subtypes. We find that TrFs MYC, nuclear factor-κB and HOXA9 have significantly lower coexpression with cell cycle arrest in HMM, co-occurring with their overactivation in HMM. In contrast, TrFs ESR1 (estrogen receptor 1), SP1 and E2F1 have significantly lower coexpression with cell cycle arrest in NHMM. SP1 chromatin immunoprecipitation targets are enriched by cell cycle arrest genes. These results motivate a cooperation model of ESR1 and SP1 in regulating cell cycle arrest, and a hypothesis that their overactivation in NHMM disrupts proper regulation of cell cycle arrest. Cotargeting ESR1 and SP1 shows a synergistic effect on inhibiting myeloma proliferation in NHMM cell lines. Therefore, studying TrF-pathway coexpression dysregulation in human cancers facilitates forming novel hypotheses toward clinical utility.

VTD consolidation, without bisphosphonates, reduces bone resorption and is associated with a very low incidence of skeletal-related events in myeloma patients post ASCT.

We prospectively evaluated the effect of bortezomib, thalidomide and dexamethasone (VTD) consolidation on bone metabolism of 42 myeloma patients who underwent an autologous stem cell transplantation (ASCT). VTD started on day 100 post ASCT; patients received four cycles of VTD (first block), were followed without treatment for 100 days and then received another four VTD cycles (second block). During this 12-month period, bisphosphonates were not administered. Best response included stringent complete remission (sCR) in 15 (35.7%) patients, complete response (CR) in 13 (30.9%), vgPR in 7 (16.6%), PR in 4 (9.5%), while 3 (7.1%) patients developed a progressive disease (PD). Importantly, 33.3% and 47.6% of patients improved their status of response after the first and second VTD block, respectively. VTD consolidation resulted in a significant reduction of circulating C-terminal cross-linking telopeptide of collagen type I (CTX), soluble receptor activator of the nuclear factor-kappa B ligand (sRANKL) and osteocalcin (OC), whereas bone-specific alkaline phosphatase (bALP) remained stable compared with pre-VTD values. During the study period, only one patient with a PD developed a skeletal-related event (that is, radiation to bone). The median time to progression (TTP) after ASCT was 34 months and the median time of next treatment was 40 months. We conclude that VTD consolidation post ASCT reduces bone resorption and is associated with a very low incidence of skeletal-related events (SREs) despite the absence of bisphosphonates; the later do not appear to be necessary in this context.

The role of novel agents on the reversibility of renal impairment in newly diagnosed symptomatic patients with multiple myeloma.

The role of thalidomide, bortezomib and lenalidomide in multiple myeloma patients presenting with renal impairment was evaluated in 133 consecutive newly diagnosed patients who were treated with a novel agent-based regimen. A significant improvement of renal function (renalPR (renal partial response)) was observed in 77% of patients treated with bortezomib, in 55% with thalidomide and in 43% with lenalidomide (P=0.011). In multivariate analysis, bortezomib-based therapy was independently associated with a higher probability of renal response compared with thalidomide- or lenalidomide-based therapy. Other important variables included eGFR (estimated glomerular filtration rate) ≥30 ml/min, age ≤65 years and myeloma response. Patients treated with bortezomib achieved at least renalPR in a median of 1.34 months vs 2.7 months for thalidomide and >6 months for lenalidomide-treated patients (P=0.028). In multivariate analysis bortezomib-based therapy, higher doses of dexamethasone (≥160 mg during the first month of treatment), an eGFR ≥30 ml/min and age ≤65 years were independently associated with shorter time to renal response. In conclusion, bortezomib-based therapies may be more appropriate for the initial management of patients with myeloma-related renal failure; however, thalidomide and lenalidomide are also associated with significant probability of improvement of their renal function.

Extensive bone marrow infiltration and abnormal free light chain ratio identifies patients with asymptomatic myeloma at high risk for progression to symptomatic disease.

Asymptomatic multiple myeloma (AMM) is characterized by a constant risk of progression to symptomatic myeloma. To evaluate previously recognized risk factors and to identify high-risk features we analyzed 96 patients with AMM and at least 18 months of follow-up. The progression rate at 1,2, and 3 years was 8%, 15% and 26%, respectively, and the projected 5-year progression rate was 38%. Extensive bone marrow (BM) infiltration, abnormal free light chain (FLC) ratio and serum monoclonal (M)-protein ≥ 3 gr/dl were the most significant factors for progression, whereas the type of heavy (IgG vs IgA) or light chain or immunoparesis of the uninvolved immunoglobulins were not. Abnormal marrow signal of magnetic resonance imaging of the spine was associated with a significant risk of progression (median 15 months, P=0.001). Extensive BM infiltration ≥ 60% (hazard ratio, HR: 13.7, P<0.001) and FLC ratio ≥ 100 (HR: 9, P=0.003) independently identified a 'very high-risk' group, which included 12.5% of patients with AMM and who progressed ≤ 18 months from initial diagnosis. Development of anemia and/or lytic bone lesions were the most common features of symptomatic progression. In conclusion, there is a subgroup of patients who have a substantial risk of progression to symptomatic disease that can be detected at diagnosis (either by extensive BM infiltration ≥ 60% or FLC ratio ≥ 100) and may be considered for immediate treatment.

High circulating sclerostin is present in patients with thalassemia-associated osteoporosis and correlates with bone mineral density.

Osteoporosis is a severe complication of thalassemia. Sclerostin is a Wnt signaling inhibitor, which is produced by osteocytes and inhibits osteoblast function. Sclerostin is implicated in the pathogenesis of osteoporosis of different etiology. The aim of the study was to evaluate circulating sclerostin in 66 patients (median age 42 years) with thalassemia and osteoporosis who participated in a phase 2, randomized study (zoledronic acid vs. placebo) and the results were compared with those of 30 healthy controls (median age 44 years) without osteopenia/osteoporosis and 62 women with postmenopausal osteoporosis (median age 63 years). At baseline, thalassemic patients with osteoporosis had elevated circulating levels of sclerostin (median: 605 pg/ml, range: 22-1,227 pg/ml) compared to healthy controls without osteopenia/osteoporosis (250 pg/ml, 0-720 pg/ml, p<0.001) and reduced levels of sclerostin compared with postmenopausal women with osteoporosis (840 pg/ml, 181-1,704 pg/ml, p<0.001). Thalassemia patients had also increased serum dickkopf-1 (Dkk-1) and high bone turnover. Circulating sclerostin levels correlated with bone mineral density in lumbar spine (r=0.619, p<0.001), distal radius (r=0.401, p=0.001) and femoral neck (r=0.301, p=0.021). Zoledronic acid did not alter sclerostin levels after 12 months of therapy, although it reduced circulating Dkk-1. We conclude that circulating sclerostin is elevated in thalassemia patients with osteoporosis and correlated with their BMD, but it was not reduced post zoledronic acid administration. These findings suggest that high sclerostin may serve as a marker of increased osteocyte activity in thalassemia patients. Drugs targeting sclerostin may also be used in this difficult to treat disorder associated with bone loss.

Circulating activin-A is elevated in patients with advanced multiple myeloma and correlates with extensive bone involvement and inferior survival; no alterations post-lenalidomide and dexamethasone therapy.

BACKGROUND: Activin-A is a transforming growth factor -ß superfamily member, which seems to be implicated in the biology of osteolytic disease in multiple myeloma. DESIGN AND METHODS: Circulating activin-A was evaluated in 98 newly diagnosed myeloma patients (85 with symptomatic disease), in 40 patients with relapsed myeloma before and after four cycles of lenalidomide and dexamethasone (RD), in 27 healthy controls and in 10 monoclonal gammopathy of undetermined significance patients. RESULTS: Patients with newly diagnosed symptomatic myeloma had increased circulating activin-A compared with controls (P < 0.001), while patients with relapsed disease had elevated activin-A even compared with symptomatic patients at diagnosis (P < 0.001). High activin-A correlated with advanced International Staging System stage (P = 0.002), increased bone resorption (P < 0.001) and extensive bone disease (P = 0.03). Low levels of activin-A (<442 pg/ml) were associated with superior median overall survival: not reached versus 59 months (P = 0.04), while activin-A inversely correlated with survival as a continuous variable (P < 0.001). RD did not alter circulating activin-A after four cycles of treatment, even in responders. CONCLUSIONS: High circulating activin-A correlates with advanced features of myeloma, supporting the rationale for the use of activin-A antagonists, such as sotatercept in myeloma. The inability of RD to reduce activin-A reveals RD as a good candidate for combination therapies with activin-A antagonists in myeloma.

Treatment of patients with relapsed/refractory multiple myeloma with lenalidomide and dexamethasone with or without bortezomib: prospective evaluation of the impact of cytogenetic abnormalities and of previous therapies.

We prospectively studied the impact of several cytogenetic abnormalities (CAs) in patients with relapsed/refractory myeloma who received lenalidomide and dexamethasone (RD) with or without the addition of bortezomib (V). On the basis of the presence of previous neuropathy, 50 patients were treated with RD and 49, without preexisting neuropathy, with VRD. The overall response rate was 63%, similar for RD and VRD. Poor risk cytogenetics were associated with lower response rates in RD (P=0.01), but not in VRD (P=0.219). The median progression-free survival (PFS) was similar for RD (9 months) and VRD (7 months). The median overall survival (OS) for all patients was 16 months, with no differences between RD or VRD regimens. Poor risk cytogenetics, especially del17p, resistance to previous thalidomide, elevated lactate dehydrogenase (LDH) and presence of extramedullary disease were associated with inferior response to therapy and shorter PFS and OS. The impact of other CAs on OS was more pronounced in RD. In conclusion, the presence of CAs is an important adverse prognostic factor for patients with relapsed/refractory myeloma, but resistance to previous thalidomide, elevated LDH and presence of extramedullary disease remain of major prognostic importance. The outcome of patients with del17p remains extremely poor even with VRD combination.