Biological effects of intraoperative radiation therapy: histopathological changes and immunomodulation in breast cancer patients.

Introduction: Intraoperative radiation therapy (IORT) delivers a single accelerated radiation dose to the breast tumor bed during breast-conserving surgery (BCS). The synergistic biologic effects of simultaneous surgery and radiation remain unclear. This study explores the cellular and molecular changes induced by IORT in the tumor microenvironment and its impact on the immune response modulation. Methods: Patients with hormone receptor (HR)-positive/HER2-negative, ductal carcinoma in situ (DCIS), or early-stage invasive breast carcinoma undergoing BCS with margin re-excision were included. Histopathological evaluation and RNA-sequencing in the re-excision tissue were compared between patients with IORT (n=11) vs. non-IORT (n=11). Results: Squamous metaplasia with atypia was exclusively identified in IORT specimens (63.6%, p=0.004), mimicking DCIS. We then identified 1,662 differentially expressed genes (875 upregulated and 787 downregulated) between IORT and non-IORT samples. Gene ontology analyses showed that IORT was associated with the enrichment of several immune response pathways, such as inflammatory response, granulocyte activation, and T-cell activation (p<0.001). When only considering normal tissue from both cohorts, IORT was associated with intrinsic apoptotic signaling, response to gamma radiation, and positive regulation of programmed cell death (p<0.001). Using the xCell algorithm, we inferred a higher abundance of γδ T-cells, dendritic cells, and monocytes in the IORT samples. Conclusion: IORT induces histological changes, including squamous metaplasia with atypia, and elicits molecular alterations associated with immune response and intrinsic apoptotic pathways. The increased abundance of immune-related components in breast tissue exposed to IORT suggests a potential shift towards active immunogenicity, particularly immune-desert tumors like HR-positive/HER2-negative breast cancer.

Does Residual Invasive Disease in Wide Local Excision after Diagnosis with Partial Biopsy Technique Influence Survival in Melanoma? Matched-Pair Analysis of Multicenter Selective Lymphadenectomy Trial I and II.

BACKGROUND: Current guidelines recommend excisional/complete biopsy for melanoma diagnosis, owing to high rates of residual disease found at wide local excision (WLE) after partial biopsy techniques. We sought to determine any survival disadvantage associated with the presence of residual invasive melanoma in the WLE after diagnosis with a partial biopsy technique. STUDY DESIGN: Data were examined from Multicenter Selective Lymphadenectomy Trials I and II (MSLT-I and -II), 2 large melanoma trials. Patients diagnosed with excisional/complete biopsy were excluded. Clinicopathologic characteristics, melanoma-specific survival (MSS), distant disease-free survival (DDFS), and disease-free survival (DFS) of those with residual invasive melanoma in the definitive WLE and those with no residual melanoma were compared. Matched pairing was used to reduce variability between groups. RESULTS: From 1994 through 2014, 3,939 patients were enrolled in these trials and 874 (22%) were diagnosed using partial biopsy techniques. Of these, 399 (46%) had residual tumor in the WLE. Only 6 patients had residual tumor in their WLE resulting in T-upstaging of their tumor. Match-pairing formed two cohorts (1:1) of patients with and without residual invasive tumor after WLE. A total of 514 patients were paired; 288 (56%) males, 148 (28.8%) aged 60 or older, 192 (37.4%) with truncal melanomas, 214 (41.6%) had Breslow thickness 2 mm or greater, and 376 (73.2%) had positive sentinel nodes. Kaplan-Meier analysis showed no statistical difference in 10-year MSS (73.6% ± 3.3% vs 73.9% ± 3.7%, p = 0.891), DDFS (68.7% ± 3.4% vs 65.3% ± 4.0%, p = 0.548), or DFS (59.6% ± 3.7% vs 59.4% ± 3.9%, p = 0.783). CONCLUSIONS: Survival in patients with primary melanoma does not appear to be worse in patients who undergo a partial biopsy technique and are later found to have residual invasive tumor in the WLE specimen.

Mitigation of Acetylcholine Esterase Activity in the 1,7-Diazacarbazole Series of Inhibitors of Checkpoint Kinase 1.

Checkpoint kinase 1 (ChK1) plays a key role in the DNA damage response, facilitating cell-cycle arrest to provide sufficient time for lesion repair. This leads to the hypothesis that inhibition of ChK1 might enhance the effectiveness of DNA-damaging therapies in the treatment of cancer. Lead compound 1 (GNE-783), the prototype of the 1,7-diazacarbazole class of ChK1 inhibitors, was found to be a highly potent inhibitor of acetylcholine esterase (AChE) and unsuitable for development. A campaign of analogue synthesis established SAR delineating ChK1 and AChE activities and allowing identification of new leads with improved profiles. In silico docking using a model of AChE permitted rationalization of the observed SAR. Compounds 19 (GNE-900) and 30 (GNE-145) were identified as selective, orally bioavailable ChK1 inhibitors offering excellent in vitro potency with significantly reduced AChE activity. In combination with gemcitabine, these compounds demonstrate an in vivo pharmacodynamic effect and are efficacious in a mouse p53 mutant xenograft model.

Identification of preferred chemotherapeutics for combining with a CHK1 inhibitor.

Here we report that GNE-783, a novel checkpoint kinase-1 (CHK1) inhibitor, enhances the activity of gemcitabine by disabling the S- and G2 cell-cycle checkpoints following DNA damage. Using a focused library of 51 DNA-damaging agents, we undertook a systematic screen using three different cell lines to determine which chemotherapeutics have their activity enhanced when combined with GNE-783. We found that GNE-783 was most effective at enhancing activity of antimetabolite-based DNA-damaging agents; however, there was a surprisingly wide range of activity within each class of agents. We, next, selected six different therapeutic agents and screened these in combination with GNE-783 across a panel of cell lines. This revealed a preference for enhanced chemopotentiation of select agents within tumor types, as, for instance, GNE-783 preferentially enhanced the activity of temozolomide only in melanoma cell lines. Additionally, although p53 mutant status was important for the overall response to combinations with some agents; our data indicate that this alone was insufficient to predict synergy. We finally compared the ability of a structurally related CHK1 inhibitor, GNE-900, to enhance the in vivo activity of gemcitabine, CPT-11, and temozolomide in xenograft models. GNE-900 significantly enhanced activity of only gemcitabine in vivo, suggesting that strong chemopotentiation in vitro can translate into chemopotentiation in vivo. In conclusion, our results show that selection of an appropriate agent to combine with a CHK1 inhibitor needs to be carefully evaluated in the context of the genetic background and tumor type in which it will be used.

Combination drug scheduling defines a "window of opportunity" for chemopotentiation of gemcitabine by an orally bioavailable, selective ChK1 inhibitor, GNE-900.

Checkpoint kinase 1 (ChK1) is a serine/threonine kinase that functions as a central mediator of the intra-S and G2-M cell-cycle checkpoints. Following DNA damage or replication stress, ChK1-mediated phosphorylation of downstream effectors delays cell-cycle progression so that the damaged genome can be repaired. As a therapeutic strategy, inhibition of ChK1 should potentiate the antitumor effect of chemotherapeutic agents by inactivating the postreplication checkpoint, causing premature entry into mitosis with damaged DNA resulting in mitotic catastrophe. Here, we describe the characterization of GNE-900, an ATP-competitive, selective, and orally bioavailable ChK1 inhibitor. In combination with chemotherapeutic agents, GNE-900 sustains ATR/ATM signaling, enhances DNA damage, and induces apoptotic cell death. The kinetics of checkpoint abrogation seems to be more rapid in p53-mutant cells, resulting in premature mitotic entry and/or accelerated cell death. Importantly, we show that GNE-900 has little single-agent activity in the absence of chemotherapy and does not grossly potentiate the cytotoxicity of gemcitabine in normal bone marrow cells. In vivo scheduling studies show that optimal administration of the ChK1 inhibitor requires a defined lag between gemcitabine and GNE-900 administration. On the refined combination treatment schedule, gemcitabine's antitumor activity against chemotolerant xenografts is significantly enhanced and dose-dependent exacerbation of DNA damage correlates with extent of tumor growth inhibition. In summary, we show that in vivo potentiation of gemcitabine activity is mechanism based, with optimal efficacy observed when S-phase arrest and release is followed by checkpoint abrogation with a ChK1 inhibitor.

The role of LAT1 in (18)F-DOPA uptake in malignant gliomas.

Positron emission tomography (PET) imaging with the amino acid tracer 6-(18)F-fluoro-L-3,4-dihydroxy-phenylalanine ((18)F-DOPA) may provide better spatial and functional information in human gliomas than CT or MRI alone. The L-type amino acid transporter 1 (LAT1) is responsible for membrane transport of large neutral amino acids in normal cells. This study assessed the relationship between LAT1 expression and (18)F-DOPA uptake in human astrocytomas. Endogenous LAT1 expression was measured in established glioblastoma (GBM) cell lines and primary GBM xenografts using Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Uptake of (18)F-DOPA was approximated in vitro using (3)H-L-DOPA as an analog. Uptake of (3)H-L-DOPA was assessed in cells expressing LAT1 shRNA or LAT1 siRNA and compared to non-targeted (NT) control shRNA or siRNA sequences, respectively. To demonstrate the clinical relevance of these findings, LAT1 immunofluorescence staining was compared with corresponding regions of (18)F-DOPA PET uptake in patients with newly diagnosed astrocytomas. LAT1 mRNA and protein expression varies in GBM, and the extent of (3)H-L-DOPA uptake was positively correlated with endogenous LAT1 expression. Stable shRNA-mediated LAT1 knockdown in T98 and GBM28 reduced (3)H-L-DOPA uptake relative to NT shRNA by 57 (P < 0.0001) and 52 % (P < 0.001), respectively. Transient siRNA-mediated LAT1 knockdown in T98 reduced (3)H-L-DOPA uptake relative to NT siRNA up to 68 % (P < 0.01). In clinical samples, LAT1 expression positively correlated with (18)F-DOPA PET uptake (P = 0.04). Expression of LAT1 is strongly associated with (3)H-L-DOPA uptake in vitro and (18)F-DOPA uptake in patient biopsy samples. These results define LAT1 as a key determinant of (18)F-DOPA accumulation in GBM.

Nasal septal perforation from bevacizumab: a discussion of outcomes, management, and pharmacovigilance.

To date, the published literature describes 18 reports of nasal septal perforation in cancer patients with the administration of bevacizumab. This complication was detected during post-marketing surveillance of bevacizumab. How should patients who develop this complication be managed? This discussion summarizes suggestions for management of bevacizumab-associated nasal septal perforation and, as relevant to healthcare providers, discusses some of the practical aspects of post-marketing pharmacovigilance.

Bevacizumab-induced nasal septal perforation: incidence of symptomatic, confirmed event(s) in colorectal cancer patients.

PURPOSE: In breast cancer patients, Mailliez and others described that 5 of 70 patients (7%) developed a bevacizumab-induced nasal septal perforation. However, to date, no studies have reported such rates in colorectal cancer patients, who derive a survival advantage with this drug. METHODS: This study examined the incidence of bevacizumab-induced, clinically symptomatic, otolaryngology specialist-confirmed nasal septal perforation among 100 patients who had been consecutively-treated for metastatic colorectal cancer. RESULTS: The incidence of nasal septal perforation was 1% (95% confidence intervals: -0.95% to 2.95%). This single adverse event was successfully managed conservatively. Within the whole group, 94 had been treated with bevacizumab at 5 mg/kg every two weeks, except for four patients treated at higher doses. The median number of bevacizumab doses (range) was seven (1-96). Concomitant chemotherapy had been prescribed to all patients, consisting of oxaliplatin, 5-fluorouracil, leucovorin, as per one of the FOLFOX regimens (44 patients); irinotecan, 5-fluorouracil, leucovorin, as per the FOLFIRI regimen (13 patients); both these regimens and no other (five patients); or a different regimen (38 patients). CONCLUSION: Nasal septal perforation from bevacizumab occurs infrequently among colorectal cancer patients.