Sister chromatid exchange and genomic instability in soft tissue sarcomas: potential implications for response to DNA-damaging treatments

Sarcomas are rare heterogenous malignancies of mesenchymal origin characterised by complex karyotypes but no specific abnormalities. Recurrence is common and metastatic disease carries poor survival despite standard DNA-damaging radiotherapy or chemotherapy. DNA double strand breaks (DSB) are either repaired by mechanisms such as homologous recombination (HR); or result in cell death by apoptosis. Endogenous γH2AX and SCE formation are early and late events, respectively and their levels are considered surrogate measures of genomic instability. Combined γH2AX and SCE analysis were used to evaluate endogenous DNA DSB levels (and their subsequent repair) in 9 primary sarcoma cell lines and compared with well-established commercial lines. All the sarcoma cell lines had elevated γH2AX and SCE levels, but there was no correlation between the DNA DSB frequency and subsequent SCE. Typically radio-resistant osteosarcoma cells had relatively low γH2AX frequency, but high SCE counts suggestive of efficient DNA repair. Conversely, liposarcoma cells derived from a radio-sensitive tumour had high H2AX but relatively lower SCE levels that may imply inefficient DNA DSB repair. To our knowledge, this is the first report that correlates H2AX and SCE levels in primary sarcoma cell lines and may provide insight into potential response to DNA damaging-treatments

Impact of pharmacodynamic biomarkers in immuno-oncology phase 1 clinical trials

Background: Phase 1 immuno-oncology (IO) trials frequently involve pharmacodynamic (PD) biomarker assessments involving tumour biopsies and/or blood collection, with increasing use of molecular imaging. PD biomarkers are set to play a fundamental role in early drug development of immuno-oncology (IO) agents. In the IO era, the impact of PD biomarkers for confirmation of biologic activity and their role in subsequent drug development have not been investigated. Methods: Phase 1 studies published between January 2014 and December 2020 were reviewed. Studies that reported on-treatment PD biomarkers [tissue-derived (tissue-PD), blood-based (blood-PD) and imaging-based (imaging-PD)] were analysed. PD biomarker results and their correlation with clinical activity endpoints were evaluated. Authors' statements on the influence of PD biomarkers on further drug development decisions, and subsequent citations of PD biomarker study results were recorded. Results: Among 386 trials, the most frequent IO agent classes evaluated were vaccines (32%) and PD-(L)1 inhibitors (25%). No PD biomarker assessments were reported in 100 trials (26%). Of the remaining 286, blood-PD, tissue-PD, and imaging-PD data were reported in 270 (94%), 94 (33%), and 12 (4%) trials, respectively. Assessments of more than one PD biomarker type were reported in 82 studies (29%). Similar proportions of blood-PD (9%), tissue-PD (7%), and imaging-PD studies (8%) had positive results that correlated with clinical activity. Results of 22 PD biomarker studies (8%) were referenced in subsequent clinical trials. Conclusions: Most phase 1 IO studies performed PD biomarker assessments. Overall, positive PD biomarker results were infrequently correlated with clinical activity or cited in subsequent trials, suggesting a limited impact on subsequent drug development. With emerging health regulatory emphasis on optimal dose selection based on PD activity, more informative and integrative multiplexed assays that capture the complexity of tumour-host immunity interactions are warranted to improve phase 1 IO trial methodology. (C) 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

High Quality Genomic Copy Number Data from Archival Formalin-Fixed Paraffin-Embedded Leiomyosarcoma: Optimisation of Universal Linkage System Labelling

Most soft tissue sarcomas are characterized by genetic instability and frequent genomic copy number aberrations that are not subtype-specific. Oligonucleotide microarray-based Comparative Genomic Hybridisation (array CGH) is an important technique used to map genome-wide copy number aberrations, but the traditional requirement for high-quality DNA typically obtained from fresh tissue has limited its use in sarcomas. Although large archives of Formalin-fixed Paraffin-embedded (FFPE) tumour samples are available for research, the degradative effects of formalin on DNA from these tissues has made labelling and analysis by array CGH technically challenging. The Universal Linkage System (ULS) may be used for a one-step chemical labelling of such degraded DNA. We have optimised the ULS labelling protocol to perform aCGH on archived FFPE leiomyosarcoma tissues using the 180k Agilent platform. Preservation age of samples ranged from a few months to seventeen years and the DNA showed a wide range of degradation (when visualised on agarose gels). Consistently high DNA labelling efficiency and low microarray probe-to-probe variation (as measured by the derivative log ratio spread) was seen. Comparison of paired fresh and FFPE samples from identical tumours showed good correlation of CNAs detected. Furthermore, the ability to macro-dissect FFPE samples permitted the detection of CNAs that were masked in fresh tissue. Aberrations were visually confirmed using Fluorescence in situ Hybridisation. These results suggest that archival FFPE tissue, with its relative abundance and attendant clinical data may be used for effective mapping for genomic copy number aberrations in such rare tumours as leiomyosarcoma and potentially unravel clues to tumour origins, progression and ultimately, targeted treatment

Autoimmune PaneLs as PrEdictors of Toxicity in Patients TReated with Immune Checkpoint InhibiTors (ALERT)

Background: Immune-checkpoint inhibitors (ICI) can lead to immune-related adverse events (irAEs) in a significant proportion of patients. The mechanisms underlying irAEs development are mostly unknown and might involve multiple immune effectors, such as T cells, B cells and autoantibodies (AutoAb). Methods: We used custom autoantigen (AutoAg) microarrays to profile AutoAb related to irAEs in patients receiving ICI. Plasma was collected before and after ICI from cancer patients participating in two clinical trials (NCT03686202, NCT02644369). A one-time collection was obtained from healthy controls for comparison. Custom arrays with 162 autoAg were used to detect IgG and IgM reactivities. Differences of median fluorescent intensity (MFI) were analyzed with Wilcoxon sign rank test and Kruskal–Wallis test. MFI 500 was used as threshold to define autoAb reactivity. Results: A total of 114 patients and 14 healthy controls were included in this study. irAEs of grade (G) ≥ 2 occurred in 37/114 patients (32%). We observed a greater number of IgG and IgM reactivities in pre-ICI collections from patients versus healthy controls (62 vs 32 p < 0.001). Patients experiencing irAEs G ≥ 2 demonstrated pre-ICI IgG reactivity to a greater number of AutoAg than patients who did not develop irAEs (39 vs 33 p = 0.040). We observed post-treatment increase of IgM reactivities in subjects experiencing irAEs G ≥ 2 (29 vs 35, p = 0.021) and a decrease of IgG levels after steroids (38 vs 28, p = 0.009). Conclusions: Overall, these results support the potential role of autoAb in irAEs etiology and evolution. A prospective study is ongoing to validate our findings (NCT04107311)

A summary of FFPE leiomyosarcoma cases included in this study.

<p>DLR Spread – Derivative Log Ratio Spread of Array Data.</p>§ -<p>Additional fresh samples obtained and frozen before fixing in formalin.</p

Correlation of Probe log₂ ratios of paired FF and FFPE samples of 3 leiomyosarcoma cases.

<p>Pearson’s Correlation, r of log₂ ratio values of all probes on tumour DNA samples was calculated using GraphPad Prism software and statistically significant (p<0.001).</p

Two-colour Interphase Fluorescence in situ Hybridisation (FISH) Images of nuclei of cultured leiomyosarcoma cells.

<p>Most cells have five or more chromosome 11 centromere (green signals), but relatively fewer copies of the ATM region 11q22 (red signals) representing copy number deletion. Nuclei are stained with DAPI (blue). Cells were derived from short-term cultures from fresh tissue (LMS 9).</p

Frequency Plot of Common Genomic Copy Number Aberrations among 22 FFPE Leiomyosarcomas.

<p>Commonly aberrant regions are plotted as a function of their chromosomal position. Red bars to the left of the chromosome represent frequency of deletions and blue bars to the right of the chromosome represent amplifications. The heights of the bars correspond to the relative frequency of aberrations among the cases. All CNAs are detected using the FASST2 algorithm.</p

Comparison of Array CGH results in paired Fresh Frozen and Formalin-fixed Paraffin -Embedded samples from LMS 9.

<p><b>Panel A:</b> Graphical whole-genome views of copy number aberrations (CNAs) identified in both sample types showing close similarities on most chromosomes. <b>Panel B:</b> Higher resolution graphical views of Chromosome 11 showing the close similarity in gain and loss patterns detected in both sample types. <b>Panel C:</b> High-resolution views showing the most dissimilar CNA pattern detected between both sample types on chromosome 4. On Panel A, aberrations called by FASST2 algorithm are represented by blue triangles to the right (amplifications) and red triangles to the left (deletions) of the chromosomes. Double blue and red triangles/lines represent high-level amplifications and two-copy deletion, respectively. On Panels B and C, dots represent individual probe log₂ ratios plotted as a function of their chromosomal position with a moving average of probe log₂ ratios (wavy dark blue line). Aberration calls are represented by thick black lines with corresponding shaded blue areas above (amplifications) and red areas below (deletions) the zero line.</p