38
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• Combined use of neo-adjuvant and adjuvant chemotherapy, applied in 10 out 38 patients (26.0%). Of 10 patients, 8 (21.0%) were RIBAS and 2 (5.0%) were sBAS.
• Surgery without chemotherapy, applied in 11 (29.0%) out of 38 patients. Of 11 patients, 8 (21.0%) were RIBAS and 3 (8.0%) were sBAS.
Regarding the chemotherapeutic regimens, however, a doublet strategy (a total of 2 chemotherapeutic drugs between neo-adjuvant and adjuvant therapy) was the most commonly used, while none of patients underwent single agent therapies.
Concerning the genomic landscape of AS, we compared our sequencing results with the two main studies on AS genomic (“The Angiosarcoma Project” by Painter et al;
“Genomic landscape of angiosarcoma: A targeted and immunotherapy biomarker analysis of 143 patients” by Espejo et al.).
Espejo et al. published preliminary data of 143 AS (from various localization) from which 53 tumors samples also underwent whole transcriptome sequencing (WTS). The primary aim of the authors was to describe molecular biomarkers which could justify the use of immunotherapy (IO-therapy) not only in the setting of cutaneous AS, but also for other localizations. They found the highest rate of IO-therapy biomarkers in HNASs while, breast and extremity AS present the highest MYC-copy number aberration (respectively in 63% breast and 40% extremity ASs). DNA-damage-repair (DDR) alterations were present in 56% of cutaneous ASs and ranged from 12-27% in other subtypes. RAS and PI3K alterations ranged from 6-27% across all subtypes. Their conclusions on overall samples underwent to WTS were:
• Canonical cancer pathways alterations were detected in 86% of AS cases.
• The most mutated genes were TP53 (29%), ARID1A (17%), POT1 (16%), and ATRX (13%).
• MYC-Amplification was found in 23% of cases.
• HNAS had more frequent markers of potential IO-therapy response (as well as DDR alterations).
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• ARID1A is possibly associated with overactive EZH2*, a target of tazemetostat.
• In Breast and Extremity ASs MYC amplification suggests a role targeting cell cycle via cyclin-dependent kinase or bromodomain inhibitors.
• RAS and PI3K are mutated in a low percentage of cases, explaining the limited benefit of tyrosine kinase inhibitors in AS.
In “The Angiosarcoma Project” (Painter et al.), 47 germinal (blood or saliva) matched tumor samples from 36 patients with various localization AS were collected. Only 2 out of 36 patients had breast AS. Genomic analyses found TP53, KDR, PIK3CA, GRIN2A and NOTCH2 as the most frequently altered genes.
PIK3CA is one of the most frequently mutated genes of the 47 samples (in 21%, i.e., 10 out of 47 samples). Nine out of 10 were Primary Breast Angiosarcomas. Although PI3K alterations were already found in previous AS studies, PIK3CA mutations are brought to light for the first time with this study.
In eight out of nine samples, PIK3CA was altered in two enriched regions with activating somatic mutations and none of them were on hotspots previously reported for AS.
Experiments in cancer cell lines demonstrated that these PIK3CA mutations were significantly more dependent on PIK3CA than lines with wild type PIK3CA.
These data strongly suggest that the PIK3CA mutations that were detected in this AS cohort are likely to be activating and sensitive to PI3Kα inhibition.
Their conclusions on overall samples underwent to WES were:
• For all 47 AS samples, the median TMB in the full cohort was 3.3 mutations per megabase (Mb).
• HNAS samples had higher median TMB than all other AS subclassifications (≥10 mutations per Mb).
• 9 out of the 10 samples with high TMB were HNFS AS (characterized by dominant signature related to damage from UV light).
• The single sample with a high TMB not characterized by UV light exposure damage was a patient with cutaneous radiation associated AS of Breast with Lynch syndrome.
• None of the other 35 non-HNFS tumor samples harbored this dominant
41 mutational signature.
• High TMB has been reported (HNFS) as a possible biomarker for response to immune checkpoint inhibition
While the aforementioned studies analyzed AS from various localization and, for breast AS, summarily evaluated genomic data from RIBAS and sBAS, we only collected breast AS samples and compared RIBAS with sBAS genomic data.
We analyzed 22 tumoral patient samples for a total of 24 tumoral samples (for one patient were available 3 tumoral samples).
About recurrent mutations in RIBAS, TTN, OBSCN, SF3B1, DNAH6, FLG2, F5, ASHL1 were the most frequent involved genes. They are all very long genes considered unconventional cancer drivers that, however, may be stochastically affected at a random point due to radiation exposure. Because all these gene are not considered actionable, biological agents may fail in RIBAS treatment due to the lack of mutated targets (such as kinases). On the other hand, since the products of these genes are principally involved in musculoskeletal and transcriptional system, in cytoskeletal arrangement, and in microtubules organization, this seems to explain why microtubule and mitotic spindle inhibitors like taxanes, eribuline, vincristine, and vinblastine are effective against RIBAS.
Actionable genes like MTOR and MAP3K21 (mutated in RIBAS and unknown origin cases; see Table 3) were mutated with a lower frequency than the above genes, but together with amplified FLT4, they may represent rational targets for drugs like everolimus, MAPK pathway inhibitors, or anti-VEGF therapies in selected cases.
Regarding CNAs:
• The most statistically significant amplification in RIBAS samples belongs to 8q21.21 region, which is related to MYC gene. The frequency of this CNA in our cohort (9 out of 14 RIBAS samples, 64.3%) is in line with those described in the literature32. Despite “WHO classification of Tumor, Breast Tumors;
IARC 2019” consider MYC-amplification by FISH and MYC overexpression by IHC as desirable pathological criteria to distinguish RIBAS form sBAS, we also found a statistically significant copy number amplification of MYC
42 in sBAS.
Combining these findings with use of MYC amplification by FISH to differentiate AVLs (benign lesions) from RIBAS (malignant lesions) we can conclude that MYC amplification seems to be associate to malignant and aggressive tumor behavior regardless of nature of lesion (sporadic or radiation-induced).
• In at least two cases of RIBAS we found 5q35.3 region amplification, in which genes encoding for VEGF3-receptor (FLT4) are located. This may be an actual target for anti-VEGF therapy and the fact that only 2 out of 14 RIBAS cases have such amplification may lead the ground for genome informed treatment decision.
• In both RIBAS and sBAS cases, the most statically significant deletions involve genes coding for collagen a1 chain (COL9A1, COL11A1, COL19A1, COL13A1, COL16A1). This event can be explained with fibroblast transdifferentiation into endothelial cells, which lose the collagen production ability. Clinical significance of abovementioned genic loss of function can related with malignant behavior of tumor because this collagen genes family may be involved with basal membrane (BM) function and cell anchorage.
When the lesion loses physiologic PM function and cells can move, the AS becomes capable of deep tissue infiltration and hematogenous spreading.
This study demonstrates that:
• Main RIBAS genes mutations involve chromosome regions coding for transcription factors and cytoskeletal, microtubules, musculoskeletal associated proteins. This seems to explain the lower effectiveness of target therapies compared with antineoplastic drugs (taxanes, eribuline, vinblastine, vincristine, anthracyclines). The RIBAS involvement of these genes is explained by radiotherapy-induced stochastic DNA damage that, for probabilistic reasons, affects the longer genes.
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• The lower mutational frequency of actionable genes (MTOR, MAP3K21), although it may justify an approach with target therapies, deserves further analysis.
• MYC CN amplification involve not only RIBAS but also sBAS.
This seems to testify that, in contrast with WHO classification of Tumor, MYC amplification is not a typically features of radiation-induced AS alone.
This CNA may be interpreted as property of malignant lesions (deep tissue infiltration and hematogenous spreading).
• The amplified region which contain VEGF3-receptor gene (FLT4) may be a target for biologic therapies with anti-VEGF drugs.
• Collagen a1 chain gene family deletions (found in both RIBAS and sBAS cases), combined with F5 (coagulation factor V) and FLG2 (Filaggrin 2) mutations and PLGLB1 and PLGLB2 (Plasminogen Like B1 and B2) CN deletion, can be interpreted as transdifferentiation feature of ASs’ cells. Such mutations cause loss of function of PM and loss of cell anchorage, and this can be related to malignant cells behavior manifested with deep tissue infiltration, hematogenous and lymphatic spreading, and microsatellite metastasis.
Our study is the first to specifically address the genomic aberration of RIBAS in the effort of linking their physiopathology with clinical outcomes. We expect that our results will shed light on novel prognostic and predictive biomarkers in this neglected but increasingly frequent tumor.
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