1 UNIVERSITÀ DEGLI STUDI DI GENOVA
SCUOLA DI SCIENZE MEDICHE E FARMACEUTICHE CORSO DI LAUREA IN MEDICINA E CHIRURGIA
TESI DI LAUREA
THE GENOMIC LANDSCAPE OF RADIOTHERAPY-INDUCED BREAST ANGIOSARCOMA: A MULTICENTRIC TRANSLATIONAL
COHORT STUDY
Relatore: Egr. Prof. Gabriele Zoppoli Correlatore: Gent.ma Dr.ssa Anna Garuti
Candidato: Nicolò Gilardi
Anno Accademico 2021-2022
2 Sommario
INTRODUCTION 4
1.1EPIDEMIOLOGY OF ANGIOSARCOMA 4
1.2ANGIOSARCOMA ETIOLOGY AND PATHOLOGY 5
1.3BREAST ANGIOSARCOMA 6
1.3.1DIFFERENTIAL DIAGNOSIS OF BREAST AS 8
1.4RADIATION-INDUCED-BREAST-ANGIOSARCOMA 9
1.4.1CLINICAL PRESENTATION 9
1.4.2PHYSIOPATHOLOGY OF RIBAS 10
1.4.3ATYPICAL VASCULAR LESION (AVL) AND ITS PATHOLOGICAL FEATURES 11 1.5DIAGNOSTIC APPROACH AND CURRENT TREATMENT STRATEGIES 14
1.5.1IMAGING TECHNIQUES AND BIOPSY 14
1.5.2PATHOLOGICAL ANALYSIS 14
1.5.3TREATMENT STRATEGIES 17
THESIS OBJECTIVE 19
2.1PRIMARY AIMS 19
2.2SECONDARY AIMS 19
MATERIAL AND METHODS 20
3.1STUDY DESIGN 20
3.2PATIENTS’ ENROLLMENT 20
3.3BIOLOGICAL SAMPLE ACCESSIONING, PROCESSING, AND ANALYSIS 21
3.3.1SECTION CUTTING: 21
3.3.2SECTIONS DEPARAFFINATION AND DNA EXTRACTION 21
3.4QUALITY CONTROL (Q.C) AND QUANTIFICATION OF THE EXTRACTED DNA 22 3.4.1FLUOROMETRIC EVALUATION WITH QUBIT DNABR OR HSASSAY 22
3.4.2CAPILLARY ELECTROPHORESIS WITH SCREENTAPE 22
3.4.3REAL TIME PCR 22
3.5LIBRARIES PREPARATION FOR CORE EXOME EF AND SEQUENCING 23
RESULTS 25
4.1DEMOGRAPHIC AND CLINICAL FEATURES OF ANALYZED CASES 25
4.2QUALITY CONTROL AND SEQUENCING RESULTS 28
4.3LIBRARY PREPARATION FOR CORE EXOME EF AND THEIR SEQUENCING 30 4.4RECURRENT MUTATIONS DEFINE THE GENOMIC LANDSCAPE OF RADIOTHERAPY
INDUCED AND SPORADIC BREAST ANGIOSARCOMA 31
DISCUSSION 38
BIBLIOGRAFIA 44
3
RINGRAZIAMENTI 47
4
INTRODUCTION
1.1 Epidemiology of Angiosarcoma
Angiosarcoma (AS) represents 1–2% of soft tissue sarcomas, which in turn comprise less than 1% of adult malignancies1.
The incidence of angiosarcoma has raised over the past 30 years but whether this is a true increase is not clear. The expansion in cases may be related to a substantial increase in the use of radiotherapy, better medical management, or a better histopathological knowledge and definition of this tumor.
AS has similar incidence between sexes and can develop at any age, but is more frequent in elder age2.
AS subtypes can further be categorized by their localization.
The cutaneous form is the most common, accounting for about half of all cases, with head and neck (HNAS) as the most frequently involved region (60% of all AS). These tumors are more frequent in elderly people and, in early stages, can be confused with cellulitis or skin trauma lesions while, in the later stages, HNAS can be misdiagnosed for squamous- cell cancer, Merkel cell carcinoma or Kaposi sarcoma.
Only 10% of all AS occur in deep soft tissues (deeper subcutaneous tissues, soft tissues of the upper and lower extremities, the abdominal and chest wall, peritoneum, retroperitoneum, and mediastinum).
This form of AS can also develop at any age but is more frequent in patients between the ages of 60 and 70 years. If untreated, these AS can enlarge and lead to ulcerations and/or hemorrhages that, especially in abdominal or thoracic locations, may cause a very poor prognosis. The remaining cases are found in parenchymal organs such as breast, heart, lung, bone, spleen, and liver. Cardiac AS is most-common differentiated malignant neoplasm of the heart and accounts for 10–15% of primary cardiac malignancies.
About AS metastases, we know that this tumor has a high tendency
for metastatic multifocal disease. The dissemination of this tumor is predominantly hematogenous, early and aggressive, with lung as favorite target of AS metastases. Other frequent sites may include the lymphatic system (lymph nodes), liver and bone3.
5
AS localization Number (%)
Head and Neck 144 (27.0)
Breast 105 (19.7)
Extremities 82 (15.3)
Trunk 51 (9.5)
Liver 32 (6.0)
Heart 25 (4.7)
Bone 19 (3.6)
Spleen 14 (2.6)
Other or unknown 62 (11.6)
Table 1: Distribution of AS for localization, pooled data from 534 patients. Courtesy of Dr Robin J Young, School of Medicine and Biomedical Sciences, UK)2
1.2 Angiosarcoma etiology and pathology
AS is a malignant, locally aggressive tumor showing morphological or immunophenotypic evidence of extensive infiltrating overgrowth of vascular endothelial cells. It often presents with lymph node infiltration and distant metastases4.
AS typically arise spontaneously, however there is evidence of malignant transformation in the context of pre-existing benign vascular lesions. Several risk factors for AS (table sotto) are well described in literature as Stewart-Treves syndrome, to date a rare form of angiosarcoma resulting from chronic lymphedema induced by total mastectomy (0.03%
of patients surviving 10 or more years after radical mastectomy)2,5.
In analogy with Stewart-Treves syndrome, which explains how lymphoedema may be one causal etiological factor in the development of breast angiosarcomas after breast cancer surgery, lymphoedema related to Milroy’s disease and chronic infections (such as filariasis) has been also linked to the development of angiosarcomas.
Milroy’s disease, a rare genetically determined disease, is commonly associated with an inheritance autosomal dominant pattern of mutated FLT4 gene. FLT4 encode, on the long arm of chromosome 5 (5q35.3) for VEGF-R3 (vascular endothelial growth factor receptor
6 3), an essential protein for proliferation, migration, and survival of lymphatic endothelial cells. FLT4 mutation cause developmental alterations in the lymphatic system with subsequent chronic and painless lymphedema, which can lead to AS6.
Radiotherapy is an independent risk factor for AS well described for BC therapy but not exclusive to that setting.
Literature also suggests that mutations in the DNA repair genes, BRCA1 and BRCA2, predispose to AS after BC treatment. In this connection, Familiar syndromes as neurofibromatosis, Maffucci syndrome and Klippel-Trenaunay syndrome are also associated with AS.
Chemicals (vinyl chloride, thorium dioxide, arsenic, radium, and anabolic steroids) also seem to be associated with the development of this tumor, particularly within hepatic AS.
The role of immunosuppression in the onset of AS remains uncertain. Epidemiological studies suggest an association between AS and Acquired Immune Deficiency Syndrome (AIDS), but this hypothesis has not yet been confirmed, additionally, the small casuistry of AIDS-related AS may be misdiagnosed Kaposi’s sarcoma. Although Human Herpes- Virus-8 (HHV-8) has a proven role in Kaposi’s sarcoma pathogenesis, evidence for his similar role in AS is lacking2.
Concerning known AS biomarkers, vascular-specific molecules as CD31, CD34, ERG, FLI1, VEGF, Factor VII are described as increased. In this regard, upregulation of overmentioned genes and overexpression of VEGFR can cause endothelial cell expansion, angiogenesis, and vascular leaks. Interestingly, KDR mutations has been seen in breast AS regardless radiation therapy exposure. On the other hand, c-MYC is typically amplified in radiation induced AS as well as in lymphedema associated AS. In addition to that, amplification of FLT4 has been detected in 25% of secondary AS4.
1.3 Breast Angiosarcoma
AS of the breast is rare, accounting for 1% of all soft tissue breast tumors7. It can be sporadic (sBAS) or secondary to certain types of injury, typically radiation therapy in the setting of adjuvant treatment for previous breast cancer (RIBAS).
7 The origin of AS might be clinically relevant because there are some evidence that tumor behavior depend on this variable2, more specifically we can distinguish:
• Cutaneous Breast AS
• Soft-Tissue Breast AS
• Lymphedema-Associated Breas AS
• Radiation-Induced Breast AS.
The clinical presentation may differ between primary and secondary breast AS. The first can occur with a palpable swelling mass which may grow rapidly. On the other hand, secondary breast AS has a wider range of possible presentations. It may present as painless bruising (usually multifocal) or a palpable mass with possible marks like purplish discoloration, hematoma-like swelling, eczematous rash and diffuse breast swelling7. Consequently, because AS may presents insidiously with purple or red skin changes, it can be easily mistaken for bruising or benign skin alterations, leading to delayed investigation and diagnosis with a subsequently worse prognosis. This scenario has, as clinical consequences, an increase size of tumor over time, deep tissue infiltration, skin ulceration, contaminated resection margins (if the patient is available to surgery) and multifocal disease2. Breast AS, as other angiosarcomas subtype, is principally characterized by hematogenous spread. Lungs are the most common site of metastases and may lead to pleural disease, hemorrhagic pleural effusion and/or pneumothorax.
Liver, bones, lymph nodes and soft-tissue are also common sites of metastatization2,3.
A: sBAS presentation; courtesy of Ifrah Ahmad Qazi, Sher-e-Kashmir Institute of Medical Sciences8 B: RIBAS after eight year after adjuvant radiation; courtesy of Tania
K. Arora, Virginia Commonwealth University School of Medicine7.
A B
8 1.3.1 Differential diagnosis of Breast AS
AS, being a tumor originating from endothelial cells, is included in the vascular tumors board2,9. Familiar and clinical history can provide useful information about the nature of breast lesion although to achieve a specific diagnosis an expert histological assessment is mandatory2. Breast AS can be misdiagnosed with Capillary Hemangiomas which are typically solitaries, well circumscribed and develop during infancy. Classic Kaposi Sarcoma generally rise in elderly man, in the Mediterranean area, characterized by multiple vasculo-cutaneous lesions on distal limbs. On the other hand, AIDS-Related- Kaposi’s Sarcoma initially presents with small pink patches before developing the classic blue-red plaques and nodules both on skin and mucous membranes. It can cause gastrointestinal, splenic and lung lesion also. Another vascular tumor that can make differential diagnosis with AS difficult is Epithelioid hemangioendotheliomas. The latter usually have a good prognosis and presents in adulthood as solitary, painful, soft-tissue lesions; it can spread, through lymphatic system, to the liver, lung, or bone.
Hemangiopericytomas instead, are a group of solitary fibrous tumors which typically comes with masses involving deep soft tissue at legs level. These tumors and hypoglycemia can occur together due to insulin-like growth factor (IGF) secretion by the tumor. In conclusion, all the above mentioned tumors are less aggressive than conventional high-grade AS, and their excision with lymph-node dissection is likely to achieve long-term control2.
Reactive and benign vascular tumors
• Capillary haemangiomas
• Juvenille haemangioma (strawberry naevus)
• Cherry angioma (Campbell de Morgan spot)
• Pyogenic granuloma
• Cavernous haemangiomas
• Epithelioid haemangioma
• Vascular ectasis (naevus flammus, spider naevus)
• Angiomatosis
• Postradiation atypical vascular lesion Intermediate grade vascular
tumors
• Kaposi’s sarcoma
• Epithelioid haemangioendothelioma
9 Malignant vascular tumors • Angiosarcoma
Tumors of perivascular cells • Haemangiopericytoma (solitary fibrous tumor)
Table 2: Classification of vascular tumors; courtesy of Dr Robin J Young, School of Medicine and Biomedical Sciences, Sheffield, S10 2RX, UK)2
1.4 Radiation-Induced-Breast-Angiosarcoma
Radiation-Induced-Breast-Angiosarcoma (RIBAS) a rare malignant tumor of the breast, due to radiation therapy, with a vascular endothelial origin. Despite its relative low incidence (1-3 cases / 1000 BC radiotherapy treatments), its absolute incidence is expected to rise over time due to the increasing use of conservative surgical approaches for early BC treatment, followed by postsurgical radiotherapy10–13. Clinically, while SBAS rises from breast parenchyma, RIBAS typically affects the part of breast dermis inside the radiation field. Only sporadically, RIBAS may first develop in the breast parenchyma and, subsequently, involve the skin14.
After radiation exposure but before AS onset, Atypical Vascular Lesions (AVLs) can arise in areas of skin contained within treatment fields; most of these lesions develop within 3 years of radiotherapy and, despite are considerate as benign, they could predict an increased risk of angiosarcoma2.
1.4.1 Clinical Presentation
The initial signs of RIBAS and its neoplastic growth may be extremely subtle. Early lesions can appear as rash or bruises or skin thickening, often on or in the proximity of BC excision site 11,14. A gradual and progressive swelling commonly follows the first presentation. In some cases, bluish nodules grossly visible at the site of the previous scar can represent the first sign of the tumor 14. The benign aspect of the initial lesions together with the non-specific radiological presentation can concur to a delayed diagnosis with subsequent poor prognosis15.
10 Figure 1: RIBAS presentation; courtesy of R. B. Cohen‐Hallaleh 16
1.4.2 Physiopathology of RIBAS
Although the association between ionizing radiation exposure and cancer rate (particularly AS) is well‐defined thanks to epidemiologic studies, the risk of developing AS onset in dose-dependent manner is still a matter of debate.
There are several mechanisms by which radiation can cause AS. The first is the DNA damage due to direct oncogenic effect of ionizing radiation; it can lead genomic instability and cancer-related mutations also. In the literature, quite a number of gene mutations present about RIBAS have been reported and some of them may be exploited to distinguish RIBAS from sBAS:
• Tumor suppressor gene p53 inactivation9 has been found in several studies related to radiation‐associated sarcomas.
• MYC, contained in 8q24 region, is recurrently amplified in RIBAS. This suggests that MYC amplification is a relevant event in AS onset9, not only in RIBAS, but also in some chronic lymphedema‐associated AS and soft tissue sarcomas (high‐grade chondrosarcomas, proximal type epithelioid sarcomas, high‐grade myxoid liposarcomas)17. In opposition, in sBAS, AVLs and few radiation‐induced sarcomas other than AS, MYC amplification is less frequently reported9,17,18.
• FLT4 amplification has been also reported, only in association with MYC amplification, in 25% of secondary AS19. FLT4 encodes for VEGFR3 as was reported above in Stewart-Treves syndrome setting.
11
• Mutations of KDR gene (encoding the VEGF2 receptor) has been described in 10% of AS patients19. This mutation appears to be specific to breast AS regardless previous radiation exposure.
• An association may exist between BRCA1/BRCA2 (breast cancer‐related tumor suppressor genes) and RIBAS but, at present, this connection and its mechanism remains debated14.
Significant germline mutations, such as those in ATM, have not been reported in the literature to date. Howevere, further mechanisms whereby radiation may lead to AS were found:
• Sustained and protracted cellular stimulation due to ischemic damage on tissue which trying to repair itself.
• Chronic lymphedema (due to radiation) which may trigger, by abundant increase of vascular growth factors, carcinogenic pathways.
• Pre-existing benign lesion that can turn malignant because of radiation.
RIBAS can arise with a median time, from radiation-therapy conclusion, of 7.5 years (range: 6 months – 26 years)9,16. These latency periods after radiation have been shown to be shorter when compared with radiation-induced sarcomas in general (onset average time of 10-12 years)9 and with chronic lymphedema AS associated (onset average time of 10 years)14.
1.4.3 Atypical Vascular Lesion (AVL) and its pathological features
As mentioned above, AS is included in vascular lesion. A personal history of previous chest irradiation in people who present to medical attention for a suspicion of vascular lesion, atypical vascular lesion (AVL) must be considered as she is classically included in differential diagnosis of RIBAS14,20.
AVLs (or atypical vascular proliferations, benign lymphangiomatous papules, lymphangioma circumscriptum, benign lymphangioendotheliomas, acquired progressive lymphangiomas, and acquired lymphangiectasis)9 are histologically characterized by relatively circumscribed and often having wedge-shaped collections of thin-walled lympho-vascular channels lined by a single layer of endothelial cells (with flattened,
12 plump or hobnailing morphology).
Their growth pattern is branching and anastomosing while their localization is in the superficial to mid-dermis (extension into the deep dermis is rare)9,20. In this way, AVL can be histologically distinguished from AS for:
• Circumscribed and dermal location associated with chronic inflammation9.
• Absence of cytologic atypia, mitoses, endothelial cell multilayering, blood lake, necrosis and diffuse infiltration into subcutaneous tissue9,20.
In addition to histologic evaluation, immunohistochemistry (IHC) has also proved useful to differentiate AVLs from post-irradiation AS. The IHC that can be employed are:
• MYC amplification test performed with fluorescence in-situ hybridization (FISH). AVLs in fact, unlike post-irradiation AS, doesn’t show overmentioned amplification18,20.
• IHC staining for c-Myc expression.
A strong and diffuse staining >80% of cells are considered as positive. The outcomes of this technique well correlates with FISH studies and can also be useful to distinguish AS from AVLs in histologic difficult cases20,21.
About these two mentioned IHC technique, although MYC amplification and its protein overexpression in AS are highly specific (92%–100%)20, the sensitivity is quietly lower (almost 86%). A negative result (might be due to sampling or technical error), does not exclude the AS diagnosis20,22.
Finally, AVL’s etiology is poorly understood. It can be considered, according the variable opinion of experts, both as a benign lesion and as a AS precursor lesion9.
13 Figure 2: A case of atypical vascular lesion of the breast skin. (A) H&E shows anastomosing lymphovascular channels in the skin dermis; (B) The endothelial cells are
plump, without cytologic atypia or multilayering; (C) The endothelial cells are positive for ERG; (D) The lymphovascular channels are negative for SMA; and (E) The
endothelial cells are negative for c-MYC. Courtesy of H. Zhang, University of Rochester Medical Center, Rochester, NY20.
Figure 3: A case of RIBAS. (A) Low power view showing a dermis-based malignancy
14 with solid growth pattern; (B) High power view showing tumor cells with marked cytologic atypia and focal vasoformative growth; (C) The tumor cells are diffusely positive for ERG; and (D) The tumor cells are positive for c-Myc by immunohistochemistry. Courtesy of H. Zhang, University of Rochester Medical Center, Rochester, NY20.
1.5 Diagnostic approach and current treatment strategies
The diagnostic work‐up for breast AS includes imaging and biopsy. Imaging techniques may involve mammogram (MMX), ultrasound (US), magnetic resonance imaging (MRI).
AS features on MMX are usually absent or non-specific; on the other hand, MRI appears to be the best imaging modalities for AS and, consequently, for RIBAS diagnosis.
Regarding treatment, however, because RIBAS is a very rare disease, to date there is not possible to rely on evidence‐based standard treatment. In fact, most data in this regard come from small retrospective case series and case reports.
Currently, the main treatment is surgical. Chemotherapy has poorly defined role due to the lack of data. Usually, the most used anti-neoplastic class are taxane, platin, CMF, ifosfamide. Radiotherapy, on the other hand, has a controversial role given that the radiotherapy is itself the cause of this neoplasm9.
1.5.1 Imaging techniques and biopsy
Because MMX doesn’t guarantee specific radiological sign of AS, US may be a supplementary imaging technique9. In this regard, in case of RIBAS, it’s possible visualize a heterogeneous, hyper-vascular mass with combined hyper and hypo echogenic areas which disrupting the architecture of the breast9,14. Comparing to MMX and US, MRI supplies a better morphologic characterization of the lesion as well as, as mentioned before, seems to be the best imaging modality. Typically, RIBAS is represented at MRI as a heterogeneous mass with low T1 and high T2 signals (in all likelihood for the vascular origin of the tumor). Another advantage of MRI is that it can also be employs to detect residual and recurrent disease after surgical excision9,23.
1.5.2 Pathological analysis
The diagnosis of RIBAS is established by histopathological confirmation through incisional or excisional biopsy of the involved skin area24.
15 Microscopically, RIBAS typically presents as a dermal-centered lesion with variable grade of infiltration of underlying adipose tissue, breast stroma an parenchyma20. The tumor is characterized by diffuse or multifocal vascular proliferation with irregular, aberrant, angulated and anastomosing vascular structures consist of endothelial cell multilayering9,20.
From a cytological point of view, malignant cells in post-radiation angiosarcoma typically show nuclei with vesicular chromatin, prominent nucleoli, and variable mitotic activity.
Poorly differentiated cases frequently show solid tumor growth, marked nuclear atypia and necrosis. Despite the vast morphological heterogeneity, in consideration of the highly aggressive clinical course, tumor grading is not recommended, and are by definition considered high grade tumors.
Figure 4: Tumor cells with large nuclei and prominent nucleoli (H&E; 40×). Courtesy of Frederico José Patrício Bonito MD, Hospital Garcia de Orta E.P.E., Portugal9.
RIBAS is consistently positive for immunohistochemical staining of CD31 and ERG, two sensitive and specific biomarker of vascular differentiation (Factor VIII, CD34 and FLI1, others vascular biomarkers, may also be positive)4,9,17,20,25. In rare cases, especially those with epithelioid features, can express EMA and cytokeratins25. Of note, tumors frequently show intense and diffuse immunoreactivity for c-MYC, consistent with MYC gene amplification17.
16 Figure 5: CD31positive cells, endothelial cell marker(4×). Courtesy of Frederico José
Patrício Bonito MD, Hospital Garcia de Orta E.P.E., Almada, Portugal9.
Figure 6: Features of a moderately differentiated cutaneous AS. The neoplasm is composed of infiltrating and anastomosing vascular structures lined by atypical
17 endothelial cells (a). Endothelial tumor cells contain enlarged and hyperchromatic nuclei. Note focal endothelial multilayering (b). Immunohistochemically, a strong nuclear staining of endothelial tumor cells for MYC is seen (c), and FISH analysis reveals MYC amplification (d). Courtesy of Paula S. Ginter MD, Weill Cornell Medical
College, NY17.
AS may be associated with their putative precursors, i.e., atypical vascular lesions, consisting in relatively circumscribed thin-walled vascular proliferation, with absent or minimal cytologic atypia and absence of infiltrative features, lacking MYC gene alterations.
RIBAS is morphologically almost indistinguishable from primary breast angiosarcoma, and the differential diagnosis is carried out by evaluating the following diagnostic criteria (“WHO classification of Tumor, Breast Tumors; IARC 2019”):
Essential:
• previous irradiation of the field (usually > 3 y)
• Predominantly located in dermis and subcutis
• Infiltrative growth, with dissection of adipose tissue and breast stroma
• At least focal atypia
Desirable:
• C-Myc overexpression by IHC
• MYC amplification by FISH
1.5.3 Treatment strategies
Surgical treatment has a leading role in the therapy of RIBAS.
In literature, surgical technique differs among the studies and consist of local excision, wide excision, and mastectomy. Mastectomy with negative margins is considered the standard procedure9. Furthermore, regarding the lower incidence of relapses, negative margins are thought to be more important than the type of surgery itself. However, despite a radical surgery with microscopic negative margins, the recurrence rate is still high (range from 54% to 92%)9,10,26. It can be explained with the vascular tumor nature which makes it typically multifocal with possible microsatellite lesions; this well-describe how
18 is difficult achieve true negative margins in AS with surgery alone. In spite of axillary dissection is the gold standard treatment (in association with mastectomy) for BC, in breast AS this is not routinely necessary because this tumor rarely involves lymph nodes9,14,27. However, lymphatic dissection should be considered if AS lesion is extensive and/or within short distance of draining lymphatics. Most tumor recurrences develop within 1 year after surgery, usually along the mastectomy scar or with other lesions arise for hematogenous dissemination2,9.
The optimal chemotherapy regimen for RIBAS is debated. Indeed, the relative efficacy and the most effective combination of drugs to be used are unclear. Furthermore, the rarity of the disease does not allow to develop a molecular tailoring of biological agents such as for more common neoplastic disorders.
A partial agreement seems to exist relating to adjuvant chemotherapeutic agents choice (after resection)28. The most common drugs are anthracyclines, taxanes, ifosfamide and cyclophosphamide, usually administered weekly. Although kinase inhibitors are not conventional drugs in AS therapy, there are three phase II trials investigating weekly paclitaxel, sorafenib and imatinib, which appear to demonstrate a possible role in the treatment of RIBAS9,27,28.
Although the AS and RIBAS pathogenic pathways are not fully understood yet, several groups have shown interest in the potential of antiangiogenic molecules, like bevacizumab. Through randomized trial has been tested he addition of bevacizumab to paclitaxel, which shown both lower response rate and higher toxicity than paclitaxel alone28; consequently, anti-VEGF/paclitaxel combination is not recommended.
Even though weekly paclitaxel has been shown to be both active and well tolerated, doxorubicin-based chemotherapy remains the first line standard regimen for metastatic or unresectable AS28.
The use of radiotherapy in RIBAS, as adjuvant or neo‐adjuvant treatment, is unclear and controversial, given the fact that the tumor itself is induced by radiation9,14. There are, in literature, few studies which have tried to show how radiation-therapy may improve recurrence rates. In one of them, a small study including thirty-five patients with both primary and radiation‐induced AS of the breast found that either conventional or hyper- fractionated adjuvant radiation, improved the rate of recurrence29. Furthermore, also one
19 systematic review suggested an improved local control through the addition of radiation- therapy to surgery although with a low evidence level9. Promising results also seem to come from the addition of hyperthermia to radiotherapy29.
Smith T. et al. provided evidence of the potential curability of RIBAS with hyper- fractionated accelerated re‐irradiation therapy (HART)9. Interesting data can be gleaned from this study: for example 79% of enrolled patients achieved the disease control with a significantly higher long‐term follow‐up compared to typical intervals to recurrence9,30; moreover, the authors also noticed that the disease progression, in the recurrent cases after HART, was documented only in areas outside the radiation field of HART, suggesting the potential benefits with more extensive irradiation9,30. According to the authors, HART should be used with larger field margins, in thrice‐daily fractions. Moreover a moderate total doses should be conveyed in small fractional doses over a short overall treatment period9,28,30.
THESIS OBJECTIVE
The thesis purpose is to characterize a large cohort of RIBAS cases from both a genomic and clinical points of view.
2.1 Primary aims
• To characterize a numerically adequate cohort of RIBAS through whole exome sequencing, comparing it in a 3:1 proportion with sBAS cases.
2.2 Secondary aims
• To correlate sequencing data with clinical outcomes
• To generate a genomic landscape of RIBAS
• To characterize potentially deleterious germline mutations of RIBAS patients
• To compare RIBAS genomics data of RIBAS with those of sBAS
20
MATERIAL AND METHODS
3.1 Study design
The present project was a translational, retrospective multicenter, international study on samples of RIBAS. The translational part was designed as case/control study in a 3:1 proportion (3 represent RIBAS samples; 1 represent sBAS samples). Tumor and germline samples were collected and, before sequencing, were subjected to quality control analyses from all enrolled patients. Specimens were obtained by surgery or biopsy. Regarding retrospective part of this study, it’s been designed to clinically characterize the breast AS and associate it with its genomic features.
3.2 Patients’ enrollment
To collect clinical data and samples we generated a survey with which we asked the other centers for their willingness to participate at this study. To be enrolled, the patients or a family member (in event of death) had to subscribe a written informed consent for the treatment of clinical data and, in case of availability, of tissue samples.
This study included both patients (of any sex and age) who presented histological diagnosis of breast AS induced by radiotherapy as adjuvant therapy for previous breast cancer (RT exposure had to be >85% of the predicted) and patients with sBAS; the diagnosis of AS had to be performed in the last 20 years. Regarding RIBAS we mean people who had a prior breast tumor (or other tumor) which request radiotherapy that involved breast; regarding SBAS, we mean people who didn’t receive chest radiotherapy previously, whether he has had a tumor or not.
We excluded patients with known presence of hereditary conditions predisposing to angiosarcoma and with Stewart-Treves Syndrome.
Retrospective data were collected in a CRF through OpenClinica, a comfortable open- source clinical data management system long used by our research group, and then we extract these data on a excel sheet that we upload on R-Studio to obtain clinical information about our cohort.
21 Concerning sample collection, we obtained formalin-fixed, paraffin-embedded (FFPE) biological samples deriving from AS biopsy and/or surgery through the collaboration between our center (IRCCS Ospedale Universitario Policlinico San Martino, Italy), IRCCS Istituto Nazionale Tumori Milano (Italy) and Katholieke Universiteit Leuven (Belgium). For each case, we obtained a source of germline material from either uninvolved breast tissue or uninvolved lymph nodes.
3.3 Biological sample accessioning, processing, and analysis
3.3.1 Section cutting:
Three 8 μm sections of tissue preserved FFPE were used for DNA extraction. The first and last sections were stained with H&E for the assessment of the percentage of tumoral infiltration. A minimal cancer cell fraction of 30% was required for sample analysis.
3.3.2 Sections deparaffination and DNA extraction
DNA was extracted using a semi-automatic extractor and the Maxwell RSC DNA FFPE AS1450 Thecnical Manual # TM437 KIT. This extraction method involves the collection in a test tube, with the aid of a scalpel blade, of the material of the sections from which the DNA must be extracted. Subsequently, mineral oil was added for dewaxing. The tubes were then placed in a preheated thermoblock at 80 ° C for 2 minutes. A solution containing 224µL of Lysis Buffer, 25 µL of Protein Kinase (PK) and 1µL of BlueDye per sample was prepared and added to the tubes and then placed in a thermoblock for 30 minutes at 56 °C. After thirty minutes, the tubes were transferred to a new thermoblock preheated to 80 ° C for 4 hours. At the end of this period the samples were left to cool at room temperature for 5 min. Subsequently, 10µl of RNaseA were added to the aqueous phase (blue) for a further room temperature incubation lasting for 5 minutes.
The extraction cartridge with the plunger in position 8 was then set up on the appropriate slide. 50µL of ultra-pure molecular biology water was dispensed into the collection tube.
The aqueous phase (blue) was taken from the Eppendorf and added to the contents of sump number 1 of the cartridge. For the extraction, the “maxwell RSC DNA FFPE kit”
protocol was chosen from the menu of the Maxwell RSC semi-automatic extractor following the instructions Maxwell RSC Instrument Operating Manual # TM411.
22 3.4 Quality control (Q.C) and quantification of the extracted DNA
The analyzed material, coming from FFPE blocks, required an accurate analysis in qualitative and quantitative terms of the sample. The DNA was therefore subjected to different methods of analysis:
• Fluorometric evaluation with Qubit RNA HS Assay Kit Q32852
• Capillary electrophoresis with ScreenTape
• Real Time PCR
3.4.1 Fluorometric evaluation with Qubit DNA BR or HS Assay
The test is highly selective for DNA and does not quantify RNA, free proteins or nucleotides, solvents, detergents, and salts. It allows the accurate quantification of concentrations of highly sensitive DNA samples (HS: between 0.005ng/µL and 120 ng/µL). For this procedure, 3µL DNA were added to a solution containing 196µL of Buffer + 1µL of dye.
3.4.2 Capillary electrophoresis with ScreenTape
DNA analysis via ScreenTape, which provides rapid, automated, and reliable electrophoretic separation of incoming genomic DNA samples, was performed on the Aglilent 2200 using the Genomic DNA ScreenTape kits (5067- 5365) with Genomic DNA Reagents (Ladder and Sample Buffer) (5067- 5366) This method allows an objective assessment of the integrity of genomic DNA (gDNA), provided by a numerical measurement of the DNA Integrity Number (DIN).
Furthermore, the percentage of fragments between 200 and 10000 base pairs (bp) were measured for each sample.
3.4.3 Real Time PCR
Samples stored in FFPE generally produce poor quality and highly degraded DNA.
Before proceeding with analyses such as NGS or array it’s necessary to evaluate the functionality of the DNA. For this investigation the FFPE QC Infinium kit code WG-321- 1001 was used on quantitative PCR (Q-PCR) 7900HT Applied Biosystem instrumentation. With Qubit DNA Genomic Assay were quantified 2 ng of DNA which were amplified in a 96-well plate using 5µL of SYBR PowerTrack and 1µL of specific
23 primers according to the protocol of the Infimium WG-321-1001 kit following the thermal cycle reported here:
The test was performed in triplicate.
At the end of the amplification, the mean of the cycles of quantification (Cq) of the replicates was calculated and the outliers were omitted. Delta Cq was calculated by subtracting the mean of the QCT Cq (control DNA supplied by the KIT) from the mean of the Cq of the samples. The selection criteria of No Template Control (NTC) wells excluded both wells with DeltaCq> 10 and wells which contain amplified material. All samples with DeltaCq less than five were considered eligible.
3.5 Libraries preparation for Core Exome EF and sequencing
The Twist Human Core Exome EF Multiplex kit was used for the project, following the protocol below:
• Fragmentation:
an enzymatic digestion of 1 minutes at 32 °C was carried out on 50 ng of gDNA and, subsequently, the fragmentation enzyme was deactivated at a temperature of 65 ° C for a time interval equal to 30 minutes.
• Ligation:
the universal twist adapters were ligated to the enzymatically fragmented DNA in a 15 min reaction at 20 ° C and then the samples were purified by a pass with magnetic beads to eliminate suboptimal sized fragments.
• Indexing:
the samples, at this point, underwent an indexing reaction or were tagged with specific Unique Dual Index (UDI), and then the material was amplified for 10 cycles in PCR. The PCR products were purified with magnetic beads.
24
• Library control:
the quality of the libraries obtained was evaluated by subjecting 1 µL of each library to an electrophoretic run-on Tape Station 2200. The purified PCR products of the samples have a size of about 400 bp, perfectly in the range described by the protocol (375-425 bp).
• Library pool preparation:
libraries were collected in 10 pools containing approximately 7 pooled samples: 5 pools with tumor tissue and 5 with the respective germinal tissue The obtained pools were lyophilized at room temperature for 30 minutes.
• Hybridization of pools and capture:
the lyophilized pools were rehydrated with 27µL of an Exome probe solution preheated to 95 ° C for 16 hours at 70 ° C in a thermal cycler.
• Bead purification:
hybridized pools were purified with a magnet pass after 30 minutes incubation with streptavidin beads. The product was then washed with Wash Buffer and left to air dry for a few minutes, avoiding the marbles to dry out.
These were then rehydrated with 45µL of ultra-pure water.
• Post capture:
the libraries then carried out 8 amplification cycles with KAPA HiFi HotStart Ready mixes and ILMN primers by Illumina. The PCR product, namely Whole Exome Capture Library, after purification, was checked with Tape Station 2200 before being sequenced.
• Sequencing:
5 µl of each pool were withdrawn and denatured with 10 µl of 0.2 N NaOH at room temperature for 5 minutes. The pool (1.5 nM) was further diluted in Hybridization Buffer (HT1) in two steps until the final molarity of 10 µM.
The libraries were loaded on MiSeq Dx (Illumina) together with a control library (PhiX Control v3, Illumina) at 1%.
25
RESULTS
4.1 Demographic and clinical features of analyzed cases
Although the study is still ongoing, from October 1st, 2020 to October 1st, 2021, 43 patients were enrolled, of which 36 from INT and 7 from HSM. Of all 43 patients, a total of 86 samples (48 tumoral, 38 germinal) were available.
Since for 5 out of 43 patients enrolled in our study, clinical data are missing, statistical analysis was performed on 38 patients. Thirty out of 38 patients were RIBAS, while 8 were sBAS, respecting the 3:1 proportion of the protocol. Median age was 68.6 (52.1 – 75.1) for all patients, 69.8 (61.4 - 76) for RIBAS patients and 41.2 (28.6 – 53.7) for sBAS patients. This data suggests a genetic predisposition for the sBAS cases we collected, based on its lower age of onset. In RIBAS patients, the median elapsed time between the previous disease which required radiotherapy and AS onset was 8.4 years (5.5 – 12.4).
RIBAS Flow-Diagram
43 patients enrolled from HSM and INT.
Clinical data and biological samples were available.
14 patients (24 samples) were excluded following quality control (QC):
• Insufficient cellularity
• Low quality of extracted DNA
• Insufficient extracted DNA 43 patients’ clinical data has been collected on OpenClinica © eCRF:
• 30 RIBAS
• 8 sBAS
• 5 of unknown origin
62 tumor/germline matched samples from 29 patients were sent to IEO for WES analysis:
• 7 HSM patients (8 tumoral samples, 7 germline samples)
• 22 INT patients (24 tumoral samples, 23 germline samples) 86 biological samples were collected from the 43 patients:
• 48 tumorali
• 38 germinali
Genomic and statistical analysis for 22 patients (24 tumoral samples) are available.
Genomic analysis for 7 patients (15 tumoral samples) is still ongoing.
Statistical analyses were performed on 38 patients (tumors of known origin).
26 Regarding the treatment, 6 patients underwent neo-adjuvant chemotherapy (4 out of 6 were RIBAS, the remaining 2 were sBAS), 2 patients underwent adjuvant chemotherapy (none of sBAS patients were subjected to this regimen), 10 patients underwent both neo- adjuvant and adjuvant chemotherapy (8 were RIBAS, 2 were sBAS) and for 9 out of 38 patients we could not retrieve data concerning their treatment.
Taxanes, anthracyclines, gemcitabine, ifosfamide, platin, as single agent or a combination of the previous drugs, are usually applied in Breast AS as treatment regimen. Treatment strategy can be stratified according to total chemotherapeutic agents administered to the individual patient, not distinguishing between neoadjuvant and adjuvant. None out of 43 patients underwent to single agent strategy while 11 out of 43 didn’t receive any therapy.
Ten patients underwent to doublet strategy (8 were RIBAS, 2 were sBAS), the triplet and quadruplet strategy have been applied in 8 out of 43 patients (respectively: 4 RIBAS patients underwent to triplet strategy while 2 RIBAS patients and 2 sBAS patients underwent to quadruplet strategy). Of 9 patients, however, information related to treatment is missing.
About histopathological data, tumor size has a relevant role.
The median size of all the cancer samples was 3.2 cm (IQR 1.4 – 5cm), in RIBAS subgroup is 3.2cm (1.5 – 5cm), in sBAS subgroup was 3.5cm (1.4 – 4.8cm). The grade of AS on the other hand, can be noted that 6 patients were G1 (3 RIBAS, 3 sBAS), 8 patients were G2 (5 RIBAS, 3 sBAS), 8 were G3 (7 RIBAS, 1 sBAS). Of 16 of the 38 patients grading data were missing (15 were RIBAS, 1 was sBAS).
Molecular markers as proliferation index also play an important role20. Although Ki-67%
may be useful to discriminate benign vascular lesions (hemangioma, angiomatosis, AVL) from well-differentiated AS20, to date, there is no consensus on its application to distinguish high form low aggressiveness of AS. Referring to literature, the percentage cutoff value of Ki-67 index assumed in this work to differentiate low from highly aggressive AS was 30%31. A Ki-67 index greater than or equal to 30% indicate a higher aggressiveness of the tumor. On the other hand, a Ki-67 index lower then 30% suggest a lesser malignant tumor behavior31. In this way, Ki-67 index was reported as <30% in 6 out of the 38 analyzed cases (5 were RIBAS, 1 was sBAS), ≥30% in 10 out of the 38 cases (8 were RIBAS, 2 was sBAS) while that information was missing for 22 patients
27 (17 were RIBAS, 5 were sBAS). The number of mitoses per high-power field (nmHPF) was used as mitotic rate for the 22 cases mentioned above. A cutoff of 7/10 mitosis per HPF was applied for differentiate higher and lower aggressive AS. Four out of 22 cases result with a nmHPF ≥7 (all RIBAS), 2 out of 22 results with a nmHPF <7 (all sBAS), for the remaining 16 both Ki-67 and nmHPF were unavailable.
Finally, regarding the involvement of resection margins, 20 out of the 38 cases were found to be not involved by AS (16 were RIBAS, 4 were sBAS), only 7 sample cases were involved by tumor (4 were RIBAS, 3 were sBAS) while the data was missing for 11 of 38 cases (10 were RIBAS, 1 was sBAS). Nevertheless, collecting data still ongoing; all the samples will be centralized and re-analyzed from an expert pathologist of INT so that more accurate information can be obtained.
All patients (N=38) RIBAS (N=30) sBAS (N=8) AGE AT DIAGNOSIS 68.6 (52.1 – 75.1) 69.8 (61.4 - 76) 41.2 (28.6 – 53.7)
TIME FROM PRIMARY NA 8.4 (5.5 – 12.4) NA
TREATMENT TYPE
Neoadjuvant 6 (16.0%) 4 (11.0%) 2 (5.0%)
Adjuvant 2 (5.0%) 2 (5.0%) 0
Both 10 (26.0%) 8 (21.0%) 2 (5.0%)
No chemotherapy 11 (29.0%) 8 (21.0%) 3 (8.0%)
NA 9 (24.0%) 8 (21.0%) 1 (3.0%)
TREATMENT REGIMEN
Single agent 0 0 0
Doublet 10 (26.3%) 8 (21%) 2 (5.2%)
Triplet 4 (10.5%) 4 (10.5%) 0
Quadruplet 4 (10.5%) 2 (5.2%) 2 (5.2%)
No 11 (28.9%) 8 (21.0%) 3 (7.9%)
NA 9 (23.6%) 8 (21.0%) 1 (2.6%)
SIZE (cm) 3.2 (1.4 – 5) 3.2 (1.5 – 5) 3.5 (1.4 – 4.8)
GRADE
1 6 (16.0%) 3 (7.9%) 3 (7.9%)
2 8 (21.0%) 5 (13.2%) 3 (7.9%)
28
3 8 (21.0%) 7 (18.4%) 1 (2.6%)
NA 16 (42.0%) 15 (39.5%) 1 (2.6%)
PROLIFERATION INDEX (Ki-67%)
< 30% 6 (15.8) 5 (13.2) 1 (2.6)
> 30% 10 (26.3) 8 (21.1) 2 (5.3)
NA 22 (57.9) 17 (44.7) 5 (13.2)
MITOSES PER HPF (for Ki-67 missing data)
< 7/10 2 (9.1%) 0 2 (9.1%)
≥ 7/10 4 (18.2%) 4 (18.2%) 0
NA 16 (72.7%) 13 (59.1%) 3 (13.6%)
MARGINS
Involved 7 (18.4%) 4 (10.5%) 3 (7.9%)
Not Involved 20 (52.6%) 16 (42.1%) 4 (10.5%)
NA 11 (28.9%) 10 (26.3%) 1 (2.6%)
All patients (N=38) RIBAS (N=30) sBAS (N=8)
4.2 Quality control and sequencing results
In the present study, a cohort of 43 patients with breast AS was analyzed. For all the patients were collected both tumoral and germinal tissue samples from two Italian breast cancer treatment centers:
• IRCCS Ospedale Policlinico San Martino (HSM) participated with 7 patients enrolled with a total of 15 samples (1 tumoral and 1 germinal for each patient, except for one patent of which were available 2 tumoral samples) which all have passed the QC tests.
• IRCCS Istituto Nazionale Tumori di Milano (INT) participated with 36 patients enrolled with a total of 71 samples. Forty out of 71 were tumoral samples (for one patient were available 3 tumoral samples while for two patients were available 2 tumoral samples). The remaining 31 were germinal samples (free from neoplasia).
29 A total of 86 samples (48 tumoral, 38 germinal), belonging to 43 patients, were collected.
RIBAS and sBAS diagnosis were performed, by an expert pathologist of the two Italian centers according to histological and immunohistochemical criteria (see: Pathological Analysis). From histopathological point of view, to distinguish high from low aggressiveness of tumor samples, was used Ki-67% index (cutoff ≥30% for high aggressiveness) if available. If Ki-67% index was missing, was employed the nmHPF (cutoff ≥7/10 mitosis per HPF for high aggressiveness). Biopsy and/or surgical samples were preserved in formalin-fixed, paraffin-embedded (FFPE).
Extracted DNA underwent quality control and quantification through distinct analysis methods:
• Fluorometric evaluation with Qubit RNA HS Assay Kit Q32852:
highly sensitive technique for an accurate quantification of extracted DNA whose results are not affected by solvent, detergent, RNA, free protein, or nucleotides. Fluorometric reading reported the following results:
Þ For tumor tissue samples, extracted DNA mean was equal to 7.29 ng/µL with a median of 3.1 ng/µL and a range between 0.073 and 37.8 ng/µL.
Þ For germinal samples, extracted DNA mean was equal to 7.07 ng/µL with a median of 1.33 ng/µL and a range between 0.045 and 72.5 ng/µL.
• Capillary electrophoresis with ScreenTape:
technique analysis who allows an objective assessment of the integrity of genomic DNA (gDNA), provided by a numerical measurement of the DNA Integrity Number (DIN). In this regard, through this technique, was measured the percentage of DNA fragments with lengths between 200 and 10’000bp.
Tumoral Samples’ DIN average was 2.1 (range 5.0 - 1.5) while the percentage of fragments with length between 200 and 10000 bp was 84.5% (range 93.4%
-73.5%). Germinal samples (control tissue) instead, had a DIN average of 0.4
30 (range 0.0 - 3.2) and a percentage of fragments with length between 200 and 10,000 bp of 82.1% (range 93% -68.9%).
• Real Time PCR:
because Samples stored in FFPE generally produce poor quality and highly degraded DNA, before proceeding with sequencing-analysis, it has been necessary to evaluate the functionality of the samples’ DNA. RT-PCR evaluation of samples allows to select the best performing biological material for sequencing. The results we have obtained are the following:
Þ For tumor samples, DeltaCq mean was 1.9 (–0.54 - 4)
Þ For germinal samples, DeltaCq mean was 2 (–0.8 - 5.8). Only one sample had a DeltaCq value> 5.
4.3 Library preparation for Core Exome EF and their sequencing
At the end of quality control, and because HSM samples are still being sequenced, only 22 patients’ matched samples (i.e., were available the tumor and germinal samples for the single patient) were available. This result in 47 samples (1 tumoral and 1 germinal for each patient, except for one patient of which were available 3 tumoral and 2 germinal samples). At this point, libraries for Core Exome EF and sequencing were prepared.
Process consisted of fragmentation, ligation, indexing, library control, library pool preparation, hybridization of pools and capture, based purification, amplification and control of libraries produces, and sequencing:
• Libraries quality was evaluated, and their amount was measured with the Qubit and the following values were obtained:
Þ Tumor tissue libraries average was 50.17 ng/µL (range 1.43-120 ng/µL) Þ Germinal libraries averaged was 46.79 ng/µL (range 1.92-110 ng/µL)
• In library pool preparation (10 pools containing approximately 7 pooled samples), 5 pools with tumor tissue at a mean concentration of 52.19 ng/µL (range 1.43-120 ng/µL) per library and 5 with the respective germinal tissues
31 at a mean concentration of 43.2 ng/µL (range 1.92-110 ng/µL) per library.
• After amplification and purification, the 5 tumor tissue pools averaged 15.27 ng/µL (range 8.73-24.2 ng/µL) while the germinal tissue pools averaged 12.52 ng/µL (range 6.7- 19 ng/µL).
4.4 Recurrent mutations define the genomic landscape of Radiotherapy induced and sporadic breast angiosarcoma
We analyzed 22 tumoral patient samples for a total of 24 tumoral samples (for one patient 3 tumoral samples were available).
TTN SF3B1 DNAH6 ASH1L VPS13D KIF14 APOB USH2A FLG2 LRRC7 MACF1 RSC1A1 OBSCN F5 SPTA1 FLG MTOR RYR2 PRRC2C ATP8A1 KDR BSN TNR RUSC1 HSPG2 HIVEP2 GBP1 TCHH ERICH3 RCC1 AOX1 OR2L13 CWF19L2
21%
21%
21%
21%
21%
21%
21%
21%
21%
21%
21%
21%
14%
14%
14%
14%
14%
14%
14%
14%
14%
14%
14%
14%
14%
14%
7%
7%
7%
0%
0%
0%
0%
0%
0%
0%
17%
0%
0%
0%
0%
0%
0%
0%
0%
0%
17%
17%
0%
0%
17%
17%
0%
0%
0%
0%
0%
0%
0%
33%
17%
17%
17%
17%
33%
17%
Alterations Inframe Ins
Stop/Gained Missense Complex Subs Inframe Del
Frameshift Elongation Frameshift Truncation
32 Figure 7: Recurrent mutations of 20 out of 24 sequenced AS (defined tumoral samples) Fourteen out of 24 were RIBAS, 6 out of 24 were sBAS while, for the remaining 4, data on the source of disease was not available.
Table 3: More frequent and relevant mutated genes All tumoral
samples (24)
All defined tumoral samples (20)
RIBAS (14) sBAS (6)
TTN 6 (25%) 3 (15%) 3 (21%) 0
SF3B1 4 (17%) 3 (15%) 3 (21%) 0
DNAH6 4 (17%) 3 (15%) 3 (21%) 0
ASH1L 4 (17%) 4 (20%) 3 (21%) 1
(17%)
F5 4 (17%) 3 (15%) 2 (14%) 1
(17%)
FLG2 3 (12%) 3 (15%) 3 (21%) 0
OBSCN 5 (21%) 2 (10%) 2 (14%) 0
MTOR 3 (12%) 2 (10%) 2 (14%) 0
MAP3K21 3 (12%) 1 (5%) 1 (7%) 0
The most mutated genes were:
• TTN (coding for Titin) and OBSCN (coding for Obscurine), genes of musculoskeletal system
• SF3B1 (coding for subunit 1 of the splicing factor 3b protein complex), involved in RNA splicing.
• DNAH6 (belonging to dynein family), which encode for microtubules- associated motor complex proteins.
• FLG2 (involved in epithelial homeostasis), which encode for Filaggrin, a protein with skin barrier function.
33
• F5 (coding for V coagulation factor), involved in endothelial homeostasis.
• ASHL1 (coding for transcriptional activators).
• MTOR (belonging to kinase family), involved in cellular response to stresses as DNA damage.
All these genes are almost uniquely mutated in RIBAS.
In summary, many of mutated genes in RIBAS are both long genes and those involved in gene transcription, cytoskeletal arrangement) and musculoskeletal system; the latter may affect cells motility. OBSC mutation was found in 3 unknown cases of AS suggesting that also these cases could be RIBAS. Therefore, we are performing mutational signature to understand whether tumors of unknown origin are more like RIBAS or SBAS.
Recurrent copy number aberrations (CNAs)
We analyzed CNAs using Log2 ratios which has been calculated from the sequencing coverage, normalized with SNP data derived from sequencing data of non-tumor DNA samples, and smoothed by GC correction. The log2 ratios were preprocessed by outlier winsorization and segmented by penalized least square regression using the R/BioConductor package copynumber. To identify recurrent focal events, the segmented data was used as input for GISTIC2.0 and run on the GenePattern cloud server of the Broad Institute. In this way we found the recurrence of both amplificated and deleted genomic regions related to the matrix of samples used as input. Obtained data represent the probability that specific gene regions, even small ones, are amplified or deleted in a statistically significant way. The more a given region has alterations in copy number, the more the algorithm identifies region-specific peaks that include genes that assume oncogenic significance by amplification or deletion. Statistical significance is represented by the qvalue whose low values represent the reliability of the data obtained.
34 In RIBAS cases we identified several significantly altered CNAs.
Regarding amplified regions:
Figure 8: Amplified regions of RIBAS cases
• 8q24.21 (qvalue = 6,98x10-21)
This is the most recurrently amplification (9 out of 14 RIBAS cases; 64,3%).
That region is related MYC gene, which when amplified, is associated to
RIBAS according to WHO criteria.
• 5q35.3 (qvalue = 1,3x10-2)
Two cases exibities very high level amplification in the genes encoding for VEGF3-receptor.
• 17q12 (qvalue = 4,4x10-4)
This region present TBC1 genes family, which encode for protein that have a role in regulation in cell growth and differentiation. These proteins are classically activated in tumorigenesis.
35
• 16p11.2 (qvalue = 6,8x10-3)
In this region we find TP53TG genes amplified. They encode for target protein of TP53. While TP53 gene is not amplified, its target protein gene are.
• 2p11.2 (qvalue = 3,7x10-3)
This region present PLGLB1 and PLGLB2 genes, which encode for Plasminogen Like B1 and B2 protein. These proteins are involved in proteolysis and seem to be associated with soft tissue tumors.
• Chromosome 1 (qvalue = 5,4x10-4/3,44x10-3)
Chromosome 1 is frequently involved in human tumors. It has a lot of gene, and their CNAs are poorly interpretable.
Regarding delated regions:
Figure 9: Deleted regions of RIBAS cases
36
• 6q13 (qvalue = 8.9x10-11)
This region contains COL9A1 and COL19A1 genes, which encode for Collagen type IX and XIX a1 chain.
• 1p21.1 (qvalue = (2,7x10-2)
This region contains COL11A1 gene, which encode for Collagen type XI a1 chain.
• Other significant delated regions are 18q11.1, 19p13.2, 2q36.3 8q24.23, 3q22.1. All of them also contain collagen related genes.
We can notice how all delated regions in RIBAS samples mainly contain collagen related genes, which may be related with basal membrane function and cellular anchorage.
In sBAS cases we found several significant CNAs both in amplification and deletion terms.
Regarding amplified regions:
Figure 10: Amplified regions of sBAS cases
37
• 8q24.21 (qvalue = 9,6x10-1)
This is the most relevant data. MYC region (8q24.21) is statistically significantly amplified in 2 out of 6 sBAS samples (33.3%).
Regarding delated regions:
Figure 11: Deleted regions of sBAS cases
• 10q22.1 (qvalue = 1,7x10-3) and 1p35.2 (qvalue = 2,3x10-1) Both chromosomal regions contain genes related to Collagen a1 chain (COL13A1 and COL16A1).
