Genomics to select treatment for patients with metastatic breast cancer.

Cancer progression is driven in part by genomic alterations1. The genomic characterization of cancers has shown interpatient heterogeneity regarding driver alterations2, leading to the concept that generation of genomic profiling in patients with cancer could allow the selection of effective therapies3,4. Although DNA sequencing has been implemented in practice, it remains unclear how to use its results. A total of 1,462 patients with HER2-non-overexpressing metastatic breast cancer were enroled to receive genomic profiling in the SAFIR02-BREAST trial. Two hundred and thirty-eight of these patients were randomized in two trials (nos. NCT02299999 and NCT03386162) comparing the efficacy of maintenance treatment5 with a targeted therapy matched to genomic alteration. Targeted therapies matched to genomics improves progression-free survival when genomic alterations are classified as level I/II according to the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT)6 (adjusted hazards ratio (HR): 0.41, 90% confidence interval (CI): 0.27-0.61, P < 0.001), but not when alterations are unselected using ESCAT (adjusted HR: 0.77, 95% CI: 0.56-1.06, P = 0.109). No improvement in progression-free survival was observed in the targeted therapies arm (unadjusted HR: 1.15, 95% CI: 0.76-1.75) for patients presenting with ESCAT alteration beyond level I/II. Patients with germline BRCA1/2 mutations (n = 49) derived high benefit from olaparib (gBRCA1: HR = 0.36, 90% CI: 0.14-0.89; gBRCA2: HR = 0.37, 90% CI: 0.17-0.78). This trial provides evidence that the treatment decision led by genomics should be driven by a framework of target actionability in patients with metastatic breast cancer.

Genomic profile analysis of leiomyomas with bizarre nuclei and fumarate hydratase deficient leiomyomas: Strengths, weaknesses, and limitations of array-CGH interpretation.

A close relationship has been demonstrated between genomic complexity and clinical outcome in uterine smooth muscle tumors. We studied the genomic profiles by array-CGH of 28 fumarate hydratase deficient leiomyomas and 37 leiomyomas with bizarre nuclei (LMBN) from 64 patients. Follow-up was available for 46 patients (from three to 249 months, mean 87.3 months). All patients were alive without evidence of disease. For 51 array-CGH interpretable tumors the mean Genomic Index (GI) was 16.4 (median: 9.8; from 1 to 57.8), significantly lower than the mean GI in LMS (mean GI 51.8, p < 0.001). We described three groups: (1) a group with FH deletion (24/58) with low GI (mean GI: 11 vs. 22,4, p = 0.02), (2) a group with TP53 deletion (17/58) with higher GI (22.4 vs. 11 p = 0.02), and (3) a group without genomic events on FH or TP53 genes (17/58) (mean GI:18.3; from 1 to 57.8). Because none of these tumors recurred and none showed morphological features of LMS we concluded that GI at the cut-off of 10 was not applicable in these subtypes of LM. By integration of all those findings, a GI <10 in LMBN remains a valuable argument for benignity. Conversely, in LMBN a GI >10 or alteration in tumor suppressor genes, should not alone warrant a diagnosis of malignancy. Nine tumors were tested with Nanocind CINSARC® signature and all were classified in low risk of recurrence. We propose, based on our observations, a diagnostic approach of these challenging lesions.

Identification of microenvironment features associated with primary resistance to anti-PD-1/PD-L1 + antiangiogenesis in gastric cancer through spatial transcriptomics and plasma proteomics.

Anti-angiogenic agents elicit considerable immune modulatory effects within the tumor microenvironment, underscoring the rationale for synergistic clinical development of VEGF and immune checkpoint inhibitors in advanced gastric cancer (AGC). Early phase studies involving Asian patients demonstrated encouraging anti-tumor efficacies. We report the results of the REGOMUNE phase II study, in which Caucasian patients were administered regorafenib, a multi-tyrosine kinase inhibitor, in combination with avelumab, a PD-L1-targeting monoclonal antibody. This therapeutic regimen resulted in deep and durable responses in 19% of patients, with the median duration of response not yet reached. Notwithstanding, a significant proportion of AGC patients exhibited no therapeutic advantage, prompting investigations into mechanisms of inherent resistance. Comprehensive biomarker profiling elucidated that non-responders predominantly exhibited an augmented presence of M2 macrophages within the tumor microenvironment and a marked overexpression of S100A10 by neoplastic cells, a protein previously implicated in macrophage chemotaxis. Additionally, peripheral biomarker assessments identified elevated levels of cytokines, including CSF-1, IL-4, IL-8, and TWEAK, correlating with adverse clinical outcomes, thereby accentuating the role of macrophage infiltration in mediating resistance. These insights furnish an invaluable foundation for elucidating, and potentially circumventing, resistance mechanisms in current AGC therapeutic paradigms, emphasizing the integral role of tumor microenvironmental dynamics and immune modulation.

Copy number alterations in metastatic and early breast tumours: prognostic and acquired biomarkers of resistance to CDK4/6 inhibitors.

BACKGROUND: Copy number alterations (CNA) are acquired during the evolution of cancers from their early stage to metastatic stage. This study aims at analysing the clinical value of the identified metastasis-associated CNAs both in metastatic breast cancers (mBCs) and early breast cancers (eBCs). METHODS: Single-nucleotide polymorphism (SNP)-array was performed on 926 biopsies from mBC patients, enrolled in SAFIR02-BREAST prospective trial. CNA profiles of eBCs from The Cancer Genome Atlas Breast Invasive Carcinoma (n = 770), Molecular Taxonomy of Breast Cancer International Consortium (n = 1620) and PACS04 trial (n = 243) cohorts were used as references for comparing mBCs and eBCs CNA profiles. Overall survival was the considered survival endpoint. RESULTS: Among the twenty-one genes frequently altered in ER + /HER2- mBCs: focal amplification of TERT was associated with poor outcome in the ER + /HER2- mBC population. Among the ER + /HER2- mBCs patients for whom CDK4/6 inhibitors information before biopsies collection was available: we identified seven genes on post-treatment biopsies, including the cyclin-dependent kinase 4 (CDK4), which was amplified in 9.8% of the ER + /HER2- mBCs pretreated population, as compared to 1.5% in the ER + /HER2- mBCs unpretreated population (P = 2.82E-04) as well as the 3 eBC populations. CDK4 amplification was associated with poor outcome in the ER + /HER2- eBCs. CONCLUSIONS: This study provides insights into the biology of mBCs and identifies clinically useful genomic features for future improvement of breast cancer patient management.

Are DNA Repair Gene Alterations Associated With the Response to Platinum-Based Regimen and Immune Checkpoint Inhibitors in Patients With Solid Tumors?

We analyzed the antitumor activity of platinum-based chemotherapies and then immune checkpoint inhibitors (ICI) in all-comers patients with solid tumors having a somatic DNA damage repair gene alteration (DDR-GA) identified through a prospective precision medicine study (NCT02534649). Each DDR-GA was classified as pathogenic (Pa), probably pathogenic (PPa), and unknown pathogenicity (UPa) according to OncoKB and ClinVAR databases. Between January 2018 and May 2020, 662 patients were screened. One hundred ninety-nine tumors with DDR-GA were found in 121 (18.3%) patients. Ninety-six patients received platinum-based chemotherapy in the advanced setting. No difference in objective response rate (ORR) under platinum regimen was observed between the 3 DDR-GA groups. The only predictor of worse progression-free survival (PFS) in Cox regression was the existence of a Pa alteration compared to the UPa group: HR = 2.11 (95% CI = 1.2-3.7), P = .009. Forty-eight patients received ICI alone or in combination. We observed a significant trend in better ORR to ICI according to the DDR-GA status: 1/11 (9%) patients in UPa, 5/17 (29.4%) patients in PPa, and 9/20 (45%) patients in Pa (P = .003, Cochran-Armitage trend test), and an increased 6-month PFS probability of 11%, 44%, and 50% in the UPa, PPa, and Pa groups, respectively (P = .37, log-rank test). Overall, somatic pathogenic DDR-GAs were not associated with ORR or PFS to platinum-based chemotherapy in patients with unselected advanced solid tumors. However, DDR-GA seemed to impact ORR and PFS to ICI, paving the way for a therapeutic combination with ICI and molecules targeting the DDR mechanisms, which are currently evaluated in ongoing clinical trials.

Atypical Spindle Cell/Pleomorphic Lipomatous Tumor With Sarcomatous Transformation: Clinicopathologic and Molecular Analysis of 4 Cases.

Atypical spindle cell/pleomorphic lipomatous tumor (ASPLT) is a recently described adipocytic tumor predominantly affecting the subcutaneous soft tissues of adults. Previous studies have shown that ASPLT follows a benign clinical course with a 4% to 12% local recurrence rate and no risk of dedifferentiation. Herein, we describe the clinicopathologic and molecular findings of 4 cases of ASPLT showing unequivocal sarcomatous transformation. Three patients were male and one was female, aged 65, 70, 74, and 78 years. Two cases presented as mass-forming lesions, while 1 case was incidentally discovered. The tumors measured 30, 55, 80, and 110 mm and occurred in the chest wall (n = 2) or arm (n = 2); all were subcutaneous. Microscopically, they showed a biphasic appearance comprising a low-grade ASPLT component and a high-grade sarcomatous component. The low-grade components showed features in the spectrum of either atypical pleomorphic lipomatous tumor (n = 2) or atypical spindle cell lipomatous tumor (n = 2). The high-grade components displayed leiomyosarcoma-like (n = 2), pleomorphic liposarcoma-like (n = 1) or undifferentiated sarcoma-like (n = 1) morphology. On immunohistochemistry, tumors were negative for MDM2 and showed loss of RB1 expression. In addition, the leiomyosarcoma-like areas seen in 2 cases were positive for smooth muscle actin and H-caldesmon. Single-nucleotide polymorphism array, performed in 3 cases, showed deletions of TP53, RB1, and flanking genes in both components. In contrast, the sarcomatous components showed more complex genomic profiles with rare segmental gains and recurrent loss of PTEN (n = 3), ATM (n = 2), and CDKN2A/B (n = 2) among other genes. Whole exome sequencing identified a TP53 variant in one case and an ATRX variant in another, each occurring in both tumor components. Limited clinical follow-up showed no recurrence or metastasis after 1 to 13 months (median, 7.5 months) postsurgical excision. Altogether, our data support that ASPLT can rarely develop sarcomatous transformation and offer insights into the molecular mechanisms underlying this event.

Pan-TRK immunohistochemistry in gynaecological mesenchymal tumours: diagnostic implications and pitfalls.

AIMS: NTRK-rearranged sarcomas of the female genital tract mainly occur in the uterus (more commonly cervix than corpus) and are characterized by a "fibrosarcoma-like" morphology and NTRK gene rearrangements. These neoplasms may exhibit histological overlap with other entities and can present diagnostic difficulties without molecular confirmation. Pan-TRK immunohistochemistry was developed to identify tumours harbouring NTRK rearrangements. The aim of this study was to characterize pan-TRK immunohistochemical expression in a large cohort of gynaecological mesenchymal neoplasms and investigate the utility of pan-TRK immunohistochemistry to distinguish NTRK-rearranged sarcoma from its mimics. METHODS AND RESULTS: A total of 473 gynaecological mesenchymal tumours (461 without known NTRK fusions and 12 NTRK-rearranged sarcomas) were selected. Pan-TRK immunohistochemistry (EPR17341, Abcam) was performed on whole tissue sections and tissue microarrays. Molecular interrogation of pan-TRK positive tumours was performed by RNA sequencing or fluorescence in situ hybridization (FISH). Of the 12 NTRK-rearranged sarcomas, 11 (92%) exhibited diffuse (≥70%) cytoplasmic pan-TRK staining with moderate/marked intensity, while the other was negative. Eleven (2.4%) additional tumours also exhibited pan-TRK immunohistochemical expression: three low-grade endometrial stromal sarcomas, seven high-grade endometrial stromal sarcomas, and an undifferentiated uterine sarcoma. Molecular confirmation of the absence of NTRK rearrangements was possible in nine of these tumours. Of these nine neoplasms, seven exhibited focal/multifocal (<70%) pan-TRK cytoplasmic staining with weak/moderate intensity. CONCLUSION: Even though pan-TRK immunohistochemical expression is not entirely sensitive or specific for NTRK-rearranged sarcomas, these neoplasms tend to exhibit diffuse staining of moderate/strong intensity, unlike its mimics. Pan-TRK should be performed in monomorphic uterine (corpus and cervix) spindle cell neoplasms that are negative for smooth muscle markers and hormone receptors and positive for CD34 and/ or S100. Ultimately, the diagnosis requires molecular confirmation.

Standardized Pathology Screening of Mature Tertiary Lymphoid Structures in Cancers.

Mature tertiary lymphoid structures (mTLSs) are organized lymphoid structures containing B lymphocytes admixed to CD23+ follicular dendritic cells. Their presence has been linked to improved survival and sensitivity to immune checkpoint inhibitors in several cancers, emerging as a promising pancancer biomarker. However, the requirements for any biomarker are clear methodology, proven feasibility, and reliability. In 357 patients' samples, we studied tertiary lymphoid structures (TLSs) parameters using multiplex immunofluorescence (mIF), hematoxylin-eosin-saffron (HES) staining, double CD20/CD23 staining, and single CD23 immunohistochemistry. The cohort included carcinomas (n = 211) and sarcomas (n = 146), gathering biopsies (n = 170), and surgical specimens (n = 187). mTLSs were defined as TLSs containing either a visible germinal center on HES staining or CD23+ follicular dendritic cells. Focusing on 40 TLSs assessed using mIF, double CD20/CD23 staining was less sensitive than mIF to assess maturity in 27.5% (n = 11/40) but was rescued by single CD23 staining in 90.9% (n = 10/11). In 97 patients, several samples (n = 240) were reviewed to characterize TLS distribution. The likelihood of finding TLSs in surgical material was 6.1 higher than in biopsy and 2.0 higher in primary samples than in metastasis after adjustment with a type of sample. Interrater agreement rates over 4 examiners were 0.65 (Fleiss kappa, 95% CI [0.46, 0.90]) for the presence of TLS and 0.90 for maturity (95% CI [0.83, 0.99]). In this study, we propose a standardized method to screen mTLSs in cancer samples using HES staining and immunohistochemistry that can be applied to all specimens.

Circulating tumor DNA landscape and prognostic impact of acquired resistance to targeted therapies in cancer patients: a national center for precision medicine (PRISM) study.

BACKGROUND: Despite the effectiveness of the various targeted therapies currently approved for solid tumors, acquired resistance remains a persistent problem that limits the ultimate effectiveness of these treatments. Polyclonal resistance to targeted therapy has been described in multiple solid tumors through high-throughput analysis of multiple tumor tissue samples from a single patient. However, biopsies at the time of acquired resistance to targeted agents may not always be feasible and may not capture the genetic heterogeneity that could exist within a patient. METHODS: We analyzed circulating tumor DNA (ctDNA) with a large next-generation sequencing panel to characterize the landscape of secondary resistance mechanisms in two independent prospective cohorts of patients (STING: n = 626; BIP: n = 437) with solid tumors who were treated with various types of targeted therapies: tyrosine kinase inhibitors, monoclonal antibodies and hormonal therapies. RESULTS: Emerging alterations involved in secondary resistance were observed in the plasma of up 34% of patients regardless of the type of targeted therapy. Alterations were polyclonal in up to 14% of patients. Emerging ctDNA alterations were associated with significantly shorter overall survival for patients with some tumor types. CONCLUSION: This comprehensive landscape of genomic aberrations indicates that genetic alterations involved in secondary resistance to targeted therapy occur frequently and suggests that the detection of such alterations before disease progression may guide personalized treatment and improve patient outcome.

Multicenter Harmonization Study of Pan-Trk Immunohistochemistry for the Detection of NTRK3 Fusions.

Pan-Trk immunohistochemistry has been described as a screening test for the detection of NTRK fusions in a broad spectrum of tumor types. However, pan-Trk testing in the clinical setting may be limited by many factors, including analytical parameters such as clones, platforms, and protocols used. This study aimed to harmonize pan-Trk testing using various clones and immunohistochemical (IHC) platforms and to evaluate the level of analytical variability across pathology laboratories. We developed several IHC pan-Trk assays using clones EPR17341 (Abcam) and A7H6R (Cell Signaling Technology) on Ventana/Roche, Agilent, and Leica platforms. To compare them, we sent unstained sections of a tissue microarray containing 9 cases with NTRK3 fusions to participating laboratories, to perform staining on Ventana/Roche (10 centers), Agilent (4 centers), and Leica (3 centers) platforms. A ready-to-use pan-Trk IVD assay (Ventana/Roche) was also performed in 3 centers. All slides were centrally and blindly reviewed for the percentage of stained tumor cells. Laboratory-developed tests with clone EPR17341 were able to detect pan-Trk protein expression in all cases, whereas lower rates of positivity were observed with clone A7H6R. Moderate to strong variability of the positive cases rate was observed with both antibodies in each IHC platforms type and each of the positivity cut points evaluated (≥1%, ≥10%, and ≥50% of stained tumor cells). The rate of false-negative cases was lower when pan-Trk staining was assessed with the lowest positivity threshold (≥1%). In conclusion, most evaluated pan-Trk IHC laboratory-developed tests were able to detect NTRK3-fusion proteins; however, a significant analytical variability was observed between antibodies, platforms, and centers.

The KAT6B::KANSL1 Fusion Defines a New Uterine Sarcoma With Hybrid Endometrial Stromal Tumor and Smooth Muscle Tumor Features.

Neoplasms harboring a KAT6B/A::KANSL1 fusion were initially reported as benign (leiomyomas) and malignant (leiomyosarcomas, low-grade endometrial stromal sarcomas [LG-ESSs]) uterine neoplasms. However, they may represent an emerging entity characterized by clinical aggressiveness contrasting with a rather reassuring microscopic appearance. Here, we aimed to confirm that this neoplasm is a distinct clinicopathologic and molecular sarcoma and identify criteria that should alert pathologists and lead to KAT6B/A::KANSL1 fusion testing in routine practice. Therefore, we conducted a comprehensive clinical, histopathologic, immunohistochemical, and molecular study, including array comparative genomic hybridization, whole RNA-sequencing, unsupervised clustering, and cDNA mutational profile analyses of 16 tumors with KAT6B::KANSL1 fusion from 12 patients. At presentation, patients were peri-menopausal (median, 47.5 years), and the primary tumors were located in the uterine corpus (12/12, 100%), with an additional prevesical location in 1 (8.3%) of 12 cases. The relapse rate was 33.3% (3/9). All tumors (16/16, 100%) showed morphologic and immunohistochemical features overlapping between leiomyoma and endometrial stromal tumors. A whirling recurrent architecture (resembling fibromyxoid-ESS/fibrosarcoma) was found in 13 (81.3%) of 16 tumors. All tumors (16/16, 100%) exhibited numerous arterioliform vessels, and 13 (81.3%) of 18 had large hyalinized central vessels and collagen deposits. Estrogen and progesterone receptors were expressed in 16 (100%) of 16 and 14 (87.5%) of 16 tumors, respectively. Array comparative genomic hybridization performed on 10 tumors classified these neoplasms as simple genomic sarcomas. Whole RNA-sequencing on 16 samples and clustering analysis on primary tumors found that the KAT6B::KANSL1 fusion always occurred between exons 3 of KAT6B and 11 of KANSL1; no pathogenic variant was identified on cDNA, all neoplasms clustered together, close to LG-ESS, and pathway enrichment analysis showed cell proliferation and immune infiltrate recruitment pathway involvement. These results confirm that the sarcomas harboring a KAT6B/A::KANSL1 fusion represent a distinct clinicopathologic entity, close to LG-ESS but different, with clinical aggressiveness despite a reassuring morphology, for which the KAT6B/A::KANSL1 fusion is the molecular driver alteration.

Recurrent YAP1::MAML2 fusions in "nodular necrotizing" variants of myxoinflammatory fibroblastic sarcoma: a comprehensive study of 7 cases.

Myxoinflammatory fibroblastic sarcoma (MIFS) is a rare soft tissue tumor with a predilection for the distal extremities and a tendency for local recurrence. Morphologically, MIFS consists of spindle and bizarre epithelioid cells resembling virocytes embedded in a fibrous to myxoid stroma with an abundant inflammatory infiltrate. Importantly, the molecular landscape of MIFS is wide and includes: VGLL3 amplification, BRAF fusion/amplification and OGA/TGFBR3 rearrangements. In this study, we describe a variant of MIFS showing a frequent nodular configuration associated with necrosis and recurrent YAP1::MAML2 fusions. The cohort consisted of 7 patients (4 females and 3 males) ranging in age from 21 to 71 years (median: 47 years). Two tumors (28%) occurred in acral locations while the remaining cases were more widely distributed (thigh, n = 2; arm, n = 1; neck; n = 1; chest-wall, n = 1). Tumor size ranged from 10 to 38 mm (median: 20 mm). Histologically, lesions frequently presented as nodules with central areas of necrosis, and were predominantly composed of sheets of epithelioid cells with large vesicular nuclei and prominent nucleoli (Reed-Sternberg-like cells or virocytes). The stroma was mostly fibrous and showed a polymorphous inflammatory infiltrate. Myxoid stromal changes were focally seen in one case, and pseudolipoblasts were absent. The immunophenotype was nonspecific, with only pan-keratin (AE1-AE3) and cyclin D1 expression in a subset of cases. RNA-Sequencing detected YAP1::MAML2 fusions in 3/7 cases; aCGH showed no significant gene copy number variations in 4 tested cases, and FISH analysis showed no VGLL3 amplification in 1 tested case. Follow-up was available for 6 cases, ranging from 7 to 63 months (median: 42 months). Local recurrence and metastasis were not seen and one tumor showed spontaneous regression following initial biopsy. In conclusion, we describe a novel variant of MIFS with distinctive clinicopathological and molecular features for which we propose the term "nodular necrotizing" MIFS.

Superficial CD34-positive fibroblastic tumor and PRDM10-rearranged soft tissue tumor are overlapping entities: a comprehensive study of 20 cases.

AIMS: Superficial CD34-positive fibroblastic tumor (SCD34FT) and PRDM10-rearranged soft tissue tumor (PRDM10-STT) are rare mesenchymal tumors. These lesions have clinicopathological similarities, but their relationship remains controversial. This study aimed to characterise a series of cases of SCD34FT and PRDM10-STT. METHODS AND RESULTS: Ten lesions each of SCD34FT and PRDM10-STT were studied using immunohistochemistry, array-comparative genomic hybridisation (aCGH), RNA sequencing and exome sequencing. Tumors mainly occurred in young adults, were generally small (< 5 cm) and arose predominantly in the superficial soft tissues of the lower extremities. Follow-up data were available in 15 cases (SCD34FT, n = 7, median 16 months; PRDM10-STT, n = 8, median 14 months), local recurrences occurred in four cases (SCD34FT, two of 10; PRDM10-STT, two of 10), while no distant spread was documented. Morphologically, tumors were relatively well-circumscribed and composed of sheets and fascicles of spindle and pleomorphic cells showing low mitotic activity (< 1/mm²) without necrosis. Other findings included: granular cell change, lipoblast-like cells, ectatic blood vessels with fibrinous material, myxoid stromal changes, metaplastic bone and increased mitotic activity (> 1/mm²). All tumors diffusely expressed CD34, while pan-keratin and desmin were commonly seen focally. SynCAM3 was diffusely expressed in 12 cases (SCD34FT, n = 5; PRDM10-STT, n = 7), independently of fusion status. aCGH profiles were 'flat' (PRDM10-STT, n = 4; SCD34FT, n = 2) and exome sequencing showed no recurrent pathogenic mutations (PRDM10-STT, n = 2; SCD34FT, n = 4). Overall, the only morphological features seen exclusively in PRDM10-STT were myxoid stromal changes (three of 10) and metaplastic bone (two of 10). CONCLUSION: We expand the current knowledge on PRDM10-STT and SCD34FT and provide additional evidence for considering them as overlapping entities.

Molecular profiling of advanced soft-tissue sarcomas: the MULTISARC randomized trial.

BACKGROUND: Soft-tissue sarcomas (STS) represent a heterogeneous group of rare tumors including more than 70 different histological subtypes. High throughput molecular analysis (next generation sequencing exome [NGS]) is a unique opportunity to identify driver mutations that can change the usual one-size-fits-all treatment paradigm to a patient-driven therapeutic strategy. The primary objective of the MULTISARC trial is to assess whether NGS can be conducted for a large proportion of metastatic STS participants within a reasonable time, and, secondarily to determine whether a NGS-guided therapeutic strategy improves participant's outcome. METHODS: This is a randomized, multicentre, phase II/III trial inspired by the design of umbrella and biomarker-driven trials. The setting plans up to 17 investigational centres across France and the recruitment of 960 participants. Participants aged at least 18 years, with unresectable locally advanced and/or metastatic STS confirmed by the French sarcoma pathological reference network, are randomized according to 1:1 allocation ratio between the experimental arm "NGS" and the standard "No NGS". NGS will be considered feasible if (i) NGS results are available and interpretable, and (ii) a report of exome sequencing including a clinical recommendation from a multidisciplinary tumor board is provided to investigators within 7 weeks from reception of the samples on the biopathological platform. A feasibility rate of more than 70% is expected (null hypothesis: 70% versus alternative hypothesis: 80%). In terms of care, participants randomized in "No NGS" arm and who fail treatment will be able to switch to the NGS arm at the request of the investigator. DISCUSSION: The MULTISARC trial is a prospective study designed to provide high-level evidence to support the implementation of NGS in routine clinical practice for advanced STS participants, on a large scale. TRIAL REGISTRATION: clinicaltrial.gov NCT03784014 .

Solid-type adenoid cystic carcinoma of the breast, a distinct molecular entity enriched in NOTCH and CREBBP mutations.

Adenoid cystic carcinoma (ACC) of the breast with a predominant solid pattern is difficult to diagnose with certainty and differentiate from more common triple-negative breast cancers (TNBCs) of basal-phenotype. To better characterize solid ACC, we performed a clinical, morphological, immunohistochemical, and molecular comparative analysis of 33 ACCs of the breast comprising 17 solid variant ACCs and 16 conventional ACCs. Solid ACCs displayed basaloid morphology with an exclusive or predominant epithelial cell population associated with decreased myoepithelial differentiation, while demonstrating MYB protein overexpression similar to the more common type of ACC. Strong and diffuse MYB expression by immunochemistry was observed in 14/17 (82%) of solid ACCs while MYB rearrangements were detected by break apart fluorescence in situ hybridization (FISH) in only 3/16 (19%) of solid ACCs. Conversely, weak MYB immunohistochemical expression was observed in only 7/204 (3%) of TNBC. Solid ACCs displayed a transcriptomic profile distinct from conventional ACCs with 549 genes showing a highly significant differential expression between conventional and solid ACC [false discovery rate (FDR) < 0.01; log2FC > |1|]. EnrichR and Kegg Pathway analyses identified PI3K-Akt and focal adhesion signaling pathways as significantly overexpressed in conventional ACCs compared with solid ACCs which significantly overexpressed the nitrogen metabolism pathway. CREBBP mutations and NOTCH activating gene mutations were only present in solid ACCs, concerning 5/16 (31%) of cases for each gene. Tumors with NOTCH activating mutations displayed a strong diffuse nuclear NICD1 staining, an established marker of Notch pathway activation. Solid ACCs also differed from basal-type TNBC, with fewer TP53 mutations and a more stable genomic profile on array comparative genomic hybridization (CGH). In summary, solid-type ACC of the breast is a distinct molecular entity within the ACC family and is different from common basal-type TNBC. MYB is a diagnostically useful biomarker of solid ACC and NOTCH could be a novel potential therapeutic target in 30% of cases.

NFATc2-rearranged sarcomas: clinicopathologic, molecular, and cytogenetic study of 7 cases with evidence of AGGRECAN as a novel diagnostic marker.

NFATc2-rearranged sarcomas (NFATc2-Sarcomas) are infrequent round cell tumors characterized by EWSR1-NFATc2 fusions and FUS-NFATc2 fusions. Although our knowledge on these neoplasms has increased recently, novel diagnostic tools and more comprehensive series are still needed. Here, we describe the features of a series of seven molecularly confirmed NFATc2-Sarcomas (EWSR1-NFATc2, n = 4; FUS-NFATc2, n = 3) and demonstrate the utility of AGGRECAN immunohistochemistry for their identification. Patients were four males and three females, ranging in age from 19 to 66 years (median: 33). All were primary bone tumors (femur, n = 4; tibia, n = 2; ilium, n = 1), frequently infiltrating the surrounding soft tissues. Treatment often consisted of neoadjuvant chemotherapy and surgery. Follow-up was available for six patients (median 18 months, range 5-102 months), three patients died of disease and four patients are currently alive. Histologically, tumors consisted of monotonous round cells growing in lobules and sheets in variable amounts of fibrous to myxoid stroma. Other findings included spindle cells, corded and trabecular architecture, nuclear pleomorphism, cartilaginous differentiation, and osteoid-like matrix. Histological response to neoadjuvant chemotherapy was poor in all resection specimens available for review (n = 4). Tumors were diffusely positive for AGGRECAN and CD99 (7/7), and a subset expressed Pan-Keratin (AE1-AE3; 3/6), S100 (2/6), BCOR (2/6), ETV-4 (2/5), WT1 (2/6), and ERG (2/5). Desmin, NKX3-1, and SATB2 were negative (0/6). Diffuse AGGRECAN staining was also seen in 8/129 round cell sarcomas used for comparison, including mesenchymal chondrosarcoma (7/26) and CIC-sarcoma (1/26). Array-CGH showed complex karyotypes with recurrent deletions of tumor suppressor genes (CDKN2A/B, TUSC7, and DMD) in three FUS-NFATC2 cases and a simpler profile without homozygous losses in one EWSR1-NFATc2 case. Segmental chromosomal gains covering the loci of the fusion genes were detected in both variants. Overall, our study confirms and expands previous observations on NFATc2-sarcomas and supports that AGGRECAN is a useful biomarker of these tumors.

Correction: NFATc2-rearranged sarcomas: clinicopathologic, molecular, and cytogenetic study of 7 cases with evidence of AGGRECAN as a novel diagnostic marker.

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

GREB1-CTNNB1 fusion transcript detected by RNA-sequencing in a uterine tumor resembling ovarian sex cord tumor (UTROSCT): A novel CTNNB1 rearrangement.

Mutations of CTNNB1 have been implicated in tumorigenesis in many organs. However, tumors harboring a CTNNB1 translocation are extremely rare and this translocation has never been reported in a uterine mesenchymal neoplasm. We report a novel translocation t(2;3)(p25;p22) involving the GREB1 (intron 8) and CTNNB1 (exon 3) in a uterine tumor resembling ovarian sex cord tumor (UTROSCT), which exhibited extrauterine metastasis. The translocation detected by RNA-sequencing was validated by RT-PCR, and resulted in nuclear expression of ß-catenin. Juxtapositioning with GREB1, which is overexpressed in response to estrogens, resulted in overexpression of a truncated and hypophosphorylated nuclear ß-catenin in the primary and recurrent tumors. This accumulation of nuclear ß-catenin results in a constitutive activation of the Wnt/ß-catenin signaling pathway with a major oncogenic effect. The CTNNB1 gene fusion, promoted by an estrogen-responsive gene (GREB1), could be a potential driver of tumorigenesis in this case and a therapeutic target with adapted inhibitors. RT-PCR and immunohistochemistry performed on 11 additional UTROSCTs showed no CTNNB1 fusion transcript or nuclear ß-catenin immunoreactivity.

Uterine and vaginal sarcomas resembling fibrosarcoma: a clinicopathological and molecular analysis of 13 cases showing common NTRK-rearrangements and the description of a COL1A1-PDGFB fusion novel to uterine neoplasms.

Mesenchymal neoplasms of the uterus (corpus and cervix) encompass a heterogeneous group of tumors with differing morphologies, immunophenotypes and molecular alterations. With the advent of modern molecular techniques, such as next generation sequencing, newly defined genetic abnormalities are being reported in this group of neoplasms. Herein we report the clinicopathological and molecular features of a series of 13 spindle cell sarcomas of the uterus and vagina (10 cervix, 2 uterine corpus, 1 vagina) with morphology resembling fibrosarcoma. After targeted RNA-sequencing, dual FISH fusion and array-CGH analysis, 7 of 13 tumors exhibited NTRK rearrangements (6 TPM3-NTRK1 and 1 EML4-NTRK3) and 3 a COL1A1-PDGFB fusion; in the other 3 neoplasms, all of which were positive with S100 (2 diffuse, 1 focal), we identified no rearrangement. All the NTRK fusion-positive sarcomas were located in the cervix and exhibited diffuse staining with Trk while all the other neoplasms were negative. CD34 was diffusely positive in all 3 of the COL1A1-PDGFB fusion sarcomas. The latter molecular abnormality is identical to that commonly found in dermatofibrosarcoma protuberans and has not been reported previously in uterine mesenchymal neoplasms. We suggest that uterine sarcomas with a morphology resembling fibrosarcoma (and in which leiomyosarcoma and the known molecularly confirmed high-grade endometrial stromal sarcomas have been excluded) can be divided into 3 groups:- an NTRK fusion group, a COL1A1-PDGFB fusion group and a group containing neither of these molecular abnormalities which, on the basis of positive staining with S100, could be tentatively classified as malignant peripheral nerve sheath tumor, although additional molecular studies may identify specific genetic alterations necessitating a nomenclature change. We suggest a diagnostic algorithm when reporting such neoplasms. Identification of these newly described fusion-associated sarcomas is important given the potential for targeted treatments.