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.

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.

SMARCB1 (INI1) Deficient Tumours of the Uterine Cervix: Report of Two Cases, Including One Associated With an NTRK Fusion.

Pathogenic variants (mutations) and other molecular events involving subunits of the SWItch/Sucrose Non-Fermentable chromatin remodelling complex are common in a wide variety of malignancies. Many of these neoplasms are characterized by undifferentiated morphology. They arise at a variety of sites in the female genital tract but have rarely been reported in the uterine cervix. We report 2 primary cervical neoplasms arising in young women (ages 28 and 29 yr) exhibiting loss of nuclear immunoreactivity with SMARCB1 (INI1). In one case, which had a mixture of epithelioid and spindle cells, molecular studies revealed no SMARCB1 pathogenic variant, but showed a SPECCL1::NTRK 3 fusion, in keeping with an NTRK fusion sarcoma. The second case exhibited rhabdoid morphology and molecular testing confirmed a SMARCB1 pathogenic variant (c.425 T>G:p.(Leu142Ter) which, interpreted in conjunction with the morphology and immunohistochemistry, resulted in classification as a proximal-type epithelioid sarcoma. To our knowledge, this is the first reported cervical neoplasm exhibiting a SMARCB1 pathogenic variant and the first NTRK fusion sarcoma showing SMARCB1 protein loss. We discuss the diagnostic challenges and complexities of the molecular findings.

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.

The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/CSDE1 and P62/SQSTM1.

Humans are frequently exposed to bacterial genotoxins of the gut microbiota, such as colibactin and cytolethal distending toxin (CDT). In the present study, whole genome microarray-based identification of differentially expressed genes was performed in vitro on HT29 intestinal cells while following the ectopic expression of the active CdtB subunit of Helicobacter hepaticus CDT. Microarray data showed a CdtB-dependent upregulation of transcripts involved in positive regulation of autophagy concomitant with the downregulation of transcripts involved in negative regulation of autophagy. CdtB promotes the activation of autophagy in intestinal and hepatic cell lines. Experiments with cells lacking autophagy related genes, ATG5 and ATG7 infected with CDT- and colibactin-producing bacteria revealed that autophagy protects cells against the genotoxin-induced apoptotic cell death. Autophagy induction could also be associated with nucleoplasmic reticulum (NR) formation following DNA damage induced by these bacterial genotoxins. In addition, both genotoxins promote the accumulation of the autophagic receptor P62/SQSTM1 aggregates, which colocalized with foci concentrating the RNA binding protein UNR/CSDE1. Some of these aggregates were deeply invaginated in NR in distended nuclei together or in the vicinity of UNR-rich foci. Interestingly, micronuclei-like structures and some vesicles containing chromatin and γH2AX foci were found surrounded with P62/SQSTM1 and/or the autophagosome marker LC3. This study suggests that autophagy and P62/SQSTM1 regulate the abundance of micronuclei-like structures and are involved in cell survival following the DNA damage induced by CDT and colibactin. Similar effects were observed in response to DNA damaging chemotherapeutic agents, offering new insights into the context of resistance of cancer cells to therapies inducing DNA damage.

Development of Radiopharmaceuticals for NPY Receptor-5 (Y5) Nuclear Imaging in Tumors by Synthesis of Specific Agonists and Investigation of Their Binding Mode.

The neuropeptide-Y (NPY) family acts through four G protein-coupled receptor subtypes in humans, namely, Y1, Y2, Y4, and Y5. A growing body of evidence suggest the involvement of the NPY system in several cancers, notably the Y5 subtype, thus acting as a relevant target for the development of radiopharmaceuticals for imaging or targeted radionuclide therapy (TRT). Here, the [cPP(1-7),NPY(19-23),Ala31,Aib32,Gln34]hPP scaffold, further referred to as sY5ago, was modified with a DOTA chelator and radiolabeled with 68Ga and 111In and investigated in vitro and in vivo using the MCF-7 model. For in vivo studies, MCF-7 cells were orthotopically implanted in female nude mice and imaging with small animal positron emission tomography/computed tomography (µPET/CT) was performed. At the end of imaging, the mice were sacrificed. A scrambled version of sY5ago, which was also modified with a DOTA chelator, served as a negative control (DOTA-[Nle]sY5ago_scrambled). sY5ago and DOTA-sY5ago showed subnanomolar affinity toward the Y5 (0.9 ± 0.1 and 0.8 ± 0.1 nM, respectively) and a single binding site at the Y5 was identified. [68Ga]Ga-DOTA-sY5ago and [111In]In-DOTA-sY5ago were hydrophilic and showed high specific internalization (1.61 ± 0.75%/106 cells at 1 h) and moderate efflux (55% of total binding externalized at 45 min). On µPET/CT images, most of the signal was depicted in the kidneys and the liver. MCF-7 tumors were clearly visualized. On biodistribution studies, [68Ga]Ga-DOTA-sY5ago was eliminated by the kidneys (∼60 %ID/g). The kidney uptake is Y5-mediated. A specific uptake was also noted in the liver (5.09 ± 1.15 %ID/g vs 1.13 ± 0.21 %ID/g for [68Ga]Ga-DOTA-[Nle]sY5ago_scrambled, p < 0.05), the lungs (1.03 ± 0.34 %ID/g vs 0.20 %ID/g, p < 0.05), and the spleen (0.85 ± 0.09%ID/g vs 0.16 ± 0.16%ID/g, p < 0.05). In MCF-7 tumors, [68Ga]Ga-DOTA-sY5ago showed 12-fold higher uptake than [68Ga]Ga-DOTA-[Nle]sY5ago_scrambled (3.43 ± 2.32 vs 0.27 ± 0.15 %ID/g, respectively, p = 0.0008) at 1 h post-injection. Finally, a proof-of-principle tissular micro-imaging study on a human primary cancer sample showed weak binding of [111In]In-DOTA-sY5ago in prostatic intra-neoplasia and high binding in the ISUP1 lesion while normal prostate was free of signal.

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.

Breast hamartoma: reassessment of an under-recognised breast lesion.

AIMS: Breast hamartomas are an under-recognised lesion because they lack a distinctive microscopic appearance. Microscopic diagnosis can often conclude 'no significant lesion' or 'normal breast tissue', leading to repeated biopsies and diagnostic delay. We describe the histological, immunohistochemical and radiological features of breast hamartomas with the aim of identifying specific signs to facilitate their diagnosis and to differentiate them from normal breast and fibroepithelial lesions. METHODS AND RESULTS: Forty-seven breast hamartomas were reassessed (histological diagnosis and imaging features). An immunohistochemical study [oestrogen receptor (ER), progesterone receptor (PR), CD34, high-mobility group A2 (HMGA2)] was performed. On breast imaging, hamartomas most often presented as probably benign solid masses with circumscribed margins and variable densities. Histologically, breast hamartomas resembled normal breast, although their stromal component was predominant, separating randomly scattered epithelial elements with areas of pure collagenous stroma. Pseudoangiomatous stromal hyperplasia (PASH) was present in 93.6% of cases and CD34 antibody highlighted intralobular, perilobular and interlobular distribution of CD34-positive fibroblasts. By comparison, CD34 was mainly expressed in the intralobular normal breast tissue stroma. Hamartoma stromal cells expressed HMGA2, ER and PR in 79%, 66% and 76.3% of our cases, respectively, compared to 7.7%, 23% and 19% in normal breast tissue, respectively (P < 0.0001; P = 0.0005; P < 0.0001). CONCLUSIONS: After ascertaining that core needle biopsy is effectively intralesional, breast hamartomas can be diagnosed with confidence by taking into account the presence of stromal changes, PASH, interlobular distribution of CD34-positive fibroblasts, HMGA2 and hormonal receptor stromal expression.

Expression of neurotensin receptor-1 (NTS1) in primary breast tumors, cellular distribution, and association with clinical and biological factors.

PURPOSE: Neurotensin receptor-1 (NTS1) is increasingly recognized as a potential target in diverse tumors including breast cancer, but factors associated with NTS1 expression have not been fully clarified. METHODS: We studied NTS1 expression using the Tissue MicroArray (TMA) of primary breast tumors from Institut Bergonié. We also studied association between NTS1 expression and clinical, pathological, and biological parameters, as well as patient outcomes. RESULTS: Out of 1419 primary breast tumors, moderate to strong positivity for NTS1 (≥ 10% of tumoral cells stained) was seen in 459 samples (32.4%). NTS1 staining was cytoplasmic in 304 tumors and nuclear in 155 tumors, a distribution which appeared mutually exclusive. Cytoplasmic overexpression of NTS1 was present in 21.5% of all breast tumors. In multivariate analysis, factors associated with cytoplasmic overexpression of NTS1 in breast cancer samples were higher tumor grade, Ki67 ≥ 20%, and higher pT stage. Cytoplasmic NTS1 was more frequent in tumors other than luminal A (30% versus 17.3%; p < 0.0001). Contrastingly, the main "correlates" of a nuclear location of NTS1 were estrogen receptor (ER) positivity, low E&E (Elston and Ellis) grade, Ki67 < 20%, and lower pT stage. In NTS1-positive samples, cytoplasmic expression of NTS1 was associated with shorter 10-year metastasis-free interval (p = 0.033) compared to NTS1 nuclear staining. Ancillary analysis showed NTS1 expression in 73% of invaded lymph nodes from NTS1-positive primaries. CONCLUSION: NTS1 overexpression was found in about one-third of breast tumors from patients undergoing primary surgery with two distinct patterns of distribution, cytoplasmic distribution being more frequent in aggressive subtypes. These findings encourage the development of NTS1-targeting strategy, including radiopharmaceuticals for imaging and therapy.

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.

Residual cancer burden index and tumor-infiltrating lymphocyte subtypes in triple-negative breast cancer after neoadjuvant chemotherapy.

PURPOSE: There is a need to refine the prognosis of triple-negative breast cancer (TNBC) patients after neoadjuvant chemotherapy (NAC) and to study the influence of the tumor microenvironment. We evaluated the prognostic value of pathological and immune markers in TNBC with residual disease (RD) after NAC. METHODS: In a series of 186 TNBC patients treated by NAC, we assessed the prognostic value of the Residual Cancer Burden (RCB) index. In 109 patients with RD, we studied the impact of clinicopathological features and tumor immune response in the residual tumor on overall survival (OS) and distant recurrence-free interval (DRFI). RESULTS: In the whole group, the OS and DRFI, at 3 years, were statistically different between the different classes of RCB (P = 0.0004 and P < 0.0001, respectively). In univariate analysis of the RD group, low RCB index and high ratios of stromal tumor-infiltrating lymphocytes (TILs), CD3 + TILs, CD4 + TILs, CD8 + TILs, and IDO1-positive cells were significant favorable prognostic factors for DRFI at 3 years. In the final multivariate model, CD4 + TILs and RCB index showed a statistically independent prognostic significance for DRFI [Hazard Ratio (HR) 2.88 (95%CI 1.34-6.17), P = 0.007 and HR 12.04 (95%CI 2.78-52.23, P < 0.0001), respectively]. The CD4 + TIL levels influenced survival in the different RCB classes with a significant effect observed in RCB-II and RCB-III classes (P = 0.05 and P = 0.05, respectively). CONCLUSIONS: These results suggest that the combination of pathological (RCB index) and tumor micro-environmental features (CD4 + TILs) help refining the prognosis of TNBC patients with RD following NAC.

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.

Neurotensin Receptor-1 Expression in Human Prostate Cancer: A Pilot Study on Primary Tumors and Lymph Node Metastases.

Neurotensin and its high-affinity receptor, NTR1, are involved in the growth of various tumors. Few data are available regarding NTR1 expression in normal and tumoral human prostate tissue samples. NTR1 expression was assessed using immunohistochemistry in 12 normal prostate tissues, 11 benign prostatic hyperplasia (BPH), 44 prostate cancers, and 15 related metastatic lymph nodes (one per patient, when available). NTR1-staining was negative in normal prostate and BPH samples. NTR1 was overexpressed in four out of 44 (9.1%) primary tumors. There was no clear association between NTR1 overexpression and age, PSA-values, Gleason score, pT-status, nodal-status, or margin. NTR1 was expressed at a high level of five out of 15 (33.3%) metastatic lymph nodes. NTR1 overexpression was thus more frequent in metastatic lymph nodes than in primary tumors (p = 0.038). In this limited series of samples, NTR1 overexpression was observed in few primary prostate cancers. Upregulation was more frequent in related lymph nodes. The presence of this target in metastatic lymph nodes may open new perspectives for imaging and radionuclide therapy of prostate cancer. Factors driving NTR1 expression in primary prostate cancer and in nodal and distant metastases still need to be characterized.

Loss of H3K27 trimethylation is not suitable for distinguishing malignant peripheral nerve sheath tumor from melanoma: a study of 387 cases including mimicking lesions.

The diagnosis of malignant peripheral nerve sheath tumor remains challenging, especially in the sporadic setting. Malignant peripheral nerve sheath tumor is a rare malignancy, and owing to the lack of specific histological criteria, immunohistochemical and molecular diagnostic markers, several differential diagnoses must be considered, in particular melanoma. Recently, inactivation of the polycomb repressive complex 2 (PRC2), induced by inactivating mutations in two of its critical constituents SUZ12 and EED, was reported in a large subset of malignant peripheral nerve sheath tumors. Homozygous PRC2 inactivation induces complete loss of trimethylation at lysine 27 of histone 3 (H3K27me3). Recent studies suggest that complete loss of H3K27me3 is highly specific for malignant peripheral nerve sheath tumor and may be a useful immunohistochemical diagnostic marker. Therefore, to determine the specificity of the complete loss of H3K27me3 expression in the context of the differential diagnosis of malignant peripheral nerve sheath tumor from melanoma (its major potential mimic), we performed H3K27me3 immunohistochemistry in a pathologically and genetically well-characterized cohort of primary (neurofibromatosis type 1 (NF1), radiation-associated and sporadic context) malignant peripheral nerve sheath tumors (n=122) and in a cohort or primary (desmoplastic) and metastatic melanomas (n=265). In total, 88 (72%) malignant peripheral nerve sheath tumors, including 46 (71%) NF1-associated, 4 (100%) radiation-associated, and 38 (72%) sporadic tumors, showed complete loss of H3K27me3. We observed increased loss of H3K27me3 with increasing histological grade. Interestingly, we found complete loss of H3K27me3 in 37% (n=98) of all melanomas, including 25% (n=9) of primary desmoplastic melanomas. Moreover, partial loss ('mosaic' pattern) was observed in 23 (19%) of all malignant peripheral nerve sheath tumors and in 136 (51%) of all melanomas. Complete loss of H3K27me3 detected by immunohistochemistry is not specific for malignant peripheral nerve sheath tumor and cannot be used safely when distinguishing malignant peripheral nerve sheath tumor from melanoma.

The mammary ducts create a favourable microenvironment for xenografting of luminal and molecular apocrine breast tumours.

There is a paucity of models for hormone receptor-positive (HR+) breast cancer because of the difficulty of establishing xenografts from these tumours. We show that this obstacle can be overcome by injecting human tumour cells directly into the mammary ducts of immunodeficient mice. Tumours from 31 patients were infected overnight with a lentiviral vector expressing tdTomato and injected through the nipple into the mammary ducts of NOD-SCID-IL2RG-/- mice. Tumours formed in the mice in 77% of cases after the first injection (6/8 luminal A, 15/20 luminal B, and 3/3 molecular apocrine). Four luminal A and one molecular apocrine graft were tested in secondary and tertiary grafts: all were successfully passaged in secondary and 4/5 in tertiary grafts. None of the samples engrafted when injected subcutaneously. The morphology, oestrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), and Ki-67 profiles of the clinical samples were maintained in the tertiary grafts. We also show that the intraductal approach can be used to test the response to targeted therapy with fulvestrant and palbociclib, using a genetically defined ER+ model. We conclude that the mammary ducts create a microenvironment that is uniquely favourable to the survival and growth of tumours derived from mammary hormone-sensing cells. This approach opens the door to testing genomically targeted treatment of HR+ tumours in precision medicine programmes. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.