The advent of digital pathology provides us with the challenging opportunity to automatically analyze whole slides of diseased tissue in order to derive quantitative profiles that can be used for ...diagnosis and prognosis tasks. In particular, for the development of interpretable models, the detection and segmentation of cell nuclei is of the utmost importance. In this paper, we describe a new method to automatically segment nuclei from Haematoxylin and Eosin (H&E) stained histopathology data with fully convolutional networks. In particular, we address the problem of segmenting touching nuclei by formulating the segmentation problem as a regression task of the distance map. We demonstrate superior performance of this approach as compared to other approaches using Convolutional Neural Networks.
Specific chromosomal translocations encoding chimeric transcription factors are considered to play crucial oncogenic roles in a variety of human cancers but the fusion proteins themselves seldom ...represent suitable therapeutic targets. Oncogenic TFE3 fusion proteins define a subset of pediatric renal adenocarcinomas and one fusion (ASPL-TFE3) is also characteristic of alveolar soft part sarcoma (ASPS). By expression profiling, we identified the MET receptor tyrosine kinase gene as significantly overexpressed in ASPS relative to four other types of primitive sarcomas. We therefore examined MET as a direct transcriptional target of ASPL-TFE3. ASPL-TFE3 binds to the MET promoter and strongly activates it. Likewise, PSF-TFE3 and NONO-TFE3 also bind this promoter. Induction of MET by ASPL-TFE3 results in strong MET autophosphorylation and activation of downstream signaling in the presence of hepatocyte growth factor (HGF). In cancer cell lines containing endogenous TFE3 fusion proteins, inhibiting MET by RNA interference or by the inhibitor PHA665752 abolishes HGF-dependent MET activation, causing decreased cell growth and loss of HGF-dependent phenotypes. MET is thus a potential therapeutic target in these cancers. Aberrant transcriptional up-regulation of MET by oncogenic TFE3 fusion proteins represents another mechanism by which certain cancers become dependent on MET signaling. The identification of kinase signaling pathways transcriptionally up-regulated by oncogenic fusion proteins may reveal more accessible therapeutic targets in this class of human cancers.
Summary Background Advances in molecular genetics of sarcoma have enabled the identification of type-specific aberrations. We aimed to assess the clinical effect of systematic implementation of ...molecular assays to improve sarcoma misdiagnosis. Methods In this multicentre, observational study, we recruited patients from 32 centres of the French Sarcoma Group/Reference Network in Pathology of Sarcomas. Eligibility criteria included: biopsy or surgical resection; suspicion of: dermatofibrosarcoma protuberans (cohort 1), dedifferentiated liposarcoma (cohort 2), Ewing's sarcoma family of tumours (cohort 3), synovial sarcoma (cohort 4), alveolar rhabdomyosarcoma (cohort 5), and myxoid or round cell liposarcoma (cohort 6); review by one sarcoma-expert pathologist; availability of frozen material (except for cohort 1 of patients with dermatofibrosarcoma protuberans because anti-CD34 immunohistochemistry is performed on paraffin-embedded tissue); and patient information. For each case, the pathologist made one primary diagnosis followed by up to two differential diagnoses, based on histological characteristics only. Each diagnosis was classified as certain, probable, or possible. For each case to determine the molecular classification, we did fluorescence in-situ hybridisation on paraffin-embedded samples. We also did comparative genomic hybridisation and quantitative PCR (cohort 2) or reverse transcriptase PCR (cohorts 3–6) on frozen and paraffin-embedded samples. We made a final diagnosis based on the molecular results. The clinical effect of diagnosis correction was assessed by a board of experts. Finding Between June 22, 2009, and Oct 30, 2012, 395 patients were enrolled in the study, of which 384 were eligible for inclusion. The diagnosis was eventually modified by molecular genetics for 53 patients: eight (16%) of 50 patients with dermatofibrosarcoma (cohort 1), seven (23%) of 30 patients with dedifferentiated liposarcoma (cohort 2), 13 (12%) of 112 with Ewing's sarcoma family of tumours (cohort 3), 16 (16%) of 97 patients with synovial sarcoma (cohort 4), seven (15%) of 46 patients with alveolar rhabdomyosarcoma (cohort 5), and two (4%) of 49 patients with myxoid or round cell liposarcoma (cohort 6), with an effect on primary management or prognosis assessment in 45 cases. Interpretation Molecular genetic testing should be mandatory for diagnostic accuracy of sarcoma and appropriate clinical management, even when histological diagnosis is made by pathologist experts in this field. Funding French National Cancer Institute and Nice University Hospital.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Morphological, immunohistochemical, and molecular methods often need to be combined for accurate diagnosis and optimal clinical management of sarcomas. Here, we have developed, a new molecular ...diagnostic assay, for the detection of gene fusions in sarcomas. This targeted multiplexed next-generation sequencing (NGS)-based method utilizes ligation dependent reverse-transcriptase polymerase chain reaction (LD-RT-PCR-NGS) to detect oncogenic fusion transcripts involving 137 genes, leading to 139 gene fusions known to be recurrently rearranged in soft-tissue and bone tumors. 158 bone and soft-tissue tumors with previously identified fusion genes by fluorescent in situ hybridization (FISH) or RT-PCR were selected to test the specificity and the sensitivity of this assay. RNA were extracted from formalin-fixed paraffin-embedded (n = 143) or frozen (n = 15) material (specimen; n = 42 or core needle biopsies; n = 116). Tested tumors encompassed 23 major translocation-related sarcomas types, including Ewing and Ewing-like sarcomas, rhabdomyosarcomas, desmoplastic small round-cell tumors, clear-cell sarcomas, infantile fibrosarcomas, endometrial stromal sarcomas, epithelioid hemangioendotheliomas, alveolar soft-part sarcomas, biphenotypic sinonasal sarcomas, extraskeletal myxoid chondrosarcomas, myxoid/round-cell liposarcomas, dermatofibrosarcomas protuberans and solitary fibrous tumors. In-frame fusion transcripts were detected in 98.1% of cases (155/158). Gene fusion assay results correlated with conventional techniques (FISH and RT-PCR) in 155/158 tumors (98.1%). These data demonstrate that this assay is a rapid, robust, highly sensitive, and multiplexed targeted RNA sequencing assay for the detection of recurrent gene fusions on RNA extracted from routine clinical specimens of sarcomas (formalin-fixed paraffin-embedded or frozen). It facilitates the precise diagnosis and identification of tumors with potential targetable fusions. In addition, this assay can be easily customized to cover new fusions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background
The optimal threshold of surgical margins for breast malignant phyllodes tumors (MPTs) and the impact of adjuvant chemotherapy and radiotherapy were investigated.
Patients and Methods
We ...conducted a multicenter nationwide retrospective study of all MPT cases with central pathological review within the French Sarcoma Group. Endpoints were local recurrence-free survival (LRFS), metastasis-free survival (MFS), and overall survival (OS) rates.
Results
Overall, 212 patients were included in the study. All non-metastatic patients underwent primary surgical treatment, including 58.6% of conservative surgeries. An R0 resection was achieved in 117 patients (59.4%: 26.9% of patients with 1–2 mm margins, 12.2% of patients with 3–7 mm margins, 20.3% of patients with ≥ 8 mm margins). Ninety-four patients (45%) underwent a second surgery (SS) to obtain R0 margins, with a final mastectomy rate of 72.6%. Radiotherapy and chemotherapy were performed in 91 (43.1%) and 23 patients (10.9%), respectively, but were not associated with better outcomes. Mastectomy was significantly associated with better LRFS (
p
< 0.001). Margins of 0, 1, or 2 mm with SS were associated with better MFS (hazard ratio HR 0.3,
p
= 0.005) and OS (HR 0.32,
p
= 0.005) compared with margins of 0–1–2 mm without SS. Wider margins (> 8 mm) were not superior to margins of 3–7 mm (3–7 mm vs. > 8 mm; HR 0.81,
p
= 0.69). Age (HR 2.14,
p
= 0.038) and tumor necrosis (HR 1.96,
p
= 0.047) were found to be poor prognostic factors and were associated with MFS.
Conclusions
This study suggests that 3 mm margins are necessary and sufficient for surgical management of MPTs, and emphasizes the importance of SS to obtain clear margins in case of 0–1–2 mm margins. No impact of adjuvant chemotherapy or radiotherapy was detected in this study.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy have a poor outcome. We developed patient-derived xenografts (PDX) from residual tumors to identify ...efficient chemotherapies and predictive biomarkers in a context of resistance to anthracyclines- and taxanes-based treatments.
PDX were established from residual tumors of primary breast cancer patients treated in neoadjuvant setting. TNBC PDX were treated by anthracyclines, taxanes, platins, and capecitabine. Predictive biomarkers were identified by transcriptomic and immunohistologic analysis. Downregulation of
was performed by siRNA in a cell line established from a PDX.
Residual TNBC PDX were characterized by a high tumor take, a short latency, and a poor prognosis of the corresponding patients. With the exception of BRCA1/2-mutated models, residual PDX were resistant to anthracyclines, taxanes, and platins. Capecitabine, the oral prodrug of 5-FU, was highly efficient in 60% of PDX, with two models showing complete responses. Prior treatment of a responder PDX with 5-FU increased expression of thymidylate synthase and decreased efficacy of capecitabine. Transcriptomic and IHC analyses of 32 TNBC PDX, including both residual tumors and treatment-naïve derived tumors, identified RB1 and TYMP proteins as predictive biomarkers for capecitabine response. Finally,
knockdown in a cell line established from a capecitabine-responder PDX decreased sensitivity to 5-FU treatment.
We identified capecitabine as efficient chemotherapy in TNBC PDX models established from residual disease and resistant to anthracyclines, taxanes, and platins. RB1 positivity and high expression of TYMP were significantly associated with capecitabine response.
.
Background
Metastatic phyllodes tumors have poor prognosis with median overall survival of 11.5 months. The objective of this study is to identify prognostic factors and the best options for ...management of metastatic malignant phyllode tumors (MMPTs).
Patients and Methods
A multicentric retrospective study, including cases of MMPT from 10 sarcoma centers, was conducted. The primary end-point was overall survival (OS), and the secondary end-point was the clinical benefit of chemotherapy (CBCT) rate.
Results
51 MMPT patients were included. Median time from diagnosis to metastatic recurrence was 13 months. Management of MMPT consisted in surgery of the metastatic disease for 16 patients (31.3%), radiation therapy of the metastatic disease for 15 patients (31.9%), and chemotherapy for 37 patients (72.5%). Median follow-up was 62.1 months 95% confidence interval (CI) 31–80 months. Median OS was 11.5 months (95% CI 7.5–18.7 months). On multivariate analysis, two or more metastatic sites hazard ratio (HR) 2.81, 95% CI 1.27–6.19;
p
= 0.01 and surgery of metastasis (HR 0.33, 95% CI 0.14–0.78;
p
= 0.01) were independently associated with OS. The CBCT rate was 31.4% and 16.7% for the first and second lines. Polychemotherapy was not superior to single-agent therapy. Alkylating-agent-based chemotherapy, possibly associated with anthracyclines, was associated with a better CBCT rate than anthracyclines alone (
p
= 0.049).
Conclusions
The results of this study emphasize the impact of the number of metastatic sites on survival of MMPT patients and the leading role of metastasis surgery in MMPT management. If systemic therapy is used, it should include alkylating agents, which are associated with a better clinical benefit.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Children who survive cancer are at more than 19-fold increased risk of developing another malignancy. Renal cell carcinoma (RCC) occurring as a secondary malignancy is uncommon. Translocation RCC, ...bearing TFE3 or TFEB gene fusions, are recently recognized entities for which risk factors have not been identified.
We describe the clinical, pathologic, cytogenetic, and molecular data on six translocation RCCs that arose in five young patients who had received chemotherapy.
The ages at time of diagnosis of the RCC ranged from 6 to 22 years. Histologically, these tumors showed typical features previously described for translocation RCCs. At the molecular level, three tumors contained the ASPL-TFE3 fusion, two contained Alpha-TFEB, and one contained PRCC-TFE3. The intervals between chemotherapy and the diagnosis of RCC ranged from 4 to 13 years. The indications for the antecedent chemotherapy were varied and included acute promyelocytic leukemia, acute myeloid leukemia with t(9;11), bilateral Wilms' tumor, systemic lupus erythematosus, and conditioning regimen of bone marrow transplant for Hurler's syndrome. Only the latter patient had also received radiation. Hence, among 39 genetically confirmed translocation RCCs in our personal experience, six (15%) have arisen in patients who had received cytotoxic chemotherapy.
Cytotoxic chemotherapy may predispose to the development of renal translocation carcinomas.
Desmoplastic small round cell tumor (DSRCT) is characterized by the t(11;22)(p13;q12) translocation, which fuses the transcriptional regulatory domain of EWSR1 with the DNA-binding domain of WT1, ...resulting in the oncogenic EWSR1-WT1 fusion protein. The paucity of DSRCT disease models has hampered preclinical therapeutic studies on this aggressive cancer. Here, we developed preclinical disease models and mined DSRCT expression profiles to identify genetic vulnerabilities that could be leveraged for new therapies. We describe four DSRCT cell lines and one patient-derived xenograft model. Transcriptomic, proteomic and biochemical profiling showed evidence of activation of the ERBB pathway. Ectopic expression of EWSR1-WT1 resulted in upregulation of ERRB family ligands. Treatment of DSRCT cell lines with ERBB ligands resulted in activation of EGFR, ERBB2, ERK1/2 and AKT, and stimulation of cell growth. Antagonizing EGFR function with shRNAs, small-molecule inhibitors (afatinib, neratinib) or an anti-EGFR antibody (cetuximab) inhibited proliferation of DSRCT cells. Finally, treatment of mice bearing DSRCT xenografts with a combination of cetuximab and afatinib significantly reduced tumor growth. These data provide a rationale for evaluating EGFR antagonists in patients with DSRCT. This article has an associated First Person interview with the joint first authors of the paper.
High levels of tumor-infiltrating lymphocytes (TIL) before neoadjuvant chemotherapy (NAC) are associated with higher pathologic complete response (pCR) rates and better survival in triple-negative ...breast cancer (TNBC) and
-positive breast cancer. We investigated the value of TIL levels by evaluating lymphocyte infiltration before and after NAC.
We assessed stromal TIL levels in 716 pre- and posttreatment matched paired specimens, according to the guidelines of the International TIL Working Group.
Pre-NAC TIL levels were higher in tumors for which pCR was achieved than in cases with residual disease (33.9% vs. 20.3%,
= 0.001). This was observed in luminal tumors and TNBCs, but not in
-positive breast cancers (
= 0.001). The association between pre-NAC TIL levels and pCR was nonlinear in TNBCs (
= 0.005). Mean TIL levels decreased after chemotherapy completion (pre-NAC TILs: 24.1% vs. post-NAC TILs: 13.0%,
< 0.001). This decrease was strongly associated with high pCR rates, and the variation of TIL levels was strongly inversely correlated with pre-NAC TIL levels (
= -0.80,
< 0.001). Pre-NAC TILs and disease-free survival (DFS) were associated in a nonlinear manner (
< 0.001). High post-NAC TIL levels were associated with aggressive tumor characteristics and with impaired DFS in
-positive breast cancers (HR, 1.04; confidence interval, 1.02-1.06;
= 0.001), but not in luminal tumors or TNBCs (
= 0.04).
The associations of pre- and post-NAC TIL levels with response to treatment and DFS differ between breast cancer subtypes. The characterization of immune subpopulations may improve our understanding of the complex interactions between pre- or post-NAC setting, breast cancer subtype, response to treatment, and prognosis.
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