Publications by authors named "Sandro Santagata"

143 Publications

Skull Base Tumors: Neuropathology and Clinical implications.

Neurosurgery 2021 Jun 23. Epub 2021 Jun 23.

Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

Tumors that arise in and around the skull base comprise a wide range of common and rare entities. Recent studies have advanced our understanding of their pathogenesis, which in some cases, have significantly influenced clinical practice. The genotype of meningiomas is strongly associated with their phenotype, including histologic subtype and tumor location, and clinical outcome. A single molecular alteration, NAB2-STAT6 fusion, has redefined the category of solitary fibrous tumors to include the previous entity hemangiopericytomas. Schwannomas, both sporadic and familial, are characterized by near ubiquitous alterations in NF2, with additional mutations in SMARCB1 or LZTR1 in schwannomatosis. In pituitary adenohypophyseal tumors, cell lineage transcription factors such as SF-1, T-PIT, and PIT-1 are now essential for classification, providing a more rigorous taxonomy for tumors that were previously considered null cell adenomas. The pituicyte lineage transcription factor TTF-1 defines neurohypophyseal tumors, which may represent a single nosological entity with a spectrum of morphologic manifestations (ie, granular cell tumor, pituicytoma, and spindle cell oncocytoma). Likewise, the notochord cell lineage transcription factor brachyury defines chordoma, discriminating them from chondrosarcomas. The identification of nonoverlapping genetic drivers of adamantinomatous craniopharyngiomas and papillary craniopharyngiomas indicates that these are distinct tumor entities and has led to successful targeted treatment of papillary craniopharyngiomas using BRAF and/or mitogen-activated protein kinase inhibitors. Similarly, dramatic therapeutic responses have been achieved in patients with Langerhans cell histiocytosis, both with BRAF-mutant and BRAF-wildtype tumors. Familiarity with the pathology of skull base tumors, their natural history, and molecular features is essential for optimizing patient care.
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http://dx.doi.org/10.1093/neuros/nyab209DOI Listing
June 2021

Activity of PD-1 blockade with Nivolumab among patients with recurrent atypical/anaplastic meningioma: Phase II trial results.

Neuro Oncol 2021 May 20. Epub 2021 May 20.

Center for Neuro-Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

Background: Programmed death-1 ligand (PD-L1) contributes to tumor immunosuppression and is upregulated in aggressive meningiomas. We performed a phase II study of nivolumab, a programmed death-1 (PD-1) blocking antibody among patients with grade ≥2 meningioma that recurred after surgery and radiation therapy.

Methods: Twenty-five patients received nivolumab (240 mg biweekly) until progression, voluntary withdrawal, unacceptable toxicity, or death. Tumor mutational burden (TMB) and quantification of tumor infiltrating lymphocytes (TIL) were evaluated as potential immunocorrelative biomarkers. Change in neurologic function was prospectively assessed using the Neurologic Assessment in Neuro-Oncology (NANO) scale.

Results: Enrolled patients had multiple recurrences including ≥3 prior surgeries and ≥2 prior courses of radiation in 60% and 72%, respectively. Nivolumab was well tolerated with no unexpected AEs. PFS-6 was 42.4% (95% CI: 22.8, 60.7) and the median OS was 30.9 months (95% CI: 17.6, NA). One patient achieved radiographic response (ongoing at 4.5 years). TMB was > 10/Mb in 2 of 15 profiled tumors (13.3%). Baseline TIL density was low but increased post-treatment in 3 patients including both patients with elevated TMB. Most patients who achieved PFS-6 maintained neurologic function prior to progression as assessed by NANO.

Conclusion: Nivolumab was well tolerated but failed to improve PFS-6, although a subset of patients appeared to derive benefit. Low levels of TMB and TIL density were typically observed. NANO assessment of neurologic function contributed to outcome assessment. Future studies may consider rationally designed combinatorial regimens.
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http://dx.doi.org/10.1093/neuonc/noab118DOI Listing
May 2021

Prognostication for meningiomas: H3K27me3 to the rescue?

Neuro Oncol 2021 Apr 3. Epub 2021 Apr 3.

Division of Neuropathology, Department of Pathology, Brigham and Women's Hospital, Boston, MA.

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http://dx.doi.org/10.1093/neuonc/noab083DOI Listing
April 2021

Targeting immunosuppressive macrophages overcomes PARP inhibitor resistance in BRCA1-associated triple-negative breast cancer.

Nat Cancer 2021 Jan 14;2(1):66-82. Epub 2020 Dec 14.

Breast Tumor Immunology Laboratory, Dana-Farber Cancer Institute, Boston, MA 02215.

Despite objective responses to PARP inhibition and improvements in progression-free survival compared to standard chemotherapy in patients with BRCA-associated triple-negative breast cancer (TNBC), benefits are transitory. Using high dimensional single-cell profiling of human TNBC, here we demonstrate that macrophages are the predominant infiltrating immune cell type in BRCA-associated TNBC. Through multi-omics profiling we show that PARP inhibitors enhance both anti- and pro-tumor features of macrophages through glucose and lipid metabolic reprogramming driven by the sterol regulatory element-binding protein 1 (SREBP-1) pathway. Combined PARP inhibitor therapy with CSF-1R blocking antibodies significantly enhanced innate and adaptive anti-tumor immunity and extends survival in BRCA-deficient tumors and is mediated by CD8 T-cells. Collectively, our results uncover macrophage-mediated immune suppression as a liability of PARP inhibitor treatment and demonstrate combined PARP inhibition and macrophage targeting therapy induces a durable reprogramming of the tumor microenvironment, thus constituting a promising therapeutic strategy for TNBC.
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http://dx.doi.org/10.1038/s43018-020-00148-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7963404PMC
January 2021

Identification and Therapeutic Targeting of GPR20, Selectively Expressed in Gastrointestinal Stromal Tumors, with DS-6157a, a First-in-Class Antibody-Drug Conjugate.

Cancer Discov 2021 Jun 12;11(6):1508-1523. Epub 2021 Feb 12.

Daiichi Sankyo, Co., Ltd., Tokyo, Japan.

Currently, the only approved treatments for gastrointestinal stromal tumor (GIST) are tyrosine kinase inhibitors (TKI), which eventually lead to the development of secondary resistance mutations in KIT or PDGFRA and disease progression. Herein, we identified G protein-coupled receptor 20 (GPR20) as a novel non-tyrosine kinase target in GIST, developed new GPR20 IHC, and assessed GPR20 expression in cell lines, patient-derived xenografts, and clinical samples from two institutes (United States and Japan). We studied GPR20 expression stratified by treatment line, KIT expression, GIST molecular subtype, and primary tumor location. We produced DS-6157a, an anti-GPR20 antibody-drug conjugate with a novel tetrapeptide-based linker and DNA topoisomerase I inhibitor exatecan derivative (DXd). DS-6157a exhibited GPR20 expression-dependent antitumor activity in GIST xenograft models including a GIST model resistant to imatinib, sunitinib, and regorafenib. Preclinical pharmacokinetics and safety profile of DS-6157a support its clinical development as a potential novel GIST therapy in patients who are refractory or have resistance or intolerance to approved TKIs. SIGNIFICANCE: GPR20 is selectively expressed in GIST across all treatment lines, regardless of / genotypes. We generated DS-6157a, a DXd-based antibody-drug conjugate that exhibited antitumor activity in GIST models by a different mode of action than currently approved TKIs, showing favorable pharmacokinetics and safety profiles..
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http://dx.doi.org/10.1158/2159-8290.CD-20-1434DOI Listing
June 2021

HAND1 and BARX1 Act as Transcriptional and Anatomic Determinants of Malignancy in Gastrointestinal Stromal Tumor.

Clin Cancer Res 2021 Mar 15;27(6):1706-1719. Epub 2021 Jan 15.

Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts.

Purpose: Gastrointestinal stromal tumor (GIST) arises from interstitial cells of Cajal (ICC) or their precursors, which are present throughout the gastrointestinal tract. Although gastric GIST is commonly indolent and small intestine GIST more aggressive, a molecular understanding of disease behavior would inform therapy decisions in GIST. Although a core transcription factor (TF) network is conserved across GIST, accessory TFs HAND1 and BARX1 are expressed in a disease state-specific pattern. Here, we characterize two divergent transcriptional programs maintained by HAND1 and BARX1, and evaluate their association with clinical outcomes.

Experimental Design: We evaluated RNA sequencing and TF chromatin immunoprecipitation with sequencing in GIST samples and cultured cells for transcriptional programs associated with HAND1 and BARX1. Multiplexed tissue-based cyclic immunofluorescence and IHC evaluated tissue- and cell-level expression of TFs and their association with clinical factors.

Results: We show that HAND1 is expressed in aggressive GIST, modulating and core TF expression and supporting proliferative cellular programs. In contrast, BARX1 is expressed in indolent and micro-GISTs. HAND1 and BARX1 expression were superior predictors of relapse-free survival, as compared with standard risk stratification, and they predict progression-free survival on imatinib. Reflecting the developmental origins of accessory TF programs, HAND1 was expressed solely in small intestine ICCs, whereas BARX1 expression was restricted to gastric ICCs.

Conclusions: Our results define anatomic and transcriptional determinants of GIST and molecular origins of clinical phenotypes. Assessment of HAND1 and BARX1 expression in GIST may provide prognostic information and improve clinical decisions on the administration of adjuvant therapy.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956056PMC
March 2021

Sporadic multiple meningiomas harbor distinct driver mutations.

Acta Neuropathol Commun 2021 01 6;9(1). Epub 2021 Jan 6.

Department of Neurosurgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Fetscherstr. 74, 01307, Dresden, Germany.

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http://dx.doi.org/10.1186/s40478-020-01113-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789520PMC
January 2021

Distinct genomic subclasses of high-grade/progressive meningiomas: NF2-associated, NF2-exclusive, and NF2-agnostic.

Acta Neuropathol Commun 2020 10 21;8(1):171. Epub 2020 Oct 21.

Translational Neuro-Oncology Laboratory, Department of Neurosurgery, Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA.

Background: Genomic studies of high-grade/progressive meningiomas have reported a heterogeneous mutation spectrum, identifying few recurrently mutated genes. Most studies have been underpowered to detect genomic subclasses of aggressive meningiomas due to relatively small number of available samples. Here, we present a genomic survey of one of the largest multi-institutional cohorts of high-grade/progressive meningiomas to date.

Methods: 850 high-grade/progressive meningiomas, including 441 WHO grade 2 and 176 WHO grade 3 meningiomas and 220 progressive WHO grade 1 meningiomas, were tested as part of a clinical testing program by hybridization capture of 406 cancer-related genes to detect base substitutions, indels, amplifications, deletions, and rearrangements. Information from pathology reports, histopathology review, and patient clinical data was assessed.

Results: Genomic analyses converged to identify at least three distinct patterns of biologically-aggressive meningiomas. The first and most common contained NF2-mutant tumors (n = 426, 50%), was associated with male sex (64.4% %, p = 0.0001) and often harbored additional mutations in CDKN2A/B (24%), and the chromatin regulators ARID1A (9%), and KDM6A (6%). A second group (NF2-agnostic) featured TERT promoter (TERTp; n = 56) or TP53 mutations (n = 25) and were either NF2-mutant or wild-type, and displayed no association with either sex (p = 0.39). The remaining group generally lacked NF2 mutations, and accounted for 40% of the cases-with three subgroups. One consistent primarily of grade 3 lesions harboring alterations in chromatin regulators BAP1 (n = 22) or PBRM1 (n = 16). A second subgroup contained AKT1 (n = 26), PIK3CA (n = 14) and SMO (n = 7) mutant skull-based meningiomas, and a third mixed subgroup included 237 meningiomas with a heterogeneous spectrum of low frequency and non-recurrent alterations.

Conclusions: Our findings indicate that the patterns of genomic alterations in high-grade/progressive meningiomas commonly group into three different categories. The most common NF2-associated canonical group frequently harbored CDKN2A/B alterations, which is potentially amenable to targeted therapies. An NF2-agnostic group harbored frequent TERTp and TP53 mutations. The final subclass, distinct from the canonical NF2 mutant associated pathway, was partly characterized by BAP1/PBRM1 alterations (rhabdoid/papillary histology) or skull-base disease. Overall, these data increase our understanding of the pathobiology of high-grade/progressive meningiomas and can guide the design of clinical trials.

Irb Approval Status: Reviewed and approved by Western IRB; Protocol No. 20152817.
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http://dx.doi.org/10.1186/s40478-020-01040-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580027PMC
October 2020

SYLARAS: A Platform for the Statistical Analysis and Visual Display of Systemic Immunoprofiling Data and Its Application to Glioblastoma.

Cell Syst 2020 09 7;11(3):272-285.e9. Epub 2020 Sep 7.

Laboratory of Systems Pharmacology, Program in Therapeutic Science, Harvard Medical School, Boston, MA 02115, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA; Ludwig Center for Cancer Research at Harvard, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Accurately profiling systemic immune responses to cancer initiation and progression is necessary for understanding tumor surveillance and, ultimately, improving therapy. Here, we describe the SYLARAS software tool (systemic lymphoid architecture response assessment) and a dataset collected with SYLARAS that describes the frequencies of immune cells in primary and secondary lymphoid organs and in the tumor microenvironment of mice engrafted with a standard syngeneic glioblastoma (GBM) model. The data resource involves profiles of 5 lymphoid tissues in 48 mice and shows that GBM causes wide-spread changes in the local and systemic immune architecture. We use SYLARAS to identify a subset of CD45R/B220 CD8 T cells that is depleted from circulation but accumulates in the tumor mass and confirm this finding using multiplexed immunofluorescence microscopy. SYLARAS is freely available for download at (https://github.com/gjbaker/sylaras). A record of this paper's transparent peer review process is included in the Supplemental Information.
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http://dx.doi.org/10.1016/j.cels.2020.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565356PMC
September 2020

Frequent inactivating mutations of the PBAF complex gene PBRM1 in meningioma with papillary features.

Acta Neuropathol 2020 07 13;140(1):89-93. Epub 2020 May 13.

Translational Neuro‑Oncology Laboratory, Department of Neurosurgery, Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA.

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http://dx.doi.org/10.1007/s00401-020-02161-7DOI Listing
July 2020

Mechanisms and therapeutic implications of hypermutation in gliomas.

Nature 2020 04 15;580(7804):517-523. Epub 2020 Apr 15.

Drug Development Department (DITEP), INSERM U1015, Université Paris Saclay, Gustave Roussy, Villejuif, France.

A high tumour mutational burden (hypermutation) is observed in some gliomas; however, the mechanisms by which hypermutation develops and whether it predicts the response to immunotherapy are poorly understood. Here we comprehensively analyse the molecular determinants of mutational burden and signatures in 10,294 gliomas. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after treatment with the chemotherapy drug temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas was recapitulated by temozolomide-induced damage in cells with MMR deficiency. MMR-deficient gliomas were characterized by a lack of prominent T cell infiltrates, extensive intratumoral heterogeneity, poor patient survival and a low rate of response to PD-1 blockade. Moreover, although bulk analyses did not detect microsatellite instability in MMR-deficient gliomas, single-cell whole-genome sequencing analysis of post-treatment hypermutated glioma cells identified microsatellite mutations. These results show that chemotherapy can drive the acquisition of hypermutated populations without promoting a response to PD-1 blockade and supports the diagnostic use of mutational burden and signatures in cancer.
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http://dx.doi.org/10.1038/s41586-020-2209-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8235024PMC
April 2020

The Human Tumor Atlas Network: Charting Tumor Transitions across Space and Time at Single-Cell Resolution.

Cell 2020 04;181(2):236-249

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Ludwig Center for Cancer Research and Laboratory of Systems Pharmacology, Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.
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http://dx.doi.org/10.1016/j.cell.2020.03.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376497PMC
April 2020

A Deregulated HOX Gene Axis Confers an Epigenetic Vulnerability in KRAS-Mutant Lung Cancers.

Cancer Cell 2020 05 2;37(5):705-719.e6. Epub 2020 Apr 2.

Genetics Division, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115, USA; Ludwig Center at Harvard, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

While KRAS mutations are common in non-small cell lung cancer (NSCLC), effective treatments are lacking. Here, we report that half of KRAS-mutant NSCLCs aberrantly express the homeobox protein HOXC10, largely due to unappreciated defects in PRC2, which confers sensitivity to combined BET/MEK inhibitors in xenograft and PDX models. Efficacy of the combination is dependent on suppression of HOXC10 by BET inhibitors. We further show that HOXC10 regulates the expression of pre-replication complex (pre-RC) proteins in sensitive tumors. Accordingly, BET/MEK inhibitors suppress pre-RC proteins in cycling cells, triggering stalled replication, DNA damage, and death. These studies reveal a promising therapeutic strategy for KRAS-mutant NSCLCs, identify a predictive biomarker of response, and define a subset of NSCLCs with a targetable epigenetic vulnerability.
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http://dx.doi.org/10.1016/j.ccell.2020.03.004DOI Listing
May 2020

Genomic characterization of human brain metastases identifies drivers of metastatic lung adenocarcinoma.

Nat Genet 2020 04 23;52(4):371-377. Epub 2020 Mar 23.

Department of Medicine I, Division of Oncology, Medical University of Vienna, Comprehensive Cancer Center Vienna, Vienna, Austria.

Brain metastases from lung adenocarcinoma (BM-LUAD) frequently cause patient mortality. To identify genomic alterations that promote brain metastases, we performed whole-exome sequencing of 73 BM-LUAD cases. Using case-control analyses, we discovered candidate drivers of brain metastasis by identifying genes with more frequent copy-number aberrations in BM-LUAD compared to 503 primary LUADs. We identified three regions with significantly higher amplification frequencies in BM-LUAD, including MYC (12 versus 6%), YAP1 (7 versus 0.8%) and MMP13 (10 versus 0.6%), and significantly more frequent deletions in CDKN2A/B (27 versus 13%). We confirmed that the amplification frequencies of MYC, YAP1 and MMP13 were elevated in an independent cohort of 105 patients with BM-LUAD. Functional assessment in patient-derived xenograft mouse models validated the notion that MYC, YAP1 or MMP13 overexpression increased the incidence of brain metastasis. These results demonstrate that somatic alterations contribute to brain metastases and that genomic sequencing of a sufficient number of metastatic tumors can reveal previously unknown metastatic drivers.
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http://dx.doi.org/10.1038/s41588-020-0592-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136154PMC
April 2020

Telomere length alterations and ATRX/DAXX loss in pituitary adenomas.

Mod Pathol 2020 08 18;33(8):1475-1481. Epub 2020 Mar 18.

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Telomeres are nucleoprotein complexes located at the termini of eukaryotic chromosomes that prevent exonucleolytic degradation and end-to-end chromosomal fusions. Cancers often have critically shortened, dysfunctional telomeres contributing to genomic instability. Telomere shortening has been reported in a wide range of precancerous lesions and invasive carcinomas. However, the role of telomere alterations, including the presence of alternative lengthening of telomeres (ALT), has not been studied in pituitary adenomas. Telomere length and the presence of ALT were assessed directly at the single cell level using a telomere-specific fluorescence in situ hybridization assay in tissue microarrays. Tumors were characterized as either ALT-positive or having short, normal, or long telomere lengths and then these categories were compared with clinicopathological characteristics. ATRX and DAXX expression was studied through immunohistochemistry. We characterized a discovery set of 106 pituitary adenomas including both functional and nonfunctional subsets (88 primary, 18 recurrent). Telomere lengths were estimated and we observed 64 (59.4%) cases with short, 39 (36.8%) cases with normal, and 0 (0%) cases with long telomeres. We did not observe significant differences in the clinicopathological characteristics of the group with abnormally shortened telomeres compared to the group with normal telomeres. However, three pituitary adenomas were identified as ALT-positive of which two were recurrent tumors. Two of these three ALT-positive cases had alterations in either of the chromatin remodeling proteins, ATRX and DAXX, which are routinely altered in other ALT-positive tumor subtypes. In a second cohort of 32 recurrent pituitary adenomas from 22 patients, we found that the tumors from 36% of patients (n = 8) were ALT-positive. This study demonstrates that short telomere lengths are prevalent in pituitary adenomas and that ALT-positive pituitary adenomas are enriched in recurrent disease.
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http://dx.doi.org/10.1038/s41379-020-0523-2DOI Listing
August 2020

Immunogenomic profiling determines responses to combined PARP and PD-1 inhibition in ovarian cancer.

Nat Commun 2020 03 19;11(1):1459. Epub 2020 Mar 19.

Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA, 02215, USA.

Combined PARP and immune checkpoint inhibition has yielded encouraging results in ovarian cancer, but predictive biomarkers are lacking. We performed immunogenomic profiling and highly multiplexed single-cell imaging on tumor samples from patients enrolled in a Phase I/II trial of niraparib and pembrolizumab in ovarian cancer (NCT02657889). We identify two determinants of response; mutational signature 3 reflecting defective homologous recombination DNA repair, and positive immune score as a surrogate of interferon-primed exhausted CD8 + T-cells in the tumor microenvironment. Presence of one or both features associates with an improved outcome while concurrent absence yields no responses. Single-cell spatial analysis reveals prominent interactions of exhausted CD8 + T-cells and PD-L1 + macrophages and PD-L1 + tumor cells as mechanistic determinants of response. Furthermore, spatial analysis of two extreme responders shows differential clustering of exhausted CD8 + T-cells with PD-L1 + macrophages in the first, and exhausted CD8 + T-cells with cancer cells harboring genomic PD-L1 and PD-L2 amplification in the second.
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http://dx.doi.org/10.1038/s41467-020-15315-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081234PMC
March 2020

HSF1 phase transition mediates stress adaptation and cell fate decisions.

Nat Cell Biol 2020 02 3;22(2):151-158. Epub 2020 Feb 3.

Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

Under proteotoxic stress, some cells survive whereas others die. The mechanisms governing this heterogeneity in cell fate remain unknown. Here we report that condensation and phase transition of heat-shock factor 1 (HSF1), a transcriptional regulator of chaperones, is integral to cell-fate decisions underlying survival or death. During stress, HSF1 drives chaperone expression but also accumulates separately in nuclear stress bodies called foci. Foci formation has been regarded as a marker of cells actively upregulating chaperones. Using multiplexed tissue imaging, we observed HSF1 foci in human tumours. Paradoxically, their presence inversely correlated with chaperone expression. By live-cell microscopy and single-cell analysis, we found that foci dissolution rather than formation promoted HSF1 activity and cell survival. During prolonged stress, the biophysical properties of HSF1 foci changed; small, fluid condensates enlarged into indissoluble gel-like arrangements with immobilized HSF1. Chaperone gene induction was reduced in such cells, which were prone to apoptosis. Quantitative analysis suggests that survival under stress results from competition between concurrent but opposing mechanisms. Foci may serve as sensors that tune cytoprotective responses, balancing rapid transient responses and irreversible outcomes.
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http://dx.doi.org/10.1038/s41556-019-0458-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135912PMC
February 2020

Minerva: a light-weight, narrative image browser for multiplexed tissue images.

J Open Source Softw 2020 15;5(54). Epub 2020 Oct 15.

Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA.

Advances in highly multiplexed tissue imaging are transforming our understanding of human biology by enabling detection and localization of 10-100 proteins at subcellular resolution (Bodenmiller, 2016). Efforts are now underway to create public atlases of multiplexed images of normal and diseased tissues (Rozenblatt-Rosen et al., 2020). Both research and clinical applications of tissue imaging benefit from recording data from complete specimens so that data on cell state and composition can be studied in the context of overall tissue architecture. As a practical matter, specimen size is limited by the dimensions of microscopy slides (2.5 × 7.5 cm or ~2-8 cm of tissue depending on shape). With current microscopy technology, specimens of this size can be imaged at sub-micron resolution across ~60 spectral channels and ~10 cells, resulting in image files of terabyte size. However, the rich detail and multiscale properties of these images pose a substantial computational challenge (Rashid et al., 2020). See Rashid et al. (2020) for an comparison of existing visualization tools targeting these multiplexed tissue images.
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http://dx.doi.org/10.21105/joss.02579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7989801PMC
October 2020

Highly multiplexed immunofluorescence images and single-cell data of immune markers in tonsil and lung cancer.

Sci Data 2019 12 17;6(1):323. Epub 2019 Dec 17.

Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, United States.

In this data descriptor, we document a dataset of multiplexed immunofluorescence images and derived single-cell measurements of immune lineage and other markers in formaldehyde-fixed and paraffin-embedded (FFPE) human tonsil and lung cancer tissue. We used tissue cyclic immunofluorescence (t-CyCIF) to generate fluorescence images which we artifact corrected using the BaSiC tool, stitched and registered using the ASHLAR algorithm, and segmented using ilastik software and MATLAB. We extracted single-cell features from these images using HistoCAT software. The resulting dataset can be visualized using image browsers and analyzed using high-dimensional, single-cell methods. This dataset is a valuable resource for biological discovery of the immune system in normal and diseased states as well as for the development of multiplexed image analysis and viewing tools.
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http://dx.doi.org/10.1038/s41597-019-0332-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917801PMC
December 2019

Localized Metabolomic Gradients in Patient-Derived Xenograft Models of Glioblastoma.

Cancer Res 2020 03 25;80(6):1258-1267. Epub 2019 Nov 25.

Department of Radiology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts.

Glioblastoma (GBM) is increasingly recognized as a disease involving dysfunctional cellular metabolism. GBMs are known to be complex heterogeneous systems containing multiple distinct cell populations and are supported by an aberrant network of blood vessels. A better understanding of GBM metabolism, its variation with respect to the tumor microenvironment, and resulting regional changes in chemical composition is required. This may shed light on the observed heterogeneous drug distribution, which cannot be fully described by limited or uneven disruption of the blood-brain barrier. In this work, we used mass spectrometry imaging (MSI) to map metabolites and lipids in patient-derived xenograft models of GBM. A data analysis workflow revealed that distinctive spectral signatures were detected from different regions of the intracranial tumor model. A series of long-chain acylcarnitines were identified and detected with increased intensity at the tumor edge. A 3D MSI dataset demonstrated that these molecules were observed throughout the entire tumor/normal interface and were not confined to a single plane. mRNA sequencing demonstrated that hallmark genes related to fatty acid metabolism were highly expressed in samples with higher acylcarnitine content. These data suggest that cells in the core and the edge of the tumor undergo different fatty acid metabolism, resulting in different chemical environments within the tumor. This may influence drug distribution through changes in tissue drug affinity or transport and constitute an important consideration for therapeutic strategies in the treatment of GBM. SIGNIFICANCE: GBM tumors exhibit a metabolic gradient that should be taken into consideration when designing therapeutic strategies for treatment..
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http://dx.doi.org/10.1158/0008-5472.CAN-19-0638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073296PMC
March 2020

Pre- and Postoperative Neratinib for HER2-Positive Breast Cancer Brain Metastases: Translational Breast Cancer Research Consortium 022.

Clin Breast Cancer 2020 04 22;20(2):145-151.e2. Epub 2019 Aug 22.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA.

Purpose: This pilot study was performed to test our ability to administer neratinib monotherapy before clinically recommended craniotomy in patients with HER2-positive metastatic breast cancer to the central nervous system, to examine neratinib's central nervous system penetration at craniotomy, and to examine postoperative neratinib maintenance.

Patients And Methods: Patients with HER2-positive brain metastases undergoing clinically indicated cranial resection of a parenchymal tumor received neratinib 240 mg orally once a day for 7 to 21 days preoperatively, and resumed therapy postoperatively in 28-day cycles. Exploratory evaluations of time to disease progression, survival, and correlative tissue, cerebrospinal fluid (CSF), and blood-based analyses examining neratinib concentrations were planned. The study was registered at ClinicalTrials.gov under number NCT01494662.

Results: We enrolled 5 patients between May 22, 2013, and October 18, 2016. As of March 1, 2019, patients had remained on the study protocol for 1 to 75+ postoperative cycles pf therapy. Two patients had grade 3 diarrhea. Evaluation of the CSF showed low concentrations of neratinib; nonetheless, 2 patients continued to receive therapy without disease progression for at least 13 cycles, with one on-study treatment lasting for nearly 6 years. Neratinib distribution in surgical tissue was variable for 1 patient, while specimens from 2 others did not produce conclusive results as a result of limited available samples.

Conclusion: Neratinib resulted in expected rates of diarrhea in this small cohort, with 2 of 5 patients receiving the study treatment for durable periods. Although logistically challenging, we were able to test a limited number of CSF- and parenchymal-based neratinib concentrations. Our findings from resected tumor tissue in one patient revealed heterogeneity in drug distribution and tumor histopathology.
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http://dx.doi.org/10.1016/j.clbc.2019.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035200PMC
April 2020

Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging.

Nat Protoc 2019 10 18;14(10):2900-2930. Epub 2019 Sep 18.

Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.
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http://dx.doi.org/10.1038/s41596-019-0206-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959005PMC
October 2019

Correction to: An update on the CNS manifestations of neurofibromatosis type 2.

Acta Neuropathol 2020 Apr;139(4):667

Division of Neuropathology, Department of Pathology, Brigham and Women's Hospital, Hale Building for Transformative Medicine, BTM8002P, 60 Fenwood Road, Boston, MA, 02115, USA.

The article An update on the CNS manifestations of neurofibromatosis type 2, written by Shannon Coy, Rumana Rashid, Anat Stemmer‑Rachamimov and Sandro Santagata, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 04 June 2019 without open access.
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http://dx.doi.org/10.1007/s00401-019-02044-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096359PMC
April 2020

Targeted treatment of papillary craniopharyngiomas harboring BRAF V600E mutations.

Cancer 2019 09 17;125(17):2910-2914. Epub 2019 Jul 17.

Divisions of Neuro-Oncology and Hematology/Oncology, Departments of Neurology and Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts.

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http://dx.doi.org/10.1002/cncr.32197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7032527PMC
September 2019

Rapid MALDI mass spectrometry imaging for surgical pathology.

NPJ Precis Oncol 2019 4;3:17. Epub 2019 Jul 4.

2Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115 USA.

Matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) is an emerging analytical technique, which generates spatially resolved proteomic and metabolomic images from tissue specimens. Conventional MALDI MSI processing and data acquisition can take over 30 min, limiting its clinical utility for intraoperative diagnostics. We present a rapid MALDI MSI method, completed under 5 min, including sample preparation and analysis, providing a workflow compatible with the clinical frozen section procedure.
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http://dx.doi.org/10.1038/s41698-019-0089-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609678PMC
July 2019

Rebalancing Protein Homeostasis Enhances Tumor Antigen Presentation.

Clin Cancer Res 2019 11 18;25(21):6392-6405. Epub 2019 Jun 18.

Whitehead Institute for Biomedical Research, Cambridge, Massachusetts.

Purpose: Despite the accumulation of extensive genomic alterations, many cancers fail to be recognized as "foreign" and escape destruction by the host immune system. Immunotherapies designed to address this problem by directly stimulating immune effector cells have led to some remarkable clinical outcomes, but unfortunately, most cancers fail to respond, prompting the need to identify additional immunomodulatory treatment options. We elucidated the effect of a novel treatment paradigm using sustained, low-dose HSP90 inhibition and in syngeneic mouse models using genetic and pharmacologic tools. Profiling of treatment-associated tumor cell antigens was performed using immunoprecipitation followed by peptide mass spectrometry.

Results: We show that sustained, low-level inhibition of HSP90 both amplifies and diversifies the antigenic repertoire presented by tumor cells on MHC-I molecules through an IFNγ-independent mechanism. In stark contrast, we find that acute, high-dose exposure to HSP90 inhibitors, the only approach studied in the clinic to date, is broadly immunosuppressive in cell culture and in patients with cancer. In mice, chronic non-heat shock-inducing HSP90 inhibition slowed progression of colon cancer implants, but only in syngeneic animals with intact immune function. Addition of a single dose of nonspecific immune adjuvant to the regimen dramatically increased efficacy, curing a subset of mice receiving combination therapy.

Conclusions: These highly translatable observations support reconsideration of the most effective strategy for targeting HSP90 to treat cancers and suggest a practical approach to repurposing current orally bioavailable HSP90 inhibitors as a new immunotherapeutic strategy..
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http://dx.doi.org/10.1158/1078-0432.CCR-19-0596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825536PMC
November 2019

An update on the CNS manifestations of neurofibromatosis type 2.

Acta Neuropathol 2020 04 4;139(4):643-665. Epub 2019 Jun 4.

Division of Neuropathology, Department of Pathology, Brigham and Women's Hospital, Hale Building for Transformative Medicine, BTM8002P, 60 Fenwood Road, Boston, MA, 02115, USA.

Neurofibromatosis type II (NF2) is a tumor predisposition syndrome characterized by the development of distinctive nervous system lesions. NF2 results from loss-of-function alterations in the NF2 gene on chromosome 22, with resultant dysfunction of its protein product merlin. NF2 is most commonly associated with the development of bilateral vestibular schwannomas; however, patients also have a predisposition to development of other tumors including meningiomas, ependymomas, and peripheral, spinal, and cranial nerve schwannomas. Patients may also develop other characteristic manifestations such as ocular lesions, neuropathies, meningioangiomatosis, and glial hamartia. NF2 has a highly variable clinical course, with some patients exhibiting a severe phenotype and development of multiple tumors at an early age, while others may be nearly asymptomatic throughout their lifetime. Despite the high morbidity associated with NF2 in severe cases, management of NF2-associated lesions primarily consists of surgical resection and treatment of symptoms, and there are currently no FDA-approved systemic therapies that address the underlying biology of the syndrome. Refinements to the diagnostic criteria of NF2 have been proposed over time due to increasing understanding of clinical and molecular data. Large-population studies have demonstrated that some features such as the development of gliomas and neurofibromas, currently included as diagnostic criteria, may require further clarification and modification. Meanwhile, burgeoning insights into the molecular biology of NF2 have shed light on the etiology and highly variable severity of the disease and suggested numerous putative molecular targets for therapeutic intervention. Here, we review the clinicopathologic features of NF2, current understanding of the molecular biology of NF2, particularly with regard to central nervous system lesions, ongoing therapeutic studies, and avenues for further research.
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http://dx.doi.org/10.1007/s00401-019-02029-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038792PMC
April 2020
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