RB1, p16, and Human Papillomavirus in Oropharyngeal Squamous Cell Carcinoma

Head and neck pathology

2021 Apr 08

Berdugo, J;Rooper, LM;Chiosea, SI;
PMID: 33830464 | DOI: 10.1007/s12105-021-01317-5

While P16 immunohistochemistry (IHC) is a well-established surrogate marker of Human Papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OSCC), Retinoblastoma 1 (RB1) loss may lead to p16 overexpression in the absence of HPV. We determined the proportion of p16-positive/HPV-negative OSCC with RB1 loss and other alterations in RB1/p16 pathway, and tested RB1 IHC as a prognostic biomarker for OSCC, along with the 8th edition of AJCC staging manual. P16 and RB1 IHC and HPV DNA in situ hybridization (ISH) were performed on 257 OSCC. High risk HPV RNA ISH, RB1 fluorescence in situ hybridization (FISH), and next generation sequencing (NGS) were done on p16-positive/HPV DNA ISH-negative OSCC. Disease free survival (DFS) was used as an endpoint. In the entire cohort and in p16-positive (n = 184) and p16-negative (n = 73) subgroups, AJCC 8th edition staging correlated with DFS (p < 0.01). RB1 IHC showed RB1 loss in p16-positive OSCC only (79/184, 43%). RB1 loss by IHC is associated with a better DFS, without providing additional prognostic information for patients with p16-positive OSCC. HPV RNA ISH was positive in 12 of 14 HPV DNA ISH-negative cases. RB1 IHC showed loss in 10 of 15 HPV DNA ISH-negative cases and in 1 of 2 HPV RNA ISH-negative cases. Overall, only one case of p16-positive/HPV RNA ISH-negative OSCC showed RB1 loss by IHC (1/184, 0.5%). Of the 10 p16-positive and HPV DNA ISH-negative cases with RB1 loss by IHC, 2 had RB1 hemizygous deletion and 3 showed Chromosome 13 monosomy by FISH. No RB1 mutations were detected by NGS. Other molecular alterations in p16-positive/HPV DNA ISH-negative cases included TP53 and TERT mutations and DDX3X loss. HPV-independent RB1 inactivation rarely results in false positive p16 IHC. RB1 inactivation by high risk HPV E7 oncoprotein may co-exist with RB1 deletion. RB1 loss is a favorable prognosticator and occurs exclusively in p16-positive OSCC. The 8th edition of the AJCC staging manual satisfactorily predicts DFS of OSCC patients.

UHRF1 overexpression promotes osteosarcoma metastasis through altered exosome production and AMPK/SEMA3E suppression

Oncogenesis

2022 Sep 06

Wu, SC;Kim, A;Gu, Y;Martinez, DI;Zocchi, L;Chen, CC;Lopez, J;Salcido, K;Singh, S;Wu, J;Nael, A;Benavente, CA;
PMID: 36068209 | DOI: 10.1038/s41389-022-00430-6

Loss-of-function mutations at the retinoblastoma (RB1) gene are associated with increased mortality, metastasis, and poor therapeutic outcome in several cancers, including osteosarcoma. However, the mechanism(s) through which RB1 loss worsens clinical outcome remains understudied. Ubiquitin-like with PHD and Ring Finger domains 1 (UHRF1) has been identified as a critical downstream effector of the RB/E2F signaling pathway that is overexpressed in various cancers. Here, we determined the role and regulatory mechanisms of UHRF1 in rendering osteosarcoma cells more aggressive. Higher UHRF1 expression correlated with malignancy in osteosarcoma cell lines, clinical samples, and genetically engineered mouse models. Gain- and loss-of-function assays revealed that UHRF1 has cell-intrinsic and extrinsic functions promoting cell proliferation, migration, invasion, angiogenesis, and metastasis. UHRF1 overexpression induced angiogenesis by suppressing AMPK activation and Semaphorin 3E (SEMA3E) expression. Further, UHRF1-mediated migration and metastasis resulted, at least in part, through altered expression of extracellular vesicles and their cargo, including urokinase-type plasminogen activator (uPA). Novel osteosarcoma genetically engineered mouse models confirmed that knocking out Uhrf1 considerably decreased metastasis and reversed the poorer survival associated with Rb1 loss. This presents a new mechanistic insight into RB1 loss-associated poor prognosis and novel oncogenic roles of UHRF1 in the regulation of angiogenesis and exosome secretion, both critical for osteosarcoma metastasis. This provides substantial support for targeting UHRF1 or its downstream effectors as novel therapeutic options to improve current treatment for osteosarcoma.

PD-L1, RB1 and mismatch repair protein immunohistochemical expression in neuroendocrine carcinoma, small cell type of the uterine cervix.

Histopathology. 2019 Jan 22.

2019 Jan 22

Morgan S, Slodkowska E, Parra-Herran C, Mirkovic J.
PMID: PMID: 30667073 | DOI: DOI:10.1111/his.13825

Abstract AIM: Neuroendocrine carcinoma, small cell type of the uterine cervix (SmCC-Cx) is a rare HPV- related tumor with limited therapeutic options. Merkel cell carcinoma, another virus-associated neuroendocrine malignancy, has significant PD-L1 expression rates. PD-L1 expression has been reported in other malignancies of the cervix. We aimed to determine the prevalence of PD-L1 in the context of mismatch repair protein (MMR) and RB1 expression status in SmCC-Cx. METHODS AND RESULTS: Ten cases of SmCC-Cx were tested for immunohistochemistry expression of PD-L1, MLH1, MSH2, MSH6, PMS2, RB1, CD3, CD20 and HPV in situ hybridization (ISH). PD-L1 expression was scored quantitatively (H-score) in tumor cells and lymphocytes (tumoral/peritumoral). PD-L1 positivity was seen in 7 cases, focal in most cases (H-scores 3 to 140). 3/9 cases showed MMR deficiency. PD-L1 expression levels correlated with MMR expression status: all 3 MLH1/PMS2-deficient cases had a ≥5% PD-L1 staining and an H-score ≥10 (p=0.01). RB1 was lost in 4/9 cases, all PD-L1+, but this correlation was not statistically significant.7/9 tumors were positive for HPV-ISH; two of these had MLH1/PMS2 loss. Among the 2 HPV-ISH negative tumors, one had MLS1/PMS2 loss. CONCLUSIONS: PD-L1 expression, predominantly focal, is seen in 70% of SmCC-Cx, while loss of MMR expression is seen in 33% of SmCC-Cx in our cohort. PD-L1 expression in >10% of tumor cells is seen in a subset of tumors in association with loss of MMR expression. These patients may be amenable to immune checkpoint inhibitor therapy as a promising alternative for this aggressive disease.

Delta-like protein 3 expression and therapeutic targeting in neuroendocrine prostate cancer

Sci Transl Med

2019 Mar 20

Puca L, Gavyert K, Sailer V, Conteduca V, Dardenne E, Sigouros M, Isse K, Kearney M, Vosoughi A, Fernandez L, Pan H, Motanagh S, Hess J, Donoghue AJ, Sboner A, Wang Y, Dittamore R, Rickman D, Nanus DM, Tagawa ST, Elemento O, Mosquera JM, Saunders L and Beltran H
PMID: 30894499 | DOI: 10.1126/scitranslmed.aav0891

Histologic transformation to small cell neuroendocrine prostate cancer occurs in a subset of patients with advanced prostate cancer as a mechanism of treatment resistance. Rovalpituzumab tesirine (SC16LD6.5) is an antibody-drug conjugate that targets delta-like protein 3 (DLL3) and was initially developed for small cell lung cancer. We found that DLL3 is expressed in most of the castration-resistant neuroendocrine prostate cancer (CRPC-NE) (36 of 47, 76.6%) and in a subset of castration-resistant prostate adenocarcinomas (7 of 56, 12.5%). It shows minimal to no expression in localized prostate cancer (1 of 194) and benign prostate (0 of 103). DLL3 expression correlates with neuroendocrine marker expression, RB1 loss, and aggressive clinical features. DLL3 in circulating tumor cells was concordant with matched metastatic biopsy (87%). Treatment of DLL3-expressing prostate cancer xenografts with a single dose of SC16LD6.5 resulted in complete and durable responses, whereas DLL3-negative models were insensitive. We highlight a patient with neuroendocrine prostate cancer with a meaningful clinical and radiologic response to SC16LD6.5 when treated on a phase 1 trial. Overall, our findings indicate that DLL3 is preferentially expressed in CRPC-NE and provide rationale for targeting DLL3 in patients with DLL3-positive metastatic prostate cancer.

Genomic Alterations in Human Papillomavirus-Positive and-Negative Conjunctival Squamous Cell Carcinomas

Investigative ophthalmology & visual science

2021 Nov 01

Ramberg, I;Vieira, FG;Toft, PB;von Buchwald, C;Funding, M;Nielsen, FC;Heegaard, S;
PMID: 34779821 | DOI: 10.1167/iovs.62.14.11

The genomic alterations contributing to the pathogenesis of conjunctival squamous cell carcinomas (SCCs) and their precursor lesions are poorly understood and hamper our ability to develop molecular therapies to reduce the recurrence rates and treatment-related morbidities of this disease. We aimed to characterize the somatic DNA alterations in human papillomavirus (HPV)-positive and HPV-negative conjunctival SCC.Patients diagnosed with conjunctival SCC in situ or SCC treated in ocular oncology referral centers in Denmark were included. HPV detection (HPV DNA PCR, p16 immunohistochemistry, and mRNA in situ hybridization) and targeted capture-based next-generation sequencing of 523 genes frequently involved in cancer were performed to describe the mutational profile based on HPV status.Tumor tissue was available in 33 cases (n = 8 conjunctival SCCs in situ, n = 25 conjunctival SCCs), constituting 25 male and 8 female patients. Nine cases were HPV positive. The HPV-positive SCCs in situ and SCCs were characterized by transcriptionally active high-risk HPV (types 16 and 39) within the tumor cells, frequent mutations in PIK3CA (n = 5/9), and wild-type TP53, CDKN2A, and RB1, while the HPV-negative counterparts harbored frequent mutations in TP53 (n = 21/24), CDKN2A (n = 7/24), and RB1 (n = 6/24).Our findings have delineated two potentially distinct distributions of somatic mutations in conjunctival SCC based on HPV status-pointing to different biological mechanisms of carcinogenesis. The present findings support a causal role of HPV in a subset of conjunctival SCC.

A proteogenomic portrait of lung squamous cell carcinoma

Cell

2021 Aug 05

Satpathy, S;Krug, K;Jean Beltran, PM;Savage, SR;Petralia, F;Kumar-Sinha, C;Dou, Y;Reva, B;Kane, MH;Avanessian, SC;Vasaikar, SV;Krek, A;Lei, JT;Jaehnig, EJ;Omelchenko, T;Geffen, Y;Bergstrom, EJ;Stathias, V;Christianson, KE;Heiman, DI;Cieslik, MP;Cao, S;Song, X;Ji, J;Liu, W;Li, K;Wen, B;Li, Y;Gümüş, ZH;Selvan, ME;Soundararajan, R;Visal, TH;Raso, MG;Parra, ER;Babur, Ö;Vats, P;Anand, S;Schraink, T;Cornwell, M;Rodrigues, FM;Zhu, H;Mo, CK;Zhang, Y;da Veiga Leprevost, F;Huang, C;Chinnaiyan, AM;Wyczalkowski, MA;Omenn, GS;Newton, CJ;Schurer, S;Ruggles, KV;Fenyö, D;Jewell, SD;Thiagarajan, M;Mesri, M;Rodriguez, H;Mani, SA;Udeshi, ND;Getz, G;Suh, J;Li, QK;Hostetter, G;Paik, PK;Dhanasekaran, SM;Govindan, R;Ding, L;Robles, AI;Clauser, KR;Nesvizhskii, AI;Wang, P;Carr, SA;Zhang, B;Mani, DR;Gillette, MA;Clinical Proteomic Tumor Analysis Consortium, ;
PMID: 34358469 | DOI: 10.1016/j.cell.2021.07.016

Lung squamous cell carcinoma (LSCC) remains a leading cause of cancer death with few therapeutic options. We characterized the proteogenomic landscape of LSCC, providing a deeper exposition of LSCC biology with potential therapeutic implications. We identify NSD3 as an alternative driver in FGFR1-amplified tumors and low-p63 tumors overexpressing the therapeutic target survivin. SOX2 is considered undruggable, but our analyses provide rationale for exploring chromatin modifiers such as LSD1 and EZH2 to target SOX2-overexpressing tumors. Our data support complex regulation of metabolic pathways by crosstalk between post-translational modifications including ubiquitylation. Numerous immune-related proteogenomic observations suggest directions for further investigation. Proteogenomic dissection of CDKN2A mutations argue for more nuanced assessment of RB1 protein expression and phosphorylation before declaring CDK4/6 inhibition unsuccessful. Finally, triangulation between LSCC, LUAD, and HNSCC identified both unique and common therapeutic vulnerabilities. These observations and proteogenomics data resources may guide research into the biology and treatment of LSCC.

Morphologic and Molecular Heterogeneity of Cervical Neuroendocrine Neoplasia: A Report of 14 Cases

The American journal of surgical pathology

2022 Sep 05

Ordulu, Z;Mino-Kenudson, M;Young, RH;Van de Vijver, K;Zannoni, GF;Félix, A;Burandt, E;Wong, A;Nardi, V;Oliva, E;
PMID: 36069807 | DOI: 10.1097/PAS.0000000000001943

Neuroendocrine neoplasms (NENs) of the cervix are rare aggressive tumors associated with poor prognosis and only limited treatment options. Although there is some literature on molecular underpinnings of cervical small cell neuroendocrine carcinomas (SCNECs), detailed morphologic and associated molecular characteristics of cervical NENs remains to be elucidated. Herein, 14 NENs (SCNEC: 6, large cell neuroendocrine carcinoma [LCNEC]: 6, neuroendocrine tumor [NET]: 2), including 5 admixed with human papillomavirus (HPV)-associated adenocarcinoma (carcinoma admixed with neuroendocrine carcinoma) were analyzed. All except 3 SCNECs were HPV16/18 positive. TP53 (3) and/or RB1 (4) alterations (3 concurrent) were only seen in SCNECs (4/6) and were enriched in the HPV16/18-negative tumors. The other most common molecular changes in neuroendocrine carcinomas (NECs) overlapping with those reported in the literature for cervical carcinomas involved PI3K/MAPK pathway (4) and MYC (4) and were seen in both SCNECs and LCNECs. In contrast, the 2 NETs lacked any significant alterations. Two LCNECs admixed with adenocarcinoma had enough material to sequence separately each component. In both pathogenic alterations were shared between the 2 components, including ERBB2 amplification in one and an MSH6 mutation with MYC amplification in the other. Overall, these findings suggest that cervical HPV-associated NETs are genomically silent and high-grade NECs (regardless of small or large cell morphology) share molecular pathways with common cervical carcinomas as it has been reported in the endometrium and are different from NECs at other sites. Molecular analysis of these highly malignant neoplasms might inform the clinical management for potential therapeutic targets.

Disruption of GMNC-MCIDAS multiciliogenesis program is critical in choroid plexus carcinoma development

Cell death and differentiation

2022 Mar 23

Li, Q;Han, Z;Singh, N;Terré, B;Fame, RM;Arif, U;Page, TD;Zahran, T;Abdeltawab, A;Huang, Y;Cao, P;Wang, J;Lu, H;Lidov, HGW;Surendran, K;Wu, L;Virga, JQ;Zhao, YT;Schüller, U;Wechsler-Reya, RJ;Lehtinen, MK;Roy, S;Liu, Z;Stracker, TH;Zhao, H;
PMID: 35322202 | DOI: 10.1038/s41418-022-00950-z

Multiciliated cells (MCCs) in the brain reside in the ependyma and the choroid plexus (CP) epithelia. The CP secretes cerebrospinal fluid that circulates within the ventricular system, driven by ependymal cilia movement. Tumors of the CP are rare primary brain neoplasms mostly found in children. CP tumors exist in three forms: CP papilloma (CPP), atypical CPP, and CP carcinoma (CPC). Though CPP and atypical CPP are generally benign and can be resolved by surgery, CPC is a particularly aggressive and little understood cancer with a poor survival rate and a tendency for recurrence and metastasis. In contrast to MCCs in the CP epithelia, CPCs in humans are characterized by solitary cilia, frequent TP53 mutations, and disturbances to multiciliogenesis program directed by the GMNC-MCIDAS transcriptional network. GMNC and MCIDAS are early transcriptional regulators of MCC fate differentiation in diverse tissues. Consistently, components of the GMNC-MCIDAS transcriptional program are expressed during CP development and required for multiciliation in the CP, while CPC driven by deletion of Trp53 and Rb1 in mice exhibits multiciliation defects consequent to deficiencies in the GMNC-MCIDAS program. Previous studies revealed that abnormal NOTCH pathway activation leads to CPP. Here we show that combined defects in NOTCH and Sonic Hedgehog signaling in mice generates tumors that are similar to CPC in humans. NOTCH-driven CP tumors are monociliated, and disruption of the NOTCH complex restores multiciliation and decreases tumor growth. NOTCH suppresses multiciliation in tumor cells by inhibiting the expression of GMNC and MCIDAS, while Gmnc-Mcidas overexpression rescues multiciliation defects and suppresses tumor cell proliferation. Taken together, these findings indicate that reactivation of the GMNC-MCIDAS multiciliogenesis program is critical for inhibiting tumorigenesis in the CP, and it may have therapeutic implications for the treatment of CPC.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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