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Introduction/Background Endocrine therapy remains the fundamental treatment for advanced HR+ breast cancer (BC). For those patients who become refractory to endocrine therapy, resistance may be associated with mutations, amplifications, and fusions in the ESR1-encoded estrogen receptor. Here we examine the frequency of ESR1 alterations and the associated genomic landscape. Methods HR+/HER2- BC samples were sequenced with the OncomapTM ExTra assay, which uses whole-exome DNA sequencing with germline subtraction to detect somatic single nucleotide substitutions, indels, and copy number alterations (CNAs), and uses RNA sequencing to detect gene fusions. Tumor mutational burden (TMB) and microsatellite instability (MSI) are also reported. For analysis, BC samples were grouped by site: local (primary breast or regional lymph node) versus metastatic. Prior treatment history was unknown. Testing for a possible association between ESR1 and other biomarkers (genes, MSI, and TMB) was done using Fisher’s Exact Test (p≤0.05). Results A total of 988 HR+HER2- breast cancer patient samples were included in the analysis. Of these, 821 (83.1%) were local samples and the remaining 167 were metastatic samples, with liver (63, 37.7%), bone (20, 12.0%), skin (16, 9.6%) and chest wall (15, 9.0%) being the most common locations. ESR1 alterations were present in 84 tumor samples, 37 local and 47 metastatic, representing 4.5% and 28.1% of samples, respectively. ESR1 alterations included missense mutations (54 samples), fusions (29 samples), and amplifications (8 samples). The most common missense mutations were Y537S (20 samples, 37.0%), D538Q (20 samples, 37.0%), and E380Q (6 samples, 11.1%), which were located in the ligand binding domain and included both clonal and subclonal events. There were 30 ESR1 fusions identified, 17 (2.1%) in local and 13 in metastatic (7.7%) samples. Most fusions (24 samples, 82.8%) involved the same partner, CCDC170, while the other 6 fusions had unique partners. Examination of the 143 other biomarkers altered among ESR1-altered samples revealed 15 genes and MSI-high status that appeared to be over-represented (Table 1). However, none of the associations were statistically significant after correcting for multiple comparisons. Further, there was no evidence that the prevalence of ERBB2, TP53, AKT1 and PIK3CA alterations differed by ESR1 status (Table 1). Conclusions ESR1 alterations were significantly more common in HR+/HER2- metastatic breast cancer samples compared to local samples. Comprehensive genomic profiling with RNA sequencing identified both common and rare ESR1 fusions, which were most frequent in the metastatic samples. No significant difference in the molecular profile of ESR1 altered vs ESR1 wildtype samples was found in this cohort. Clinical trials with novel selective ER degraders (SERDs) to target these ESR1 alterations are ongoing. Table 1. Biomarkers that showed the highest association with ESR1 and other notable breast cancer biomarkers. Citation Format: Gargi Basu, Sameer S. Udhane, Susan Dombrowski, Lenny Hong, Fadel Alyaqoub, Michelle Barbi de Moura, Thiruppavai Chandrasekaran, Turgut Dogruluk, David Driscoll, Aimee Jalkanen, Pawan Noel, Szabolcs Szelinger, Min Wang, David Hall, Jess Hoag, Janine Lobello, Frederick Baehner, Snehal Thakkar, Joyce O’Shaughnessy. ESR1-alterations in HR+HER2- breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-14-12.

Supplementary Figure 7: Schematic representation of structure of wildtype Stat5a and Stat5b, dominant-negative Stat5 (DN-Stat5) and constitutively-active Stat5 (CA-Stat5) proteins.

Clinical Characteristics of Outcome Cohorts 1 and 2.

Purpose:Parathyroid hormone-related protein (PTHrP) is required for normal mammary gland development and biology. A PTHLH gene polymorphism is associated with breast cancer risk, and PTHrP promotes growth of osteolytic breast cancer bone metastases. Accordingly, current dogma holds that PTHrP is upregulated in malignant primary breast tumors, but solid evidence for this assumption is missing.Experimental Design:We used quantitative IHC to measure PTHrP in normal and malignant breast epithelia, and correlated PTHrP levels in primary breast cancer with clinical outcome.Results:PTHrP levels were markedly downregulated in malignant compared with normal breast epithelia. Moreover, low levels of nuclear localized PTHrP in cancer cells correlated with unfavorable clinical outcome in a test and a validation cohort of breast cancer treated at different institutions totaling nearly 800 cases. PTHrP mRNA levels in tumors of a third cohort of 737 patients corroborated this association, also after multivariable adjustment for standard clinicopathologic parameters. Breast cancer PTHrP levels correlated strongly with transcription factors Stat5a/b, which are established markers of favorable prognosis and key mediators of prolactin signaling. Prolactin stimulated PTHrP transcript and protein in breast cancer cell lines in vitro and in vivo, effects mediated by Stat5 through the P2 gene promoter, producing transcript AT6 encoding the PTHrP 1-173 isoform. Low levels of AT6, but not two alternative transcripts, correlated with poor clinical outcome.Conclusions:This study overturns the prevailing view that PTHrP is upregulated in primary breast cancers and identifies a direct prolactin–Stat5–PTHrP axis that is progressively lost in more aggressive tumors.

PURPOSE T-cell–mediated cytotoxicity is suppressed when programmed cell death-1 (PD-1) is bound by PD-1 ligand-1 (PD-L1) or PD-L2. Although PD-1 inhibitors have been approved for triple-negative breast cancer, the lower response rates of 25%-30% in estrogen receptor–positive (ER+) breast cancer will require markers to identify likely responders. The focus of this study was to evaluate whether PD-L2, which has higher affinity than PD-L1 for PD-1, is a predictor of early recurrence in ER+ breast cancer. METHODS PD-L2 protein levels in cancer cells and stromal cells of therapy-naive, localized or locoregional ER+ breast cancers were measured retrospectively by quantitative immunofluorescence histocytometry and correlated with progression-free survival (PFS) in the main study cohort (n = 684) and in an independent validation cohort (n = 273). All patients subsequently received standard-of-care adjuvant therapy without immune checkpoint inhibitors. RESULTS Univariate analysis of the main cohort revealed that high PD-L2 expression in cancer cells was associated with shorter PFS (hazard ratio [HR], 1.8; 95% CI, 1.3 to 2.6; P = .001), which was validated in an independent cohort (HR, 2.3; 95% CI, 1.1 to 4.8; P = .026) and remained independently predictive after multivariable adjustment for common clinicopathological variables (HR, 2.0; 95% CI, 1.4 to 2.9; P < .001). Subanalysis of the ER+ breast cancer patients treated with adjuvant chemotherapy (n = 197) revealed that high PD-L2 levels in cancer cells associated with short PFS in univariate (HR, 2.5; 95% CI, 1.4 to 4.4; P = .003) and multivariable analyses (HR, 3.4; 95% CI, 1.9 to 6.2; P < .001). CONCLUSION Up to one third of treatment-naive ER+ breast tumors expressed high PD-L2 levels, which independently predicted poor clinical outcome, with evidence of further elevated risk of progression in patients who received adjuvant chemotherapy. Collectively, these data warrant studies to gain a deeper understanding of PD-L2 in the progression of ER+ breast cancer and may provide rationale for immune checkpoint blockade for this patient group.

Context Mutations in cytochrome P450 oxidoreductase (POR) cause a form of congenital adrenal hyperplasia (CAH). We report a novel R550W mutation in POR identified in a 46,XX patient with signs of aromatase deficiency. Objective Analysis of aromatase deficiency from the R550W mutation in POR. Design, setting, and patient Both the child and the mother had signs of virilization. Ultrasound revealed the presence of uterus and ovaries. No defects in CYP19A1 were found, but further analysis with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform in Madrid and Barcelona, Spain, revealed compound heterozygous mutations c.73_74delCT/p.L25FfsTer93 and c.1648C > T/p.R550W in POR. Wild-type and R550W POR were produced as recombinant proteins and tested with multiple cytochrome P450 enzymes at University Children’s Hospital, Bern, Switzerland. Main outcome measure and Results POR-R550W showed 41% of the WT activity in cytochrome c and 7.7% activity for reduction of MTT. Assays of CYP19A1 showed a severe loss of activity, and CYP17A1 as well as CYP21A2 activities were also lost by more than 95%. Loss of CYP2C9, CYP2C19, and CYP3A4 activities was observed for the R550W-POR. Predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed. Conclusions Pathological effects due to POR-R550W were identified, expanding the knowledge of molecular pathways associated with aromatase deficiency. Screening of the POR gene may provide a diagnosis in CAH without defects in genes for steroid metabolizing enzymes.

806 Background: BRAF V600E mutations can lead to activation of the MEK/ERK pathway. The combination of dabrafenib plus trametinib was recently FDA-approved for use in these BRAF V600E altered unresectable or metastatic cancers. BRAF non-V600 alterations also increase phosphorylation of MEK and ERK and are potential candidates for MEK/ERK inhibitors. Here we examine the frequency of BRAF mutations and their genomic background across GI cancers. Methods: The Oncomap ExTra genomic profiling assay utilizes tumor-normal, whole-exome, whole-transcriptome DNA and RNA sequencing to identify somatic alterations in tumors. The assay detects single-nucleotide substitutions, indels, copy number alterations, alternative transcripts, and gene fusions. Tumor mutational burden (TMB) and microsatellite instability (MSI) are also assessed. The frequency of BRAF and other gene alterations were identified across all GI cancers. Reported BRAF alterations were limited to those linked to an FDA approved therapy, clinical trial eligibility, and those deemed significant from review of the literature. Results: A total of 1927 patients with GI cancers were assayed between April 2018 to July 2022. The BRAF gene was mutated in 85 (4.4%) patients, which included 53 (2.8%) that were V600E and 32 (1.5%) that were non-V600, representing 37.6% of BRAF mutations. Colorectal and small bowel cancers had the highest frequencies of BRAF mutations at 7.6% and 7.1% respectively, and the frequency was lower in other GI cancers (0-2.3%). In addition to BRAF V600E, other mutations occurred across the kinase domain, including D594 (0.6%), K601E (0.3%), G466 (0.3%) and G469 (0.2%). BRAF fusions were found in 1 CRC and 2 pancreatic cancer cases. BRAF mutational status (altered versus not) was significantly associated with mutations in several other genes. In addition, BRAF status was significantly associated with TMB-high and MSI-high. Conclusions: Analysis of whole-exome and whole transcriptome sequencing data found actionable BRAF mutations in 4.4% of GI cancer patients, 37.6% of whom had BRAF mutations not at V600. Several biomarkers were significantly associated with BRAF alterations. Assays that are limited to DNA sequencing, or to targeted gene panels, may not detect mutations and fusions that could aid in therapy selection and prognosis. [Table: see text]

Most breast cancer deaths are caused by estrogen receptor-α–positive (ER+) disease. Preclinical progress is hampered by a shortage of therapy-naive ER+ tumor models that recapitulate metastatic pro...

Background: Immune-oncology (I-O) drugs show promise across multiple tumor types, and several biomarkers (BM) predict responsiveness to I-O therapy. In this descriptive study of clinical lab experience (2018-2021), we investigated the prevalence of genomic BMs associated with sensitivity and resistance to I-O drugs across solid tumors. Methods: The GEM ExTra® assay was used to exome-sequence 2,359 paired samples at 180X and 400X coverage for germline and tumor DNA respectively. Reportable alterations were single base substitutions, indels, copy number alterations, fusions, alternate transcripts, as well as tumor mutational burden (TMB) and microsatellite instability (MSI) status. For I-O sensitive BMs we recorded MSI-High status, TMB-High (> 10 mut/Mb) status, and mutations in POLE/POLD1, MLH1, MSH2/3/6, PMS1/2, PD-L1/-L2, CTLA4, CD28, ARID2, and PBRM1. For I-O resistance BMs, IDO1 amplification and mutations in B2M, JAK1/2, IDO1, KRAS and STK11 were tallied. Results: I-O sensitive BMs varied from 0% to 58% (results in Table 1 for selected tumor types). Overall, TMB-High was in 6% of samples and MSI-High in 3%; all MSI-High were also TMB-High. In the 46% of renal cell carcinoma samples with I-O-sensitive BMs, PBRM1 mutation was common (94%). In 3 TMB-High endometrial samples, POLE/POLD1 was mutated, indicative of an ultra-mutated phenotype. Of note, 35% of colorectal cancer and 8% of melanoma samples with I-O sensitive mutations also had loss-of-function mutations in JAK1/2 and B2M, suggestive of I-O resistance. In non-small cell lung cancer, co-mutation of STK11 and KRAS, suggesting inferior response to I-O therapy, was present in 4% of samples with I-O BMs. IDO1 amplification was present in less than 1% of entire cohort. Conclusions: Our study found I-O sensitive BMs in common and in rare aggressive cancers. The GEM ExTra assay can stratify cancer patients for likely responsiveness to immunotherapy. Table 1. Immune-oncology sensitive biomarkers observed across select tumors Solid tumor type % of samples with signature All (n = 2,360) 13% Melanoma (n = 50) 54% Other skin cancers (n = 13) 62% Renal cell carcinoma (n = 200) 46% Urothelial (n = 86) 22% Endometrial (n = 88) 20% Gastro-intestinal (n = 360) 12% Sarcoma (n = 90) 11% Prostate (n = 151) 6% Lung (n = 100) 4% Breast (n = 297) 2% Central nervous system (n = 60) 0% Citation Format: Pawan Noel, David W. Hall, Sameer S. Udhane, Min Wang, Fadel S. Alyaqoub, Szabolcs Szelinger, Audrey A. Ozols, Janine R. LoBello, Frederick L. Baehner, Gargi D. Basu. Comprehensive genomic profiling to identify biomarkers predictive of response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1263.

3124 Background: Deleterious events in DNA damage response (DDR) are hallmarks of cancer associated with sensitivity to PARP inhibitors (PARPi) and immune checkpoint inhibitors (CPI). This study investigated DDR pathway alterations across solid tumors. Methods: Samples were sequenced with the Oncomap ExTra assay using tumor-normal paired whole-exome DNA sequencing to detect single base substitutions, indels, and copy number alterations that were clinically actionable, defined as associated with FDA approved drugs or clinical trial enrollment. Here, we report the frequency of MSI-high and TMB-high (>10 mutations/Mb), and clinically-actionable alterations for the following 49 DDR genes: ARID1A, ATM, ATR, ATRX, BAP1, BARD1, BLM, BRCA1/2, BRIP1, CDK12, CHEK1/2, EPCAM, ERCC1/2/3/4/5, FANCA/C/D2/E/F/G/I/L/M, MLH1, MRE11A, MSH2/6, MUTYH, NBN, PALB2, PMS2, PPP2R2A, PTEN, RAD21/50/51/51B/51C/51D/52/54L, XRCC1/2/3. Results: Of the 6055 patient samples profiled, 1633 (27.0%) had clinically actionable alterations in DDR genes; DDR alterations varied from 0-64.3% across tumor types. For the 5657 samples with TMB/MSI data, 429 (7.6%) had high TMB and 193 (3.4%) were MSI-high; MSI-high samples were usually TMB-high (n = 180; 93.2%). The percent of cancers that were TMB-high or MSI-high varied from 0-64.8% and 0-20.8% respectively. Actionable BRCA1/2 gene alterations were present in 319 patients (5.3%, range 0-15.3%). Three cancers (biliary, brain and liver) had BRCA1/2 alterations in less than 2% of patients but across the 49 DDR genes alterations were present in more than 20% of patients, representing a greater than 10-fold difference. Across solid tumors, this analysis identifies a group of 1314 patients (21.7%) who harbor a DDR gene alteration other than BRCA1/2. Conclusions: Defective DNA repair as detected by deleterious alterations in DDR genes along with TMB/MSI status has the potential to guide clinicians to FDA-approved therapy or clinical trial enrollment in a large percentage of patients across solid tumor types.[Table: see text]

1096 Background: The use of targeted therapies identified using genetic and genomic approaches is now routine in breast cancer (BC). In this clinical lab experience study the frequency of actionable somatic alterations in DNA repair pathway genes associated with the use of PARP inhibitors (PARPi) is described. Methods: BC samples were sequenced with the Oncomap ExTra assay, which uses whole-exome DNA sequencing with germline subtraction to detect somatic single base substitutions, indels, and copy number alterations, and RNA sequencing to detect gene fusions. Clinically actionable alterations were defined as associated with FDA approved drugs or clinical trial enrollment. Here, the focus is on 49 repair genes associated with PARPi response: ARID1A, ATM, ATR, ATRX, BAP1, BARD1, BLM, BRCA1/2, BRIP1, CDK12, CHEK1/2, EPCAM, ERCC1/2/3/4/5, FANCA/C/D2/E/F/G/I/L/M, MLH1, MRE11A, MSH2/6, MUTYH, NBN, PALB2, PMS2, PPP2R2A, PTEN, RAD21/50/51/51B/51C/51D/52/54L, XRCC1/2/3. Results: Of 1103 BCs, 246 (22.3%) had mutations in repair genes; 69 (6.3%) were in BRCA1/2. Repair gene mutations were less common in HER2+ cancers (n=27, 14.3%) compared to HR+ HER2- (n=156, 23.9%) or TN cancers (n=49, 26.1%) (p+ cancers (p- germline BRCA1/2 mutated BC patients. Recent evidence suggests somatic BRCA1/2 mutations predict PARPi benefit (Tung, NM. J Clin Oncol 2020). In addition to BRCA1/2 alterations, our study also highlights the importance of alterations in other DNA repair genes associated with response to PARPi. Trials are ongoing to determine if these genes predict for PARPi benefit.[Table: see text]

Context: Mutations in Cytochrome P450 oxidoreductase (POR) cause a form of congenital adrenal hyperplasia (CAH). We are reporting a novel R550W mutation in POR identified in a 46, XX patient with signs of aromatase deficiency. Objective: Analysis of aromatase deficiency from R550W mutation in POR. Design, Setting, and Patient: Both the child and the mother had signs of virilization. Ultrasound revealed the presence of uterus and ovaries. No defects in CYP19A1 were found, but further analysis with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform in Madrid and Barcelona, Spain, revealed compound heterozygous mutations c.73_74delCT/p.L25FfsTer93 and c.1648C>T/p.R550W in POR. WT and R550W POR were produced as recombinant proteins and tested with multiple cytochrome P450 enzymes at University Children’s Hospital, Bern, Switzerland. Main Outcome Measure and Results: R550W POR showed 41% of the WT activity in cytochrome c and 7.7% activity for reduction of MTT. Assays of CYP19A1 showed a severe loss of activity and CYP17A1, as well as CYP21A2 activities, were also lost by more than 95%. Loss of CYP2C9, CYP2C19, and CYP3A4 activities was observed for the R550W-POR. Predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed. Conclusions: Pathological effects due to POR R550W were identified, expanding the knowledge of molecular pathways associated with aromatase deficiency. Screening of the POR gene may provide a diagnosis in CAH without defects in genes for steroid metabolizing enzymes.

Purpose: Parathyroid hormone-related protein (PTHrP) is required for normal mammary gland development and biology. A PTHLH gene polymorphism is associated with breast cancer risk, and PTHrP promotes growth of osteolytic breast cancer bone metastases. Accordingly, current dogma holds that PTHrP is upregulated in malignant primary breast tumors, but solid evidence for this assumption is missing. Experimental Design: We used quantitative IHC to measure PTHrP in normal and malignant breast epithelia, and correlated PTHrP levels in primary breast cancer with clinical outcome. Results: PTHrP levels were markedly downregulated in malignant compared with normal breast epithelia. Moreover, low levels of nuclear localized PTHrP in cancer cells correlated with unfavorable clinical outcome in a test and a validation cohort of breast cancer treated at different institutions totaling nearly 800 cases. PTHrP mRNA levels in tumors of a third cohort of 737 patients corroborated this association, also after multivariable adjustment for standard clinicopathologic parameters. Breast cancer PTHrP levels correlated strongly with transcription factors Stat5a/b, which are established markers of favorable prognosis and key mediators of prolactin signaling. Prolactin stimulated PTHrP transcript and protein in breast cancer cell lines in vitro and in vivo, effects mediated by Stat5 through the P2 gene promoter, producing transcript AT6 encoding the PTHrP 1-173 isoform. Low levels of AT6, but not two alternative transcripts, correlated with poor clinical outcome. Conclusions: This study overturns the prevailing view that PTHrP is upregulated in primary breast cancers and identifies a direct prolactin–Stat5–PTHrP axis that is progressively lost in more aggressive tumors.

OBJECTIVES: Assess the pathologic changes in the lungs of COVID-19 decedents and correlate these changes with demographic data, clinical course, therapies, and duration of illness. METHODS: Lungs of 12 consecutive COVID-19 decedents consented for autopsy were evaluated for gross and histopathologic abnormalities. A complete Ghon "en block" dissection was performed on all cases; lung weights and gross characteristics recorded. Immunohistochemical studies were performed to characterize lymphocytic infiltrates and to assess SARS-CoV-2 capsid protein. RESULTS: Two distinct patterns of pulmonary involvement were identified. Three of 12 cases demonstrated a predominance of acute alveolar damage (DAD) while 9 of 12 cases demonstrated a marked increase in intra-alveolar macrophages in a fashion resembling desquamative interstitial pneumonia or macrophage activation syndrome (DIP/MAS). Two patterns were correlated solely with a statistically significant difference in the duration of illness. The group exhibiting DAD had duration of illness of 5.7 days while the group with DIP/MAS had duration of illness of 21.5 days (t-test p = 0.014). CONCLUSIONS: The pulmonary pathology of COVID-19 patients demonstrates a biphasic pattern, an acute phase demonstrating DAD changes while the patients with a more prolonged course exhibit a different pattern that resembles DIP/MAS-like pattern. The potential mechanisms and clinical significance are discussed.

Human coronavirus disease 2019 (COVID-19) is a life-threatening and highly contagious disease caused by coronavirus SARS-CoV-2. Sensitive and specific detection of SARS-CoV-2 viral proteins in tissues and cells of COVID-19 patients will support investigations of the biologic behavior and tissue and cell tropism of this virus. We identified commercially available affinity-purified polyclonal antibodies raised against nucleocapsid and spike proteins of SARS-CoV-2 that provide sensitive and specific detection of the virus by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Two immunohistochemistry protocols are presented that are mutually validated by the matched detection patterns of the two distinct viral antigens in virus-infected cells within autopsy lung tissue of COVID-19 deceased patients. Levels of nucleocapsid protein in the lungs of COVID-19 decedents, as measured by quantitative histo-cytometry of immunohistochemistry images, showed an excellent log–linear relationship with levels of viral nucleocapsid RNA levels, as measured by qRT-PCR. Importantly, since the nucleocapsid protein sequence is conserved across all known viral strains, the nucleocapsid immunohistochemistry protocol is expected to recognize all common variants of SARS-CoV-2. Negative controls include autopsy lung tissues from patients who died from non-COVID-19 respiratory disease and control rabbit immunoglobulin. Sensitive detection of SARS-CoV-2 in human tissues will provide insights into viral tissue and cell distribution and load in patients with active infection, as well as provide insight into the clearance rate of virus in later COVID-19 disease stages. The protocols are also expected to be readily transferable to detect SARS-CoV-2 proteins in tissues of experimental animal models or animals suspected to serve as viral reservoirs.

Context: Mutations in Cytochrome P450 oxidoreductase (POR) cause a form of congenital adrenal hyperplasia (CAH). We are reporting a novel R550W mutation in POR identified in a 46, XX patient with signs of aromatase deficiency. Objective: Analysis of aromatase deficiency from R550W mutation in POR. Design, Setting, and Patient: Both the child and the mother had signs of virilization. Ultrasound revealed the presence of uterus and ovaries. No defects in CYP19A1 were found, but further analysis with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform in Madrid and Barcelona, Spain, revealed compound heterozygous mutations c.73_74delCT/p.L25FfsTer93 and c.1648C>T/p.R550W in POR. WT and R550W POR were produced as recombinant proteins and tested with multiple cytochrome P450 enzymes at University Children’s Hospital, Bern, Switzerland. Main Outcome Measure and Results: R550W POR showed 41% of the WT activity in cytochrome c and 7.7% activity for reduction of MTT. Assays of CYP19A1 showed a severe loss of activity and CYP17A1, as well as CYP21A2 activities, were also lost by more than 95%. Loss of CYP2C9, CYP2C19, and CYP3A4 activities was observed for the R550W-POR. Predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed. Conclusions: Pathological effects due to POR R550W were identified, expanding the knowledge of molecular pathways associated with aromatase deficiency. Screening of the POR gene may provide a diagnosis in CAH without defects in genes for steroid metabolizing enzymes.

Cytochromes P450 located in the endoplasmic reticulum require NADPH cytochrome P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause multiple disorders in humans related to the biosynthesis of steroid hormones and also affect drug-metabolizing cytochrome P450 activities. Here we are reporting the effects of a POR genetic variant P284T which is located in the hinge region of POR that is necessary for the flexibility of domain movements. Human wild-type and P284T mutant of POR, as well as cytochrome P450 proteins, were expressed in bacteria, purified and then reconstituted in liposomes for enzyme kinetic assays. Quality of POR proteins was checked by cytochrome c, ferricyanide and tetrazolium dye reduction assay and measurements flavin content. We found that for the P284T variant of POR the cytochrome c reduction activity was reduced to 47% of the WT and MTT reduction was reduced to only 15% of the WT. No impact on ferricyanide reduction activity was observed, but a severe loss of CYP19A1 (aromatase) activity was observed (9% of WT). In the assays of drug metabolizing cytochrome P450 enzymes, the P284T variant of POR showed 26% activity for CYP2C9, 44% activity for CYP2C19, 23% activity for CYP3A4 and 44% activity in CYP3A5 assays compared to the WT POR. These results indicate a severe effect on several cytochrome P450 activities due to the P284T variation in POR which suggests a negative impact on both the steroid as well as drug metabolism in the individuals carrying this variation.

A broad spectrum of human diseases are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR). Cytochrome P450 proteins perform several reactions, including the metabolism of steroids, drugs, and other xenobiotics. In 2004 the first human patients with defects in POR were reported, and over 250 variations in POR are known. Information about the effects of POR variants on drug metabolizing enzymes is limited and has not received much attention. By analyzing the POR sequences from genomics databases, we identified potentially disease-causing variations and characterized these by in vitro functional studies using recombinant proteins. Proteins were expressed in bacteria and purified for activity assays. Activities of cytochrome P450 enzymes were tested in vitro using liposomes prepared with lipids into which P450 and P450 reductase proteins were embedded. Here we are reporting the effect of POR variants on drug metabolizing enzymes CYP2C9, CYP2C19, and CYP3A5 which are responsible for the metabolism of many drugs. POR Variants A115V, T142A, A281T, P284L, A287P, and Y607C inhibited activities of all P450 proteins tested. Interestingly, the POR variant Q153R showed a reduction of 20-50% activities with CYP2C9 and CYP2C19 but had a 400% increased activity with CYP3A5. The A287P is most common POR mutation found in patients of European origin, and significantly inhibited drug metabolism activities which has important consequences for monitoring and treatment of patients. In vitro, functional assays using recombinant proteins provide a useful model for establishing the metabolic effect of genetic mutations. Our results indicate that detailed knowledge about POR variants is necessary for correct diagnosis and treatment options for persons with POR deficiency and the role of changes in drug metabolism and toxicology due to variations in POR needs to be addressed.

Androgens are precursors for sex steroids and are predominantly produced in the human gonads and the adrenal cortex. They are important for intrauterine and postnatal sexual development and human reproduction. Although human androgen biosynthesis has been extensively studied in the past, exact mechanisms underlying the regulation of androgen production in health and disease remain vague. Here, the knowledge on human androgen biosynthesis and regulation is reviewed with a special focus on human adrenal androgen production and the hyperandrogenic disorder of polycystic ovary syndrome (PCOS). Since human androgen regulation is highly specific without a good animal model, most studies are performed on patients harboring inborn errors of androgen biosynthesis, on human biomaterials and human (tumor) cell models. In the past, most studies used a candidate gene approach while newer studies use high throughput technologies to identify novel regulators of androgen biosynthesis. Using genome wide association studies on cohorts of patients, novel PCOS candidate genes have been recently described. Variant 2 of the DENND1A gene was found overexpressed in PCOS theca cells and confirmed to enhance androgen production. Transcriptome profiling of dissected adrenal zones established a role for BMP4 in androgen synthesis. Similarly, transcriptome analysis of human adrenal NCI-H295 cells identified novel regulators of androgen production. Kinase p38α (MAPK14) was found to phosphorylate CYP17 for enhanced 17,20 lyase activity and RARB and ANGPTL1 were detected in novel networks regulating androgens. The discovery of novel players for androgen biosynthesis is of clinical significance as it provides targets for diagnostic and therapeutic use.

Classic 3β-hydroxysteroid dehydrogenase type 2 (3β-HSD II) deficiency causes congenital adrenal hyperplasia with glucocorticoid, mineralocorticoid, and sex steroid deficiency. We present a female patient with congenital adrenal hyperplasia detected in newborn screening due to elevated 17OH-progesterone. Female external genitalia and non-measurable androgen levels elicited the suspicion of a defect early in the steroid cascade. Two loss-of-function HSD3B2 mutations (1 novel) were detected and confirmed in silico. We argue that in a girl with glucocorticoid and mineralocorticoid deficiency without virilization, 3β-HSD II deficiency is an important differential diagnosis. 17OH-progesterone may initially be elevated due to placental and peripheral activity of 3β-HSD I, whereas dehydroepiandrosterone may not be increased.

Context3β-hydroxysteroid dehydrogenase deficiency (3βHSD) is a rare disorder of sexual development and steroidogenesis. There are two isozymes of 3βHSD,HSD3B1andHSD3B2. Human mutations are known for theHSD3B2gene which is expressed in the gonads and the adrenals. Little is known about testis histology, fertility and malignancy risk.ObjectiveTo describe the molecular genetics, the steroid biochemistry, the (immuno-)histochemistry and the clinical implications of a loss-of-functionHSD3B2mutation.MethodsBiochemical, genetic and immunohistochemical investigations on human biomaterials.ResultsA 46,XY boy presented at birth with severe undervirilization of the external genitalia. Steroid profiling showed low steroid production for mineralocorticoids, glucocorticoids and sex steroids with typical precursor metabolites for HSD3B2 deficiency. The genetic analysis of theHSD3B2gene revealed a homozygous c.687del27 deletion. At pubertal age, he showed some virilization of the external genitalia and some sex steroid metabolites appeared likely through conversion of precursors secreted by the testis and converted by unaffectedHSD3B1in peripheral tissues. However, he also developed enlarged breasts through production of estrogens in the periphery. Testis histology in late puberty revealed primarily a Sertoli-cell-only pattern and only few tubules with arrested spermatogenesis, presence of few Leydig cells in stroma, but no neoplastic changes.ConclusionsThe testis with HSD3B2 deficiency due to the c.687del27 deletion does not express the defective protein. This patient is unlikely to be fertile and his risk for gonadal malignancy is low. Further studies are needed to obtain firm knowledge on malignancy risk for gonads harboring defects of androgen biosynthesis.

All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause metabolic disorders of steroid hormone biosynthesis and affect certain drug metabolizing P450 activities. We studied mutations A115V, T142A, Q153R identified in the flavin mononucleotide (FMN) binding domain of POR that interacts with partner proteins and P284L located in the hinge region that is required for flexibility and domain movements in POR. Human wild type and mutant POR as well as CYP3A4 and CYP19A1 proteins in recombinant form were expressed in bacteria, and purified proteins were reconstituted in liposomes for enzyme kinetic assays. Quality of POR protein was checked by cytochrome c reduction assay as well as flavin content measurements. We found that proteins carrying mutations A115V, T142A located close to the FMN binding site had reduced flavin content compared to wild type POR and lost almost all activity to metabolize androstenedione via CYP19A1 and showed reduced CYP3A4 activity. The variant P284L identified from apparently normal subjects also had severe loss of both CYP19A1 and CYP3A4 activities, indicating this to be a potentially disease causing mutation. The mutation Q153R initially identified in a patient with disordered steroidogenesis showed remarkably increased activities of both CYP19A1 and CYP3A4 without any significant change in flavin content, indicating improved protein-protein interactions between POR Q153R and some P450 proteins. These results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to accurately determine the genotype-phenotype correlations of POR variants.

OBJECTIVE The steroidogenic acute regulatory protein (StAR) transports cholesterol to the mitochondria for steroidogenesis. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH) which is characterized by impaired synthesis of adrenal and gonadal steroids causing adrenal insufficiency, 46,XY disorder of sex development (DSD) and failure of pubertal development. Partial loss of StAR activity may cause adrenal insufficiency only. PATIENT A newborn girl was admitted for mild dehydration, hyponatremia, hyperkalemia and hypoglycaemia and had normal external female genitalia without hyperpigmentation. Plasma cortisol, 17OH-progesterone, DHEA-S, androstendione and aldosterone were low, while ACTH and plasma renin activity were elevated, consistent with the diagnosis of primary adrenal insufficiency. Imaging showed normal adrenals, and cytogenetics revealed a 46,XX karyotype. She was treated with fluids, hydrocortisone and fludrocortisone. DESIGN, METHODS AND RESULTS Genetic studies revealed a novel homozygous STAR mutation in the 3' acceptor splice site of intron 4, c.466-1G>A (IVS4-1G>A). To test whether this mutation would affect splicing, we performed a minigene experiment with a plasmid construct containing wild-type or mutant StAR gDNA of exons-introns 4-6 in COS-1 cells. The splicing was assessed on total RNA using RT-PCR for STAR cDNAs. The mutant STAR minigene skipped exon 5 completely and changed the reading frame. Thus, it is predicted to produce an aberrant and shorter protein (p.V156GfsX19). Computational analysis revealed that this mutant protein lacks wild-type exons 5-7 which are essential for StAR-cholesterol interaction. CONCLUSIONS STAR c.466-1A skips exon 5 and causes a dramatic change in the C-terminal sequence of the protein, which is essential for StAR-cholesterol interaction. This splicing mutation is a loss-of-function mutation explaining the severe phenotype of our patient. Thus far, all reported splicing mutations of STAR cause a severe impairment of protein function and phenotype.

Classic 3β-hydroxysteroid dehydrogenase type 2 (3β-HSD II) deficiency causes congenital adrenal hyperplasia with glucocorticoid, mineralocorticoid, and sex steroid deficiency. We present a female patient with congenital adrenal hyperplasia detected in newborn screening due to elevated 17OH-progesterone. Female external genitalia and non-measurable androgen levels elicited the suspicion of a defect early in the steroid cascade. Two loss-of-function HSD3B2 mutations (1 novel) were detected and confirmed in silico. We argue that in a girl with glucocorticoid and mineralocorticoid deficiency without virilization, 3β-HSD II deficiency is an important differential diagnosis. 17OH-progesterone may initially be elevated due to placental and peripheral activity of 3β-HSD I, whereas dehydroepiandrosterone may not be increased.

Cytochrome P450 oxidoreductase (POR) is required for metabolic reactions of steroid and drug metabolizing cytochrome P450 proteins located in endoplasmic reticulum. Mutations in POR cause a complex set of disorders resembling combined deficiencies of multiple steroid metabolizing enzymes. The P450 oxidoreductase deficiency (PORD) was first reported in patients with symptoms of defects in steroidogenic cytochrome P450 enzymes and ambiguous genitalia, and bone malformation features resembling Antley-Bixler syndrome. POR is now classified as a separate and rare form of congenital adrenal hyperplasia (CAH), which may cause disorder of sexual development (DSD). Since the initial description of PORD in 2004, a large number of POR mutations and polymorphisms have been described. In this report we have performed computational analysis of mutations and polymorphisms in POR linked to metabolism of steroids and xenobiotics and pathology of PORD from the reported cases. The mutations in POR that were identified in patients with disruption of steroidogenesis also have severe effects on cytochrome P450 proteins involved in metabolism of drugs. Different variations in POR show a range of diverse effects on different partner proteins that are often linked to the location of the particular variants. The variations in POR that cause defective binding of co-factors always have damaging effects on all partner proteins, while the mutations causing subtle structural changes may lead to altered interaction with partner proteins and the overall effect may be different for each individual partner. Computational analysis of available sequencing data and mutation analysis shows that Japanese (R457H), Caucasian (A287P) and Turkish (399-401) populations can be linked to unique founder mutations. Other mutations identified so far were identified as rare alleles or in single isolated reports. The common polymorphism of POR is the variant A503V which can be found in about 27% of alleles in general population but there are remarkable differences among different sub populations.

Myxoid sarcoma (MLS) is one of the most common types of malignant soft tissue tumors. MLS is characterized by the FUS-DDIT3 or EWSR1-DDIT3 fusion oncogenes that encode abnormal transcription factors. The receptor tyrosine kinase (RTK) encoding RET was previously identified as a putative downstream target gene to FUS-DDIT3 and here we show that cultured MLS cells expressed phosphorylated RET together with its ligand Persephin. Treatment with RET specific kinase inhibitor Vandetanib failed to reduce RET phosphorylation and inhibit cell growth, suggesting that other RTKs may phosphorylate RET. A screening pointed out EGFR and ERBB3 as the strongest expressed phosphorylated RTKs in MLS cells. We show that ERBB3 formed nuclear and cytoplasmic complexes with RET and both RTKs were previously reported to form complexes with EGFR. The formation of RTK hetero complexes could explain the observed Vandetanib resistence in MLS. EGFR and ERBB3 are clients of HSP90 that help complex formation and RTK activation. Treatment of cultured MLS cells with HSP90 inhibitor 17-DMAG, caused loss of RET and ERBB3 phosphorylation and lead to rapid cell death. Treatment of MLS xenograft carrying Nude mice resulted in massive necrosis, rupture of capillaries and hemorrhages in tumor tissues. We conclude that complex formation between RET and other RTKs may cause RTK inhibitor resistance. HSP90 inhibitors can overcome this resistance and are thus promising drugs for treatment of MLS/RCLS. ispartof: Oncotarget vol:7 issue:1 pages:433-45 ispartof: location:United States status: published

Fused in sarcoma (FUS) is a multifunctional protein involved in transcriptional control, pre-mRNA processing, RNA transport and translation. The domain structure of FUS reflects its functions in gene regulation and its ability to interact with other proteins, RNA and DNA. By use of a recombinant fragment of FUS in pull-down experiments followed by mass spectrometry analysis we have identified a novel interaction between the FUS N-terminal and the cytolinker plectin. An in situ proximity ligation assay confirmed that FUS-plectin interactions take place in the cytoplasm of cells. Furthermore, plectin deficient cells showed an altered subcellular localization of FUS and a deregulated expression of mRNAs bound to FUS. Our results show that plectin is important for normal FUS localization and function. Mutations involving FUS are causative factors in sarcomas and leukemias and also hereditary forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Plectin deficiency causes epidermolysis bullosa, a disease involving the skin and neuromuscular system. The novel FUS-plectin interaction offers new perspectives for understanding the role of FUS and plectin mutations in the pathogenesis of these diseases.

Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs). Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute) versus long-term (chronic) stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

The orteronel, abiraterone and galeterone, which were developed to treat castration resistant prostate cancer, inhibit 17,20 lyase activity but little is known about their effects on adrenal androgen biosynthesis. We studied the effect of several inhibitors and found that orteronel was selective towards 17,20 lyase activity than abiraterone and galeterone. Gene expression analysis showed that galeterone altered the expression of HSD3B2 but orteronel did not change the expression of HSD3B2, CYP17A1 and AKR1C3. The CYP19A1 activity was not inhibited except by compound IV which lowered activity by 23%. Surprisingly abiraterone caused complete blockade of CYP21A2 activity. Analysis of steroid metabolome by gas chromatography - mass spectrometry revealed changes in steroid levels caused by different inhibitors. We can conclude that orteronel is a highly specific inhibitor of 17,20 lyase activity. The discovery of these specific drug actions on steroidogenic enzyme activities would be valuable for understanding the regulation of androgens.