A multi-institutional survey of apheresis services among institutions in the United States.

INTRODUCTION: Apheresis practices in the United States (US) have not been comprehensively characterized to date. This study aimed to address this gap by evaluating apheresis therapy through a national survey. METHODS: A multi-institutional survey was conducted between April and July 2023. The survey, comprising 54 questions, focused on institutional demographics, procedures, equipment, staffing, training, and impacts of the Coronavirus Disease 2019 (COVID-19) pandemic. Responses from 22 institutions, primarily academic medical centers, were analyzed. RESULTS: Therapeutic plasma exchange (TPE) was the most common procedure, followed by hematopoietic progenitor cell collection (HPC-A) and red blood cell exchange (RCE). CAR-T cell collections were widespread, with some institutions supporting over 30 protocols concurrently. Most sites used the Spectra Optia Apheresis System, were managed by a transfusion medicine service, and employed internal apheresis providers. Insufficient staffing levels, exacerbated by the COVID-19 pandemic, were common and most often addressed using overtime. DISCUSSION: The survey highlighted the ubiquity of TPE, expanding cellular collections and staffing challenges. The role of apheresis in supporting cellular therapy, particularly in newly developing cell and gene therapies and clinical trials, was evident. Staffing issues during the pandemic emphasized the need for innovative recruitment strategies. CONCLUSION: This nationwide survey provides the most comprehensive analysis to date of apheresis practices in large US academic centers.

Targeting oncogenic microRNAs from the miR-371~373 and miR-302/367 clusters in malignant germ cell tumours causes growth inhibition through cell cycle disruption.

BACKGROUND: MiR-371~373 and miR-302/367 cluster over-expression occurs in all malignant germ cell tumours (GCTs), regardless of age (paediatric/adult), site (gonadal/extragonadal), or subtype [seminoma, yolk sac tumour (YST), embryonal carcinoma (EC)]. Six of eight microRNAs from these clusters contain the seed sequence 'AAGUGC', determining mRNA targeting. Here we sought to identify the significance of these observations by targeting these microRNAs functionally. METHODS: We targeted miR-371~373 and/or miR-302/367 clusters in malignant GCT cell lines, using CRISPR-Cas9, gapmer primary miR-302/367 transcript inhibition, and peptide nucleic acid (PNA) or locked nucleic acid (LNA)-DNA inhibition targeting miR-302a-d-3p, and undertook relevant functional assays. RESULTS: MiR-302/367 cluster microRNAs made the largest contribution to AAGUGC seed abundance in malignant GCT cells, regardless of subtype (seminoma/YST/EC). Following the unsuccessful use of CRISPR-Cas9, gapmer, and PNA systems, LNA-DNA-based targeting resulted in growth inhibition in seminoma and YST cells. This was associated with the de-repression of multiple mRNAs targeted by AAGUGC seed-containing microRNAs, with pathway analysis confirming predominant disruption of Rho-GTPase signalling, vesicle organisation/transport, and cell cycle regulation, findings corroborated in clinical samples. Further LNA-DNA inhibitor studies confirmed direct cell cycle effects, with an increase of cells in G0/G1-phase and a decrease in S-phase. CONCLUSION: Targeting of specific miR-371~373 and miR-302/367 microRNAs in malignant GCTs demonstrated their functional significance, with growth inhibition mediated through cell cycle disruption.

Blood conservation strategies at United States hospitals during the COVID-19 pandemic: Findings from a multi-institutional analysis - International Society of Blood Transfusion survey.

BACKGROUND: Due to the coronavirus disease 2019 (COVID-19) pandemic, the transfusion medicine community has experienced unprecedented blood supply shortages since March 2020. As such, numerous changes to everyday practice have occurred with a specific emphasis on blood conservation. We sought to determine the strategies used to mitigate blood shortages and promote blood conservation during the pandemic. METHODS: An anonymous, 37-question survey was developed using Research Electronic Data Capture and distributed via e-mail to transfusion medicine specialists across the US obtained via publicly available databases. RESULTS: Amongst surveyed [41.1% response rate (51/124 institutions)], 98.0% experienced a product shortage, with the greatest number reporting red blood cell (RBC) shortages (92.0%). This led to 35.3% of institutions altering the composition and/or number of blood product suppliers, including a 100% increase in the number of institutions acquiring blood from organizations that connect hospital transfusion services with blood collection centers (e.g., Blood Buy) compared to before March 2020. Prospective triaging of blood products was the most common blood conservation strategy (68.1%), though 35.4% altered their RBC exchange or transfusion program for patients receiving chronic RBC transfusion/exchange. As a result of these changes, 78.6% of institutions reported that these changes resulted in a reduction in blood product usage, and 38.1% reported a decrease in product wastage. CONCLUSIONS: Most hospitals experienced the effects of the supply shortage, and many of them implemented blood conserving measures. Conservation strategies were associated with decreased blood utilization and waste, and future studies could evaluate whether these changes persist.

Myeloproliferative Neoplasms, Version 3.2022, NCCN Clinical Practice Guidelines in Oncology.

The classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) consist of myelofibrosis, polycythemia vera, and essential thrombocythemia and are a heterogeneous group of clonal blood disorders characterized by an overproduction of blood cells. The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for MPN were developed as a result of meetings convened by a multidisciplinary panel with expertise in MPN, with the goal of providing recommendations for the management of MPN in adults. The Guidelines include recommendations for the diagnostic workup, risk stratification, treatment, and supportive care strategies for the management of myelofibrosis, polycythemia vera, and essential thrombocythemia. Assessment of symptoms at baseline and monitoring of symptom status during the course of treatment is recommended for all patients. This article focuses on the recommendations as outlined in the NCCN Guidelines for the diagnosis of MPN and the risk stratification, management, and supportive care relevant to MF.

Pathogen-reduced platelets in pediatric and neonatal patients: Demographics, transfusion rates, and transfusion reactions.

BACKGROUND: Pathogen-reduced platelets (PR PLT) are the emerging standard for proactive transfusion-transmitted infection (TTI) mitigation. There is, however, continued hesitation to transfuse PR PLT in children due to limited published data. We report demographics, rates of transfusion, and transfusion reactions (TR) associated with FDA-approved PR PLT in pediatric and neonatal patients at an academic medical center. METHODS: Retrospective review was performed for patients <18 years receiving at least one platelet over a 300-day period at a large, tertiary care hospital. Patients were transfused PR or conventional (CONV) PLT, based on inventory availability. Statistical analysis was performed using Fisher Exact Test. RESULTS: During the study period, 191 patients received 1010 platelet transfusions (892 units). Sixty-eight patients received PR PLT only (1.3 units/patient, 95% confidence interval [CI] 1.1-1.5; 1.8 transfusions/patient, 95% CI 1.4-2.2), and 56 patients received CONV PLT only (1.4 units/patient, 95% CI 1.1-1.7; 1.6 transfusions/patient, 95% CI 1.3-1.9). Patients with hematologic malignancies undergoing chemotherapy/radiation and allogeneic hematopoietic stem cell transplant received the most platelet transfusions and more commonly received both platelet types. Of 506 PR PLT units, 5 TRs occurred; 386 CONV PLT resulted in two TRs (p = .7052). Of 51 neonates, 37 received PR PLT without adverse events, including 13 receiving phototherapy. No TTIs were identified in any group. CONCLUSION: There was no significant difference in rates of transfusion or TRs between PR and CONV PLT. Our study provides additional evidence that PR PLT can be transfused to pediatric and neonatal patients without increasing the risk of acute adverse events.

A pilot study of stored low titer group O whole blood + component therapy versus component therapy only for civilian trauma patients.

BACKGROUND: This pilot assessed transfusion requirements during resuscitation with whole blood followed by standard component therapy (CT) versus CT alone, during a change in practice at a large urban Level I trauma center. METHODS: This was a single-center prospective cohort pilot study. Male trauma patients received up to 4 units of cold-stored low anti-A, anti-B group O whole blood (LTOWB) as initial resuscitation followed by CT as needed (LTOWB + CT). A control group consisting of women and men who presented when LTOWB was unavailable, received CT only (CT group). Exclusion criteria included antiplatelet or anticoagulant medication and death within 24 hours. The primary outcome was total transfusion volume at 24 hours. Secondary outcomes were mortality, morbidity, and intensive care unit- and hospital-free days. RESULTS: Thirty-eight patients received LTOWB, with a median of 2.0 (interquartile range [IQR] 1.0-3.0) units of LTOWB transfused. Thirty-two patients received CT only. At 24 hours after presentation, the LTOWB +CT group had received a median of 2,138 mL (IQR, 1,275-3,325 mL) of all blood products. The median for the CT group was 4,225 mL (IQR, 1,900-5,425 mL; p = 0.06) in unadjusted analysis. When adjusted for Injury Severity Score, sex, and positive Focused Assessment with Sonography for Trauma, LTOWB +CT group patients received 3307 mL of blood products, and CT group patients received 3,260 mL in the first 24 hours (p = 0.95). The adjusted median ratio of plasma to red cells transfused was higher in the LTOWB + CT group (0.85 vs. 0.63 at 24 hours after admission; p = 0.043. Adjusted mortality was 4.4% in the LTOWB + CT group, and 11.7% in the CT group (p = 0.19), with similar complications, intensive care unit-, and hospital-free days in both groups. CONCLUSION: Beginning resuscitation with LTOWB results in equivalent outcomes compared with resuscitation with CT only. LEVEL OF EVIDENCE: Therapeutic (Prospective study with 1 negative criterion, limited control of confounding factors), level III.

Circulating microRNAs as biomarkers to assist the management of the malignant germ-cell-tumour subtype choriocarcinoma.

Germ-cell-tumours (GCTs) are heterogeneous and management is complex. The current conventional biomarkers, alpha-fetoprotein and human-chorionic-gonadotropin (HCG), have limited utility for diagnosis/follow-up as secretion is restricted to specific malignant-GCT subtypes and long half-life can make interpretation and clinical decision-making challenging. We sought to identify circulating microRNAs that reflected choriocarcinoma disease activity more accurately than HCG in a metastatic primary mediastinal nonseminomatous-GCT (PMNSGCT) case with elevated diagnostic serum HCG (>250,000 U/L), consistent with pure choriocarcinoma. We undertook comprehensive microRNA profiling (n = 754 microRNAs) using two 384-well TaqMan Low-Density-Array cards in 16 serum samples; 10 from PMNSGCT diagnosis/follow-up and six controls. Key findings underwent confirmatory qRT-PCR. We identified a serum panel of choriocarcinoma-specific 'chromosome-19-microRNA-cluster' (C19MC) microRNAs that were highly elevated at diagnosis but fell rapidly on treatment and normalised before the second full chemotherapy course. We also re-confirmed serum elevation of the previously identified malignant-GCT marker miR-371a-3p at diagnosis. These circulating microRNA markers reflected choriocarcinoma disease activity more accurately than serum HCG and real-time knowledge would have assisted clinical decision-making. With further study, these microRNA markers will facilitate future management of such patients and are likely to result in improved outcomes.

Myeloid/Lymphoid Neoplasms with Eosinophilia and TK Fusion Genes, Version 3.2021, NCCN Clinical Practice Guidelines in Oncology.

Eosinophilic disorders and related syndromes represent a heterogeneous group of neoplastic and nonneoplastic conditions, characterized by more eosinophils in the peripheral blood, and may involve eosinophil-induced organ damage. In the WHO classification of myeloid and lymphoid neoplasms, eosinophilic disorders characterized by dysregulated tyrosine kinase (TK) fusion genes are recognized as a new category termed, myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB or FGFR1 or with PCM1-JAK2. In addition to these aforementioned TK fusion genes, rearrangements involving FLT3 and ABL1 genes have also been described. These new NCCN Guidelines include recommendations for the diagnosis, staging, and treatment of any one of the myeloid/lymphoid neoplasms with eosinophilia (MLN-Eo) and a TK fusion gene included in the 2017 WHO Classification, as well as MLN-Eo and a FLT3 or ABL1 rearrangement.

Clinical utility of circulating miR-371a-3p for the management of patients with intracranial malignant germ cell tumors.

BACKGROUND: The current biomarkers alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) have limited sensitivity/specificity for diagnosing malignant germ cell tumors (GCTs) and "marker-negative" patients require histological confirmation for diagnosis. However, GCTs at intracranial sites are surgically relatively inaccessible and biopsy carries risks. MicroRNAs from the miR-371~373 and miR-302/367 clusters are over-expressed in all malignant GCTs and, in particular, miR-371a-3p shows elevated serum levels at diagnosis for testicular disease. METHODS: Using our robust preamplified qRT-PCR methodology, we quantified miR-371a-3p levels in serum and cerebrospinal fluid (CSF) in a series of 4 representative clinical cases, 3 with intracranial malignant GCT and 1 with Langerhans cell histiocytosis (LCH), compared with appropriate control cases. RESULTS: Serum and/or CSF miR-371a-3p levels distinguished those with intracranial malignant GCTs from LCH and, if known in real time, could have helped clinical management. The benefits would have included (1) the only confirmatory evidence of an intracranial malignant GCT in 1 case, supporting clinical decision making; (2) early detection of intracranial malignant GCT in another, where an elevated CSF miR-371a-3p level preceded the histologically confirmed diagnosis by 2 years; and (3) confirmation of an intracranial malignant GCT relapse with an elevated serum miR-371a-3p level, where serum and CSF AFP and HCG levels were below thresholds for such a diagnosis. CONCLUSIONS: This series highlights the potential for microRNA quantification to assist the noninvasive diagnosis, prognostication, and management for patients with intracranial malignant GCTs. Serum and CSF should be collected routinely as part of future studies to facilitate the extension of these findings to larger patient cohorts.

Comparison of human bone marrow stromal cells cultured in human platelet growth factors and fetal bovine serum.

BACKGROUND: Bone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with fetal bovine serum (FBS). As an alternative to FBS, pooled solvent detergent apheresis platelets, HPGF-C18, was evaluated for BMSC culture. METHODS: A comparison of passage 2 BMSC growth revealed that 10% HPGF-C18 produced similar cell numbers as 20% FBS. Marrow aspirates from 5 healthy subjects were cultured for 4 passages in 10% HPGF-C18 or 20% FBS and were analyzed for proliferation, colony formation efficiency (CFE), surface marker expression, suppression of mixed lymphocyte reactions (MLRs), global gene and microRNA expression analysis. BMSC supernatant cytokine and growth factor concentrations were also compared. RESULTS: Primary cultures of marrow aspirates in 10% HPGF-C18 and 20% FBS yielded similar numbers and CFE. After 4 passages, 10% HPGF-C18 and 20% FBS yielded similar numbers of BMSCs, surface marker expression patterns and immunosuppression effects. Gene and microRNA expression analysis revealed that BMSCs cultured under the two conditions had distinct expression profiles. Gene Set Enrichment Analysis (GSEA) revealed HPGF-C18-cultured BMSCs were enriched in metabolic processing and biosynthetic pathways, cell proliferation and cell cycle pathways, and immune response pathways. FBS-cultured BMSCs were enriched in MAPK signaling, TGF-beta signaling, cell adhesion and extracellular matrix pathways. Differently expressed microRNAs were related to the osteogenesis of BMSCs. The supernatant of HPGF-C18 BMSCs had higher levels of PEDF and TGFB1 and lower levels of IL6, VEGF, SDF1 and PLGF. CONCLUSIONS: Traditional measures, expansion, surface marker expression and inhibition of MLRs suggest that BMSC cultured in HPGF-C18 and FBS were similar, but analysis at the molecular level revealed many differences. BMSCs cultured in HPGF-C18 should be assessed in specific functional assays that reflect application-specific potency before substituting FBS with HPGF-C18.

"Future-Proofing" Blood Processing for Measurement of Circulating miRNAs in Samples from Biobanks and Prospective Clinical Trials.

Background: Quantifying circulating nucleic acids is an important new approach to cancer diagnosis/monitoring.Methods: We compared the suitability of serum versus plasma for measuring miRNAs using qRT-PCR and assessed how preanalytic variables that can affect circulating tumor DNA (ctDNA) quantification in plasma also influence miRNA levels.Results: Across 62 blood-derived specimens, plasma samples in EDTA, Streck-DNA, and Streck-RNA tubes showed significantly higher Ct values for multiple housekeeping miRNAs, compared with serum samples. For the EDTA-plasma tubes, this difference was only seen when including the high-speed centrifugation protocol used to optimize ctDNA extraction. In plasma samples derived from blood stored at room temperature for up to 14 days (conditions that typically apply to samples processed for biobanking), levels of endogenous housekeeping miRNAs gradually increased, in parallel with the hemolysis marker hsa-miR-451a, consistent with release from blood cells/platelets. It was necessary to normalize levels of the housekeeping miRNAs to those of hsa-miR-451a, to obtain the stable values needed for referencing test miRNA levels.Conclusions: Our data indicate that plasma samples prepared for ctDNA extraction are suboptimal for miRNA quantification and require the incorporation of multiple data normalization steps. For prospective studies designed to measure both miRNAs and ctDNA, the most suitable approach would be to obtain both serum (for miRNAs) and plasma (for ctDNA). If only plasma can be collected, we recommend an initial low-speed centrifugation step, followed by aliquoting the supernatant into parallel samples, one for direct miRNA quantification, and the other for a further high-speed centrifugation step to optimize ctDNA retrieval.Impact: These recommendations will help "future-proof" clinical studies in which quantification of circulating miRNAs is a component. Cancer Epidemiol Biomarkers Prev; 27(2); 208-18. ©2017 AACR.

A quality initiative of postoperative radiographic imaging performed on mastectomy specimens to reduce histology cost and pathology report turnaround time.

Breast pathology relies on gross dissection for accurate diagnostic work, but challenges can necessitate submission of high tissue volumes resulting in excess labor, laboratory costs, and delays. To address these issues, a quality initiative was created through implementation of the Faxitron PathVision specimen radiography system as part of the breast gross dissection protocol; this report documents its impact on workflow and clinical care. Retrospective data from 459 patients who underwent simple or modified radical mastectomy at our institution between May 2012 and December 2014 were collected. Comparison was made between the mastectomy specimen control group before radiography use (233 patients, 340 breasts) and Faxitron group that underwent postoperative radiography (226 patients, 338 breasts). We observed a statistically significant decrease in mean number of blocks between control and Faxitron groups (47.0 vs 39.7 blocks; P<.0001), for calculated cost savings of US $146 per mastectomy. A statistically significant decrease in pathology report turnaround time was also observed (4.2 vs 3.8days; P=.038). Postoperative mastectomy specimen radiography has increased workflow efficiency and decreased histology costs and pathology report turnaround time. These findings may underestimate actual benefits and highlight the importance of quality improvement projects in anatomical pathology.

Virus transcript levels and cell growth rates after naturally occurring HPV16 integration events in basal cervical keratinocytes.

Cervical carcinogenesis is characterized by a clonal selection process in which the high-risk human papillomavirus (HRHPV) genome usually changes from the extra-chromosomal (episomal) state seen in productive infections to DNA that is integrated into host chromosomes. However, it is not clear whether all HRHPV integration events provide cells with a selective growth advantage compared with the episome-containing cells from which they originate. It is also unclear whether selection of cells containing a particular integrant from a mixed population simply reflects the highest levels of virus oncogene expression or has additional determinants. These early events in cervical carcinogenesis cannot readily be addressed by cross-sectional studies of clinical samples. We used the W12 model system to generate a panel of cervical squamous cell clones that were derived from an identical background under non-competitive conditions and differed only by the genomic site of HPV16 integration. Compared with the 'baseline' episome-containing cells from which they were isolated, only 9/17 clones (53%) showed significantly greater growth rates and only 7/17 (41%) showed significantly greater expression of the major virus oncogenes E7/E6. There were significant variations in levels of HPV16 transcription per DNA template, changes that were associated with histone modifications in the integrated virus chromatin. Cell growth rates showed only weak and non-significant associations with protein and mRNA levels for E7, E6, and the mean E7/E6 values. We conclude that HPV16 integration in basal cervical cells does not necessarily lead to increased levels of virus oncogenes, or to a competitive growth advantage, when compared with the initiating episome-containing cells.

Depletion of HPV16 early genes induces autophagy and senescence in a cervical carcinogenesis model, regardless of viral physical state.

In cervical carcinomas, high-risk human papillomavirus (HR-HPV) may be integrated into host chromosomes or remain extra-chromosomal (episomal). We used the W12 cervical keratinocyte model to investigate the effects of HPV16 early gene depletion on in vitro cervical carcinogenesis pathways, particularly effects shared by cells with episomal versus integrated HPV16 DNA. Importantly, we were able to study the specific cellular consequences of viral gene depletion by using short interfering RNAs known not to cause phenotypic or transcriptional off-target effects in keratinocytes. We found that while cervical neoplastic progression in vitro was characterized by dynamic changes in HPV16 transcript levels, viral early gene expression was required for cell survival at all stages of carcinogenesis, regardless of viral physical state, levels of early gene expression or histology in organotypic tissue culture. Moreover, HPV16 early gene depletion induced changes in host gene expression that were common to both episome-containing and integrant-containing cells. In particular, we observed up-regulation of autophagy genes, associated with enrichment of senescence and innate immune-response pathways, including the senescence-associated secretory phenotype (SASP). In keeping with these observations, HPV16 early gene depletion induced autophagy in both episome-containing and integrant-containing W12 cells, as evidenced by the appearance of autophagosomes, punctate expression of the autophagy marker LC3, conversion of LC3B-I to LC3B-II, and reduced levels of the autophagy substrate p62. Consistent with the reported association between autophagy and senescence pathways, HPV16 early gene depletion induced expression of the senescence marker beta-galactosidase and increased secretion of the SASP-related protein IGFBP3. Together, these data indicate that depleting HR-HPV early genes would be of potential therapeutic benefit in all cervical carcinogenesis pathways, regardless of viral physical state. In addition, the senescence/SASP response associated with autophagy induction may promote beneficial immune effects in bystander cells.

LIN28 Expression in malignant germ cell tumors downregulates let-7 and increases oncogene levels.

Despite their clinicopathologic heterogeneity, malignant germ cell tumors (GCT) share molecular abnormalities that are likely to be functionally important. In this study, we investigated the potential significance of downregulation of the let-7 family of tumor suppressor microRNAs in malignant GCTs. Microarray results from pediatric and adult samples (n = 45) showed that LIN28, the negative regulator of let-7 biogenesis, was abundant in malignant GCTs, regardless of patient age, tumor site, or histologic subtype. Indeed, a strong negative correlation existed between LIN28 and let-7 levels in specimens with matched datasets. Low let-7 levels were biologically significant, as the sequence complementary to the 2 to 7 nt common let-7 seed "GAGGUA" was enriched in the 3' untranslated regions of mRNAs upregulated in pediatric and adult malignant GCTs, compared with normal gonads (a mixture of germ cells and somatic cells). We identified 27 mRNA targets of let-7 that were upregulated in malignant GCT cells, confirming significant negative correlations with let-7 levels. Among 16 mRNAs examined in a largely independent set of specimens by quantitative reverse transcription PCR, we defined negative-associations with let-7e levels for six oncogenes, including MYCN, AURKB, CCNF, RRM2, MKI67, and C12orf5 (when including normal control tissues). Importantly, LIN28 depletion in malignant GCT cells restored let-7 levels and repressed all of these oncogenic let-7 mRNA targets, with LIN28 levels correlating with cell proliferation and MYCN levels. Conversely, ectopic expression of let-7e was sufficient to reduce proliferation and downregulate MYCN, AURKB, and LIN28, the latter via a double-negative feedback loop. We conclude that the LIN28/let-7 pathway has a critical pathobiologic role in malignant GCTs and therefore offers a promising target for therapeutic intervention.

In vitro progression of human papillomavirus 16 episome-associated cervical neoplasia displays fundamental similarities to integrant-associated carcinogenesis.

An important event in the development of cervical squamous cell carcinoma (SCC) is deregulated expression of high-risk human papillomavirus (HR-HPV) oncogenes, most commonly related to viral integration into host DNA. Mechanisms of development of the approximately 15% of SCCs that contain extrachromosomal (episomal) HR-HPV are poorly understood due to limited longitudinal data. We therefore used the W12 model to study mechanisms of cervical carcinogenesis associated with episomal HPV16. In vitro progression of W12 normally occurs through selection of cells containing integrated HPV16. However, in one long-term culture, keratinocytes developed a selective growth advantage and invasive phenotype while retaining HPV16 episomes at increased copy number in the absence of transcriptionally active integrants. Longitudinal investigations revealed similarities between the episome- and integrant-associated routes of neoplastic progression. Most notable were dynamic changes in viral early gene expression in episome-retaining cells, consistent with continually changing selective pressures. An early increase in viral transcription preceded elevated episome copy number and was followed by a reduction to near baseline after the development of invasiveness. Episomal transcriptional deregulation did not require selection of a specific sequence variant of the HPV16 upstream regulatory region, although increased levels of acetylated histone H4 around the late promoter implicated a role for altered chromatin structure. Interestingly, invasive episome-retaining cells showed high levels of HPV16 E2/E6 proteins (despite decreased transcript levels) and reduced expression of IFN-stimulated genes, adaptations that support viral persistence and cell survival. Our findings suggest a unified working model for events important in cervical neoplastic progression regardless of HR-HPV physical state.