Immunoexpression of Ki67, P16 and Beta-catenin in precursor lesions of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (CSCC) is the second most common type of skin cancer, after basal cell carcinoma, representing about 10-20% of all malignant skin tumors. The mortality rates of CSCC approach those of renal and oropharyngeal carcinomas, as well as melanoma, with the increasing of the risk once metastases and perineural invasion occur. Both actinic keratosis (AK) and Bowen's disease (BD) are direct precursors with the potential for progression to CSCC. In this study, we analyzed the expression of Ki67, P16 and Beta-catenin in the precursor lesions of CSCC in relation to histological prognostic parameters, respectively between them, with the aim of identifying possible correlations with a role in prognosis. Ki67 and P16 presented higher scores in advanced precancerous lesions, such as keratinocyte intraepithelial neoplasia (KIN) III and BD and low scores in seborrheic keratosis (SK). The immunoreactivity to the investigated markers confirms the multistage skin carcinogenesis, and their involvement starting from the initiation phase of the cancer process. The importance of the studied markers in the evolution and prognosis of precancerous lesions of CSCC is also supported by the linear correlations revealed between the immunoexpressions of P16, Ki67 and the membranous immunoexpression of Beta-catenin in AK.

Identification and validation of EMT-immune-related prognostic biomarkers CDKN2A, CMTM8 and ILK in colon cancer.

Colon cancer (CC) is a disease with high incidence and mortality rate. The interaction between epithelial-mesenchymal transition (EMT) and immune status has important clinical significance. We aim to identify EMT-immune-related prognostic biomarkers in colon cancer. The GEO2R and GEPIA 2.0 were utilized to calculate the differential expression genes between CC and normal mucosa. Immport, InnateDB and EMTome databases were used to define EMT-immune-related genes. We conducted batch prognostic analysis by TCGA data. The expression patterns were verified by multiple datasets and lab experiments. GEPIA 2.0 and TIMER 2.0 were utilized to analyze the correlation of the hub genes with EMT markers and immune infiltration. GeneMANIA, STRING, and Metascape were used for co-expression and pathway enrichment analysis. Finally, we established a signature by the method of multivariate Cox regression analysis. CDKN2A, CMTM8 and ILK were filtered out as prognostic genes. CDKN2A and CMTM8 were up-regulated, while ILK was down-regulated in CC. CDKN2A was positively correlated with infiltration of macrophages, Th2 cells, Treg cells, and negatively correlated with NK cells. CMTM8 was negatively correlated with CD8+ T cells, dendritic cells, and NK cells. ILK was positively correlated with CD8+ T cells and dendritic cells. Moreover, CDKN2A, CMTM8 and ILK were significantly correlated with EMT markers. The three genes could participate in the TGF-ß pathway. The prognosis model established by the three hub genes was an independent prognosis factor, which can better predict the prognosis. CDKN2A, CMTM8 and ILK are promising prognostic biomarkers and may be potential therapeutic targets in colon cancer.

Generation of mice for evaluating endogenous p16Ink4a protein expression.

The cyclin-dependent kinase inhibitor p16Ink4a plays a central role in cellular senescence in vitro. Although previous studies suggested cellular senescence is integrated in the systemic mechanisms of organismal aging, the localization and the dynamics of p16Ink4a in tissues remain poorly understood, which hinders uncovering the role of p16Ink4a under the in vivo context. One of the reasons is due to the lack of reliable reagents; as we also demonstrate here that commonly used antibodies raised against human p16INK4A barely recognize its murine ortholog. Here we generated a mouse model, in which the endogenous p16Ink4a is HA-tagged at its N-terminus, to explore the protein expression of p16Ink4a at the organismal level. p16Ink4a was induced at the protein level along the course of senescence in primary embryonic fibroblasts derived from the mice, consistently to its transcriptional level. Remarkably, however, p16Ink4a was not detected in the tissues of the mice exposed to pro-senescence conditions including genotoxic stress and activation of oncogenic signaling pathways, indicating that there is only subtle p16Ink4a proteins induced. These results in our mouse model highlight the need for caution in evaluating p16Ink4a protein expression in vivo.

An instructive role for Interleukin-7 receptor α in the development of human B-cell precursor leukemia.

Kinase signaling fuels growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Yet its role in leukemia initiation is unclear and has not been shown in primary human hematopoietic cells. We previously described activating mutations in interleukin-7 receptor alpha (IL7RA) in poor-prognosis "ph-like" BCP-ALL. Here we show that expression of activated mutant IL7RA in human CD34+ hematopoietic stem and progenitor cells induces a preleukemic state in transplanted immunodeficient NOD/LtSz-scid IL2Rγnull mice, characterized by persistence of self-renewing Pro-B cells with non-productive V(D)J gene rearrangements. Preleukemic CD34+CD10highCD19+ cells evolve into BCP-ALL with spontaneously acquired Cyclin Dependent Kinase Inhibitor 2 A (CDKN2A) deletions, as commonly observed in primary human BCP-ALL. CRISPR mediated gene silencing of CDKN2A in primary human CD34+ cells transduced with activated IL7RA results in robust development of BCP-ALLs in-vivo. Thus, we demonstrate that constitutive activation of IL7RA can initiate preleukemia in primary human hematopoietic progenitors and cooperates with CDKN2A silencing in progression into BCP-ALL.

Response and recurrence correlates in individuals treated with neoadjuvant anti-PD-1 therapy for resectable oral cavity squamous cell carcinoma.

Neoadjuvant PD-1 blockade may be efficacious in some individuals with high-risk, resectable oral cavity head and neck cancer. To explore correlates of response patterns to neoadjuvant nivolumab treatment and post-surgical recurrences, we analyzed longitudinal tumor and blood samples in a cohort of 12 individuals displaying 33% responsiveness. Pretreatment tumor-based detection of FLT4 mutations and PTEN signature enrichment favors response, and high tumor mutational burden improves recurrence-free survival. In contrast, preexisting and/or acquired mutations (in CDKN2A, YAP1, or JAK2) correlate with innate resistance and/or tumor recurrence. Immunologically, tumor response after therapy entails T cell receptor repertoire diversification in peripheral blood and intratumoral expansion of preexisting T cell clones. A high ratio of regulatory T to T helper 17 cells in pretreatment blood predicts low T cell receptor repertoire diversity in pretreatment blood, a low cytolytic T cell signature in pretreatment tumors, and innate resistance. Our study provides a molecular framework to advance neoadjuvant anti-PD-1 therapy for individuals with resectable head and neck cancer.

p16INK4a Regulates Cellular Senescence in PD-1-Expressing Human T Cells.

T-cell dysfunction arising upon repeated antigen exposure prevents effective immunity and immunotherapy. Using various clinically and physiologically relevant systems, we show that a prominent feature of PD-1-expressing exhausted T cells is the development of cellular senescence features both in vivo and ex vivo. This is associated with p16INK4a expression and an impaired cell cycle G1 to S-phase transition in repeatedly stimulated T cells. We show that these T cells accumulate DNA damage and activate the p38MAPK signaling pathway, which preferentially leads to p16INK4a upregulation. However, in highly dysfunctional T cells, p38MAPK inhibition does not restore functionality despite attenuating senescence features. In contrast, p16INK4a targeting can improve T-cell functionality in exhausted CAR T cells. Collectively, this work provides insights into the development of T-cell dysfunction and identifies T-cell senescence as a potential target in immunotherapy.

B-cell receptor signaling and genetic lesions in TP53 and CDKN2A/CDKN2B cooperate in Richter transformation.

B-cell receptor (BCR) signals play a critical role in the pathogenesis of chronic lymphocytic leukemia (CLL), but their role in regulating CLL cell proliferation has still not been firmly established. Unlike normal B cells, CLL cells do not proliferate in vitro upon engagement of the BCR, suggesting that CLL cell proliferation is regulated by other signals from the microenvironment, such as those provided by Toll-like receptors or T cells. Here, we report that BCR engagement of human and murine CLL cells induces several positive regulators of the cell cycle, but simultaneously induces the negative regulators CDKN1A, CDKN2A, and CDKN2B, which block cell-cycle progression. We further show that introduction of genetic lesions that downregulate these cell-cycle inhibitors, such as inactivating lesions in CDKN2A, CDKN2B, and the CDKN1A regulator TP53, leads to more aggressive disease in a murine in vivo CLL model and spontaneous proliferation in vitro that is BCR dependent but independent of costimulatory signals. Importantly, inactivating lesions in CDKN2A, CDKN2B, and TP53 frequently co-occur in Richter syndrome (RS), and BCR stimulation of human RS cells with such lesions is sufficient to induce proliferation. We also show that tumor cells with combined TP53 and CDKN2A/2B abnormalities remain sensitive to BCR-inhibitor treatment and are synergistically sensitive to the combination of a BCR and cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor both in vitro and in vivo. These data provide evidence that BCR signals are directly involved in driving CLL cell proliferation and reveal a novel mechanism of Richter transformation.

Prognostic role of immunohistochemical overexpression of the p16 protein in women under the age of 35 and diagnosed with HSIL (CIN2) subjected to "cervix sparing" excision.

OBJECTIVE: To evaluate the role of immunohistochemical staining overexpression of p16 protein (p16 IHC) as a prognostic factor of persistence or recurrence of intraepithelial disease after excision procedure in young women diagnosed with HSIL (CIN2). PATIENTS AND METHODS: 62 women with a histological diagnosis of HSIL (CIN2) subjected to "cervix sparing" excisional procedure were included in this retrospective study. All had age less than or equal to 35 years, negative history of immunosuppression, available follow-up, and assessment of the resection margins state. Immunohistochemical staining for the p16 protein was evaluated on reviewed and confirmed HSIL (CIN2) histological specimens with negative resection margins. The post-treatment follow-up, including cytology, colposcopy, and histology, ranged from a minimum of 6 months to a maximum of 60 months. The persistence or recurrence of SIL during the follow-up period was based on histologic referral and defined as "the presence of SIL", "the presence of HSIL" and "progression to HSIL (CIN3)". RESULTS: 31/62 patients were positive for immunostaining (p16 IHC+), and 31/62 were negative (p16 IHC-). Persistence or recurrence after excision occurred more frequently within the p16 IHC+ than in p16 IHC- group, both as SIL (29% p16 IHC- vs. 32.3% p16 IHC+, p = 0.783) and HSIL (6.5% p16 IHC- vs. 12.9% p16 IHC+, p = 0.671). None of the patients in the p16 IHC- group showed progression to CIN3 for the entire observation period, whereas 9.7% of p16 IHC+ women progressed to CIN3 lesion (p = 0.042). The p16 IHC positivity showed a significant association with progression to CIN3 in 5 years of follow-up (p = 0.029) and with the presence of SIL after two years of follow-up (p = 0.031). The differences between the two groups increased after two years post-treatment: the p16 IHC- patients still had SIL only in 3.2% of cases and no longer had HSIL, while the p16 IHC+ women still showed SIL in 19.4% and HSIL in 6.5% of cases. The negative predictive value (NPV) of p16 IHC in predicting SIL's presence after treatment increased with the severity of the lesion (NPV for SIL 70.97%, for HSIL 93.55%, for CIN3 100%). CONCLUSIONS: The study suggests that young patients with p16 IHC- HSIL (CIN2) have a better post-excisional course of the cervical intraepithelial disease compared to p16 IHC+ women and that p16 IHC could have prognostic utility during the long-term follow-up, especially in forecasting progression to CIN3 in consideration of the high NPV (up to 100%). The efficacy of the adjuvant HPV vaccination in the management of HSIL (CIN2) p16+ young women is to be evaluated as part of the fertility-sparing treatment.

Integrative molecular characterization of sarcomatoid and rhabdoid renal cell carcinoma.

Sarcomatoid and rhabdoid (S/R) renal cell carcinoma (RCC) are highly aggressive tumors with limited molecular and clinical characterization. Emerging evidence suggests immune checkpoint inhibitors (ICI) are particularly effective for these tumors, although the biological basis for this property is largely unknown. Here, we evaluate multiple clinical trial and real-world cohorts of S/R RCC to characterize their molecular features, clinical outcomes, and immunologic characteristics. We find that S/R RCC tumors harbor distinctive molecular features that may account for their aggressive behavior, including BAP1 mutations, CDKN2A deletions, and increased expression of MYC transcriptional programs. We show that these tumors are highly responsive to ICI and that they exhibit an immune-inflamed phenotype characterized by immune activation, increased cytotoxic immune infiltration, upregulation of antigen presentation machinery genes, and PD-L1 expression. Our findings build on prior work and shed light on the molecular drivers of aggressivity and responsiveness to ICI of S/R RCC.

[Clinical value of p16INK4a immunocytochemistry in cervical cancer screening].

Objective: To evaluate the value of p16INK4a detected by p16INK4a immunostaining as a new generation of cervical cytology for primary screening and secondary screening in population-based cervical cancer screening, and in improving cytological diagnosis. Methods: Between 2016 and 2018, 5 747 non-pregnant women aged 25-65 years with sexual history were recruited and underwent cervical cancer screening via high-risk (HR)-HPV/liquid-based cytological test (LCT) test in Shenzhen and surrounding areas. All slides were immuno-stained using p16INK4a technology, among them, 902 cases were offered p16INK4a detection during primary screening, and the remaining 4 845 cases were called-back by the virtue of abnormal HR-HPV and LCT results for p16INK4a staining. Participants with complete LCT examination, HR-HPV test, p16INK4a staining and histopathological examination results were included in this study. The performance of p16INK4a in primary and secondary screening, and in assisting cytology to detect high grade squamous intraepithelial lesion [HSIL, including cervical intraepithelial neoplasia (CIN) Ⅱ or Ⅲ] or worse [HSIL (CIN Ⅱ)+ or HSIL (CIN Ⅲ)+] were analyzed. Results: (1) One-thousand and ninety-seven cases with complete data of p16INK4a and histology were included. Pathological diagnosis: 995 cases of normal cervix, 37 cases of low grade squamous intraepithelial lesion (LSIL), 64 cases of HSIL and one case of cervical cancer were found. Among them, 65 cases of HSIL (CIN Ⅱ)+ and 34 cases of HSIL (CIN Ⅲ)+ were detected. The positive rate of p16INK4a in HSIL (CIN Ⅱ)+ was higher than that in CINⅠ or normal pathology (89.2% vs 10.2%; P<0.01). (2) p16INK4a as primary screening for HSIL (CIN Ⅱ)+ or HSIL (CIN Ⅲ)+ was equally sensitive to primary HR-HPV screening (89.2% vs 95.4%, 94.1% vs 94.1%; P>0.05), but more specific than HR-HPV screening (89.8% vs 82.5%, 87.7% vs 80.2%; P<0.05). p16INK4a was equally sensitive and similarly specific to cytology (≥LSIL; P>0.05). (3) The specificity of LCT adjunctive p16INK4a for detecting HSIL (CIN Ⅱ)+ or HSIL (CIN Ⅲ)+ were higher than that of LCT alone or adjunctive HR-HPV (P<0.01), while the sensitivity were similar (P>0.05). (4) p16INK4a staining as secondary screening: p16INK4a was significantly more specific (94.1% vs 89.7%, 91.9% vs 87.4%; P<0.01) and comparably sensitive (84.6% vs 90.8%, 88.2% vs 91.2%; P>0.05) to cytology for triaging primary HR-HPV screening. HPV 16/18 to colposcopy and triage other HR-HPV with p16INK4a was equally sensitive (88.2% vs 94.1%; P=0.500) and more specific (88.3% vs 83.0%; P<0.01) than HPV 16/18 to colposcopy and triage other HR-HPV with LCT≥ atypical squamous cells of undetermined significance (ASCUS), and the referral rate decreased (14.0% vs 19.4%; P=0.005). Conclusions: For primary screening, p16INK4a is equally specific to cytology and equally sensitive to HR-HPV screening. p16INK4a alone could be an efficient triage after primary HR-HPV screening. In addition, p16INK4a immunostaining could be used as an ancillary tool to cervical cytological diagnosis, and improves its accuracy in cervical cancer screening.

Conservative management of CIN2 p16 positive lesions in women with multiple HPV infection.

BACKGROUND: According to the 2006 American Society for Colposcopy and Cervical Pathology guidelines, positive CIN2 p16 in women over the age of 25 should be managed with excisional treatment. However, excisional treatment is associated with physical, psychological and obstetric morbidity and can have a negative impact on sexual function. In our study we sought to identify a clear management strategy, addressing the impact of routine use of p16 immunohistochemistry in this population and identify appropriate criteria for patient selection with the aim of reducing over-treatment. METHOD: We studied the medical records of 130 patients who had undergone laser therapy for CIN2. Each patient underwent colposcopy, biopsy and HPV test and were tested for p16 protein,. Patients were divided based on HPV infection into: single infections, multiple infections. All patients underwent ZTA laser therapy with follow-up (2-year follow-up). STATISTICAL ANALYSIS: Contingency tables were created to evaluate the correlation between single, multiple and CIN2+ infections. Values with p < 0.05 were considered statistically significant. RESULTS: Single infections had a histological regression of 61.8% (21/34) and a histological persistence rate of 35.3% (12/34), which was greater than the multiple infection rate. The common characteristic that the women with persistence and progression had was the dimension of the lesion and the genotype 16. Ten cases of histological persistence and the only case of progression had one lesion greater than three quarters of the cervix. CONCLUSIONS: With the progress of our understanding of the natural history of infection from human papillomavirus and the increasing use of colposcopy, thanks to the addition of HPV genotyping and the technique of immunohistochemistry, conservative management of these lesions is now possible.

HPV-related carcinoma of the oropharynx: challenges on small biopsy specimens.

Human papillomavirus (HPV)-related oropharyngeal carcinoma can morphologically mimic many processes from benign to malignant and lead pathologists to incorrectly diagnose them and/or use inappropriate diagnostic terminology. Recognition of HPV-related oropharyngeal carcinoma and its variants is critical for appropriate prognostic and therapeutic considerations in small biopsy material. Various pitfalls in diagnosing these tumors on limited biopsies are discussed herein.

Concordance of tumor infiltrating lymphocytes, PD-L1 and p16 expression in small biopsies, resection and lymph node metastases of oropharyngeal squamous cell carcinoma.

OBJECTIVE: The incidence of oropharyngeal squamous cell carcinoma (OPSCC), especially human papillomavirus (HPV)-associated, is increasing worldwide. Immunotherapy become available for patients with carcinomas in the head and neck region, however without ideal biomarker. Markers like PD-L1 vary in the clone of the antibody used, and the method of evaluation. Adequate and reliable immune cells characterization and evaluation is still not found. Furthermore, studies analyzing representativeness of different tissue samples are scarce. We analyzed small biopsy, lymph node (LN) metastasis and resected OPSCC, in regards of tumor infiltrating lymphocyte (TIL) density, PD-L1 and p16 expression. MATERIAL AND METHODS: Patients with OPSCC diagnosed from 2000 to 2016, with small biopsy, resection specimen and LN metastasis samples were selected. We analyzed TILs on hematoxylin-eosin stain, and PD-L1 and p16 expression in tumor cells. Concordance between different tumor locations was evaluated. RESULTS: 93 patients, with 65 small biopsies, 72 resection specimens, and 70 LN metastases were included. TILs, p16 and PD-L1 demonstrated very high concordance. Additionally, PD-L1 expression in the small biopsies was more representative of the PD-L1 expression in the resection specimens, than the LN samples. CONCLUSION: TILs density can be reliably assessed using hematoxylin-eosin stain with high concordance between the small biopsy, resection specimen and LN metastasis. Evaluation of concordance of p16 expression is very high, nevertheless some cases might be misdiagnosed on a small biopsy or lymph node metastasis. Evaluation of PD-L1 expression is very reliable on the biopsy specimen. Different PD-L1 clones and methods of evaluation still remain to be addressed.

Detection of Circulating Antibodies to p16 Protein-Derived Peptides in Hepatocellular Carcinoma.

OBJECTIVE: This study aimed at confirming the alteration of circulating anti-p16 immunoglobulin G (IgG) levels in hepatocellular carcinoma (HCC). METHODS: An in-house-developed enzyme-linked immunosorbent assay was used for determining plasma IgG antibodies against p16-derived antigens in 122 HCC patients and 134 healthy controls. RESULTS: Plasma anti-p16 IgG levels were significantly higher in HCC patients than in the controls (Z = 3.51, P = 0.0004), with no difference between males and females. A trend of increasing plasma anti-p16 IgG levels was associated with increasing HCC stage, with group 3 patients having the highest anti-p16 IgG levels (Z = 3.38, P = 0.0008). Group 3 exhibited the best sensitivity (19.6%) and specificity (95%) for plasma anti-p16 IgG detection, with an area under the receiver operating characteristic curve of 0.659 (95% confidence interval, 0.564-0.754). CONCLUSION: Circulating IgG antibody to p16 protein might be a useful biomarker for HCC prognosis assessment rather than for early malignancy diagnosis.

P16 as a marker of carcinoma in effusions and peritoneal washing.

BACKGROUND: Considering the potential of p16 as a marker for diagnosis, prognosis and therapeutic response, the aim of this study was to assess its presence, via immunocytochemistry, in metastatic carcinoma of different primary sites and histological types obtained from effusions and peritoneal washings. A total of 118 samples including 85 of metastatic carcinoma and 33 samples of benign effusion/peritoneal washing were prepared by the plasma/thromboplastin method. Immunocytochemistry reactions were performed on cell block sections using antibodies against p16, claudin-4, MOC-31, calretinin, HBME and CD68. RESULTS: P16 overexpression was observed in 88.23% of all carcinoma samples. All cervix adenocarcinoma samples showed p16 overexpression. Overexpression in adenocarcinomas of ovary, lung and breast was observed in 93.75, 93.10 and 75% of the samples, respectively. Overexpression was observed in all different histological types analyzed: small cell carcinoma (lung), squamous cell carcinoma (cervical) and urothelial carcinoma (bladder). The specificity of p16 for carcinoma detection was of 96.96%. CONCLUSION: Overexpression of p16 was observed in most metastatic carcinoma, from different primary sites and histological types, obtained from effusions and peritoneal washings. Due to its high frequency of overexpression in metastatic carcinoma, p16 may play a possible role in tumor progression and it may be considered as a complementary diagnostic marker depending on histological type and primary site of carcinoma.

Threshold for interpretation of p16 immunostaining in fine-needle aspirate specimens of metastatic oropharyngeal squamous cell carcinoma.

INTRODUCTION: Human papilloma virus (HPV)-associated oropharyngeal squamous cell carcinoma (HPV-OPSCC) has been recognized to have an excellent response to treatment and has a distinct pathologic staging. For this reason, HPV testing is recommended in cytology specimens of metastatic OPSCC, although the guidelines for testing are not clearly defined. The aim of the current study was to establish a threshold for interpretation of p16 in aspirates from metastatic OPSCC. MATERIALS AND METHODS: Cases diagnosed as metastatic SCC by cytology in neck lymph nodes with concurrent p16 on cytology and on paired surgical specimen or an in situ hybridization (ISH) for HPV were included in the study. Stain intensity and percentage positive cells for p16 was compared with p16 on paired surgical pathology specimens and/or ISH RNA for HPV on cytology specimens. RESULTS: Of the 52 cases diagnosed as metastatic SCC on neck aspirates, paired surgical pathology specimens and/or ISH HPV was available in 17 cases. A p16 expression in ≥10%-15% cells resulted in a sensitivity and negative predictive value of 66% and 37%, respectively. However, when even minimal expression in tumor fragments is considered positive, the negative predictive value increases to 100%. CONCLUSIONS: We recommend that even minimal nuclear expression for p16 in viable tumor fragments must be considered as positive in cytology specimens. Expression limited to only background single tumor cells or in a necrotic specimen must be interpreted with caution.

Flow Cytometry Characterization of Pluripotent Transmembrane Glycoproteins on Resident Cervix Uteri Cells in Patients Screened for Cervical Cancer.

The aim of this study was to characterize both by flow cytometry analysis and immunohistochemistry cervix uteri cells of nulliparous women screened for cervical intraepithelial neoplasia (CIN) in comparison to a group without CIN by using mesenchymal stem cell-like and hematopoietic lineage markers. A significant expression for CD29, CD38, HLA-I, and HLA-II was correlated positively to the CIN degree and it was more relevant in patients positive for human papilloma virus (HPV). Thus, identification and detailed characterization of pluripotent resident in uteri cells could be a promising therapeutic target.

Hhex regulates murine lymphoid progenitor survival independently of Stat5 and Cdkn2a.

The transcription factor Hhex (hematopoietically expressed homeobox gene) is critical for development of multiple lymphoid lineages beyond the common lymphoid progenitor. In addition, Hhex regulates hematopoietic stem cell (HSC) self-renewal, emergency hematopoiesis, and acute myeloid leukemia initiation and maintenance. Hhex mediates its effects on HSCs and acute myeloid leukemia stem cells via repression of the Cdkn2a tumor suppressor locus. However, we report here that loss of Cdkn2a does not rescue the failure of lymphoid development caused by loss of Hhex. As loss of Hhex causes apoptosis of lymphoid progenitors associated with impaired Bcl2 expression and defective Stat5b signaling, we tested the effects of rescuing these pathways using transgenic mice. Expression of the anti-apoptotic factor Bcl2, but not activated Stat5, rescued the development of T-, B-, and NK-cell lineages in the absence of Hhex. These results indicate that Bcl2 expression, but not Stat5b signaling or loss of Cdkn2a, can overcome the lymphoid deficiencies caused by the absence of Hhex, suggesting that the primary role of this transcription factor is to promote survival of lymphoid progenitors during early lymphoid development.

Comparison of Molecular Assays for HPV Testing in Oropharyngeal Squamous Cell Carcinomas: A Population-Based Study in Northern Ireland.

BACKGROUND: Determination of human papillomavirus (HPV) status has become clinically relevant for patient stratification under UICC TNM8 staging. Within the United Kingdom, a combination of p16 IHC and HPV DNA-ISH is recommended for classifying HPV status. This study will assess a series of clinically applicable second-line molecular tests to run in combination with p16 IHC to optimally determine HPV status. METHODS: The ability of HPV RNA-ISH, HPV DNA-ISH, and HPV DNA-PCR to identify p16-positive/HPV-positive patients was investigated in a population-based oropharyngeal squamous cell carcinoma (OPSCC) cohort of patients diagnosed in Northern Ireland from 2000 to 2011. RESULTS: Only 41% of the Northern Irish OPSCC patient population was associated with HPV-driven carcinogenesis. Both ISH assays were more specific than the DNA-PCR assay (100% and 95% vs. 67%) and were less likely to be affected by preanalytic factors such as increasing block age. A pooled HPV genotype probe for RNA-ISH was found to be the most accurate molecular assay assessed (95% accuracy) when compared with p16 positivity. CONCLUSIONS: Our study demonstrates the advantage of tissue-based molecular assays when determining HPV status in retrospective samples. Specifically, we demonstrate the enhanced sensitivity and specificity of ISH techniques compared with PCR-based methodology when working with formalin-fixed paraffin-embedded tissue, and found HPV RNA-ISH to be the most effective assay for determining HPV status. IMPACT: As p16 IHC is a relatively inexpensive, accessible, and sensitive test for stratifying patients by HPV status, this study finds that more patients would benefit from first-line p16 IHC followed by specific HPV testing using HPV RNA-ISH to confirm HPV status.

Prognostic and diagnostic validity of p16/Ki-67, HPV E6/E7 mRNA, and HPV DNA in women with ASCUS: a follow-up study.

BACKGROUND: We evaluated the prognostic and diagnostic ability of p16/Ki-67 immunocytochemistry, HPV E6/E7 mRNA testing and HPV DNA assay in triaging ASCUS to find a way to manage cervical lesions more effectively. METHODS: We conducted a prospective study through follow-up. The detection methods of the three factors: p16/Ki-67 immunocytochemistry conducted by using the CINtec® Plus Kit, E6/E7 mRNA testing by QuantiVirus®HPV E6/E7 mRNA assay and DNA by Hybrid Capture 2 assay. RESULTS: One hundred three women with ASCUS satisfied requirements and completed the entire follow-up process. All CIN2+ occurred in women who were mRNA positive at baseline, none in mRNA negative. 100% (6/6) patients with CIN2+ were HPV DNA assay positive, 100% (6/6) were HPV E6/E7 mRNA testing positive and 50.0% (3/6) were p16/Ki-67 immunocytochemistry positive. The risk ratio of E6/E7 mRNA test was 57.306 (95% CI 0.077-42,400.545). For endpoint of CIN2+, the sensitivity between HPV DNA assay and HPV E6/E7 mRNA testing is no statistical difference, but statistical difference exists between HPV E6/E7 mRNA testing vs. p16/Ki-67 immunocytochemistry (χ2 = 5.718, P = 0.023) and HPV DNA assay vs. p16/Ki-67 immunocytochemistry (χ2 = 5.718, P = 0.023). The specificity of E6/E7 mRNA testing, p16/Ki-67 and DNA assay in triaging ASCUS was 44.33, 75.26 and 11.34% respectively and is all statistical difference (χ2 = 26.277, P < 0.001(HPV DNA assay vs. HPV E6/E7 mRNA testing), χ2 = 19.297, P < 0.001(HPV E6/E7 mRNA testing vs. p16/Ki-67 immunocytochemistry), χ2 = 80.707, P < 0.001(HPV DNA assay vs. p16/Ki-67 immunocytochemistry). The expression level of 2097.09 copies/ml was the optimal cut-off value for HPV E6/E7 mRNA testing to diagnose CIN2+, the sensitivity and specificity was 61.1 and 68.2%. CONCLUSIONS: High expression of HPV E6/E7 mRNA could be a good candidate as a diagnostic biomarker to triage ASCUS superseding HPV DNA. p16/Ki-67 immunocytochemistry is suggested to be a good tool to triage ASCUS, but it reduced the sensitivity of diagnosis when improves the diagnostic specificity.