Non-redundant activity of GSK-3α and GSK-3ß in T cell-mediated tumor rejection.

Glycogen synthase kinase-3 (GSK-3) is a positive regulator of PD-1 expression in CD8+ T cells and GSK-3 inhibition enhances T cell function and is effective in the control of tumor growth. GSK-3 has two co-expressed isoforms, GSK-3α and GSK-3ß. Using conditional gene targeting, we demonstrate that both isoforms contribute to T cell function to different degrees. Gsk3b-/- mice suppressed tumor growth to the same degree as Gsk3a/b-/- mice, whereas Gsk3a-/- mice behaved similarly to wild-type, revealing an important role for GSK-3ß in regulating T cell-mediated anti-tumor immunity. The individual GSK-3α and ß isoforms have differential effects on PD-1, IFNγ, and granzyme B expression and operate in synergy to control PD-1 expression and the infiltration of tumors with CD4 and CD8 T cells. Our data reveal a complex interplay of the GSK-3 isoforms in the control of tumor immunity and highlight non-redundant activity of GSK-3 isoforms in T cells, with implications for immunotherapy.

The genomic road to invasion-examining the similarities and differences in the genomes of associated oral pre-cancer and cancer samples.

BACKGROUND: It is frequently assumed that pre-invasive lesions are simpler precursors of cancer and will contain a limited subset of the genomic changes seen in their associated invasive disease. Driver mutations are thought to occur early, but it is not known how many of these are present in pre-invasive lesions. These assumptions need to be tested with the increasing focus on both personalised cancer treatments and early detection methodologies. METHODS: We examined genomic copy number changes in 256 pre-invasive and invasive samples from 69 oral cancer patients. Forty-eight samples from 16 patients were further examined using exome sequencing. RESULTS: Evidence of a shared ancestor of both dysplasia and carcinoma was seen in all but one patient. One-third of dysplasias showed independent copy number events. The remainder had a copy number pattern that was similar to or simpler than that of the carcinoma. All dysplasias examined contained somatic mutations absent in the related carcinoma. Previously observed copy number changes and TP53 mutations were very frequently observed, and almost always shared between dysplasia and carcinoma. Other gene changes were more sporadic. Pathway analysis confirmed that each patient's disease developed in a different way. Examining the numbers of shared mutations and the rate of accumulation of mutations showed evidence that all samples contain a population of sub-clones, with little evidence of selective advantage of a subset of these. CONCLUSIONS: These findings suggest that most of the genomic changes driving oral cancer occur in the pre-cancerous state by way of gradual random accumulation rather than a dramatic single event.

Non-Hodgkin lymphoma in Romania: a single-centre experience.

Epidemiologic studies of non-Hodgkin lymphoma (NHL) in Eastern Europe are scarce in the literature. We report the experience of the "Ion Chiricuta" Institute of Oncology in Cluj-Napoca (IOCN), Romania, in the diagnosis and outcome of patients with NHL. We studied 184 consecutive NHL patients diagnosed in the Pathology Department of IOCN during the years 2004-2006. We also obtained epidemiological data from the Northwestern (NW) Cancer Registry. In the IOCN series, the most common lymphoma subtype was diffuse large B-cell lymphoma (43.5%), followed by the chronic lymphocytic leukaemia/small lymphocytic lymphoma (21.2%). T-cell lymphomas represented a small proportion (8.2%). The median age of the patients was 57 years, with a male-to-female ratio of 0.94. Patients with indolent B-cell lymphomas had the best overall survival, whereas those with mantle cell lymphoma had the worst survival. The NW Cancer Registry data showed that the occurrence of NHL in the NW region of Romania was higher in men [world age-standardized incidence rate/100 000 (ASR)-5.9; 95% CI 5.1-6.6] than in women (ASR-4.1; 95% CI 3.5-4.7) with age-standardized male-to-female ratio of 1.44 (p = 0.038). Chronic lymphocytic leukaemia/small lymphocytic lymphoma was the most common NHL in the NW region of Romania, accounting for 43% of all cases, followed by diffuse large B-cell lymphoma (36%). The 5-year, age-standardized cumulative relative survival for NHL in the County of Cluj in NW Romania, for the period of 2006-2010, was 51.4%, with 58.4% survival for men and 43.2% for women. Additional studies of NHL in Eastern Europe are needed. Copyright © 2015 John Wiley & Sons, Ltd.

Segmental differences in upregulated apical potassium channels in mammalian colon during potassium adaptation.

Rat proximal and distal colon are net K+ secretory and net K+ absorptive epithelia, respectively. Chronic dietary K+ loading increases net K+ secretion in the proximal colon and transforms net K+ absorption to net K+ secretion in the distal colon, but changes in apical K+ channel expression are unclear. We evaluated expression/activity of apical K+ (BK) channels in surface colonocytes in proximal and distal colon of control and K+-loaded animals using patch-clamp recording, immunohistochemistry, and Western blot analyses. In controls, BK channels were more abundant in surface colonocytes from K+ secretory proximal colon (39% of patches) than in those from K+-absorptive distal colon (12% of patches). Immunostaining demonstrated more pronounced BK channel α-subunit protein expression in surface cells and cells in the upper 25% of crypts in proximal colon, compared with distal colon. Dietary K+ loading had no clear-cut effects on the abundance, immunolocalization, or expression of BK channels in proximal colon. By contrast, in distal colon, K+ loading 1) increased BK channel abundance in patches from 12 to 41%; 2) increased density of immunostaining in surface cells, which extended along the upper 50% of crypts; and 3) increased expression of BK channel α-subunit protein when assessed by Western blotting (P < 0.001). Thus apical BK channels are normally more abundant in K+ secretory proximal colon than in K+ absorptive distal colon, and apical BK channel expression in distal (but not proximal) colon is greatly stimulated as part of the enhanced K+ secretory response to dietary K+ loading.

Non-Hodgkin lymphoma in the developing world: review of 4539 cases from the International Non-Hodgkin Lymphoma Classification Project.

The distribution of non-Hodgkin lymphoma subtypes varies around the world, but a large systematic comparative study has never been done. In this study, we evaluated the clinical features and relative frequencies of non-Hodgkin lymphoma subtypes in five developing regions of the world and compared the findings to the developed world. Five expert hematopathologists classified 4848 consecutive cases of lymphoma from 26 centers in 24 countries using the World Health Organization classification, and 4539 (93.6%) were confirmed to be non-Hodgkin lymphoma, with a significantly greater number of males than females in the developing regions compared to the developed world (P<0.05). The median age at diagnosis was significantly lower for both low- and high-grade B-cell lymphoma in the developing regions. The developing regions had a significantly lower frequency of B-cell lymphoma (86.6%) and a higher frequency of T- and natural killer-cell lymphoma (13.4%) compared to the developed world (90.7% and 9.3%, respectively). Also, the developing regions had significantly more cases of high-grade B-cell lymphoma (59.6%) and fewer cases of low-grade B-cell lymphoma (22.7%) compared to the developed world (39.2% and 32.7%, respectively). Among the B-cell lymphomas, diffuse large B-cell lymphoma was the most common subtype (42.5%) in the developing regions. Burkitt lymphoma (2.2%), precursor B- and T-lymphoblastic leukemia/lymphoma (1.1% and 2.9%, respectively) and extranodal natural killer/T-cell lymphoma (2.2%) were also significantly increased in the developing regions. These findings suggest that differences in etiologic and host risk factors are likely responsible, and more detailed epidemiological studies are needed to better understand these differences.

Non-Hodgkin lymphoma in Southern Africa: review of 487 cases from The International Non-Hodgkin Lymphoma Classification Project.

Comparative data on the distribution of non-Hodgkin lymphoma (NHL) subtypes in Southern Africa (SAF) is scarce. In this study, five expert haematopathologists classified 487 consecutive cases of NHL from SAF using the World Health Organization classification, and compared the results to North America (NA) and Western Europe (WEU). Southern Africa had a significantly lower proportion of low-grade (LG) B-NHL (34·3%) and a higher proportion of high-grade (HG) B-NHL (51·5%) compared to WEU (54·5% and 36·4%) and NA (56·1% and 34·3%). High-grade Burkitt-like lymphoma was significantly more common in SAF (8·2%) than in WEU (2·4%) and NA (2·5%), most likely due to human immunodeficiency virus infection. When SAF patients were divided by race, whites had a significantly higher frequency of LG B-NHL (60·4%) and a lower frequency of HG B-NHL (32·7%) compared to blacks (22·5% and 62·6%), whereas the other races were intermediate. Whites and other races had a significantly higher frequency of follicular lymphoma and a lower frequency of Burkitt-like lymphoma compared to blacks. The median ages of whites with LG B-NHL, HG B-NHL and T-NHL (64, 56 and 67 years) were significantly higher than those of blacks (55, 41 and 34 years). Epidemiological studies are needed to better understand these differences.

Past and future impact of next-generation sequencing in head and neck cancer.

Progress in sequencing technology is intrinsically linked to progress in understanding cancer genomics. The purpose of this review was to discuss the development from Sanger sequencing to next-generation sequencing (NGS) technology. We highlight the technical considerations for understanding reports using NGS. We discuss the findings of studies in head and neck cancer using NGS as well as The Cancer Genome Atlas. Finally we discuss future routes for research utilizing this methodology and the potential impact of this. © 2015 Wiley Periodicals, Inc. Head Neck 38: E2395-E2402, 2016.

Relative frequency of non-Hodgkin lymphoma subtypes in selected centres in North Africa, the middle east and India: a review of 971 cases.

Comparative data regarding the distribution of non-Hodgkin lymphoma (NHL) subtypes in North Africa, the Middle East and India (NAF/ME/IN) is scarce in the literature. In this study, we evaluated the relative frequencies of NHL subtypes in this region. Five expert haematopathologists classified 971 consecutive cases of newly-diagnosed NHL from five countries in NAF/ME/IN. After review, 890 cases (91·7%) were confirmed to be NHL and compared to 399 cases from North America (NA). The male-to-female ratio was significantly higher in NAF/ME/IN (1·8) compared to NA (1·1; P< 0·05). The median ages of patients with low-grade (LG) and high-grade (HG) B-NHL in NAF/ME/IN (56 and 52 years, respectively) were significantly lower than in NA (64 and 68 years, respectively). In NAF/ME/IN, a significantly lower proportion of LG B-NHL (28·4%) and a higher proportion of HG B-NHL (58·4%) were found compared to NA (56·1% and 34·3%, respectively). Diffuse large B-cell lymphoma was more common in NAF/ME/IN (49·4%) compared to NA (29·3%), whereas follicular lymphoma was less common in NAF/ME/IN (12·4%) than in NA (33·6%). In conclusion, we found significant differences in NHL subtypes and clinical features between NAF/ME/IN and NA. Epidemiological studies are needed to better understand the pathobiology of these differences.

Non-Hodgkin lymphoma in the Far East: review of 730 cases from the international non-Hodgkin lymphoma classification project.

Large and systematic studies of non-Hodgkin lymphoma (NHL) in the Far East (FE) with good comparative data are scarce in the literature. In this study, five expert hematopathologists classified 730 consecutive cases of newly-diagnosed NHL from four sites in the FE (excluding Japan) using the World Health Organization classification. The results were compared to 399 cases from North America (NA). We found a significantly higher male to female ratio in the FE compared to NA (1.7 versus 1.1; p < 0.05). The median ages of patients with low-grade (LG) and high-grade (HG) B-NHL in the FE (58 and 51 years, respectively) were significantly lower than in NA (64 and 68 years, respectively). The FE had a significantly lower relative frequency of B-NHL and a higher frequency of T-NHL (82 vs. 18 %) compared to NA (90.5 vs. 9.5 %). Among mature B cell lymphomas, the FE had a significantly higher relative frequency of HG B-NHL (54.8 %) and a lower frequency of LG B-NHL (27.2 %) than NA (34.3 and 56.1 %, respectively). Diffuse large B cell lymphoma was more common in the FE (49.4 %) compared to NA (29.3 %), whereas the relative frequency of follicular lymphoma was lower in the FE (9.4 %) compared to NA (33.6 %). Among T-NHL, nasal NK/T cell NHL was more frequent in the FE (5.2 %) compared to NA (0 %). Peripheral T cell lymphoma was also more common in the FE (9.1 %) than in NA (5.3 %). Further epidemiologic studies are needed to better understand the pathobiology of these differences.

Classification of non-Hodgkin lymphoma in South-eastern Europe: review of 632 cases from the international non-Hodgkin lymphoma classification project.

The distribution of non-Hodgkin lymphoma (NHL) subtypes varies around the world, but a systematic study of South-eastern Europe (SEEU) has never been done. Therefore, we evaluated the relative frequencies of NHL subtypes in three SEEU countries--Croatia, Romania and Macedonia. Five expert haematopathologists reviewed 632 consecutive cases of newly diagnosed NHL from the three SEEU countries using the World Health Organization classification. The results were compared to 399 cases from North America (NA) and 580 cases from Western Europe (WEU). The proportions of B- and T-cell NHL and the sex distribution in SEEU were similar to WEU and NA. However, the median ages of patients with low- and high-grade B-NHL in SEEU (60 and 59 years, respectively) were significantly lower than in NA (64 and 68 years, respectively; P < 0·05). SEEU had a significantly lower proportion of low-grade B-NHL (46·6%) and higher proportion of high-grade B-NHL (44·5%) compared to both WEU (54·5% and 36·4%, respectively) and NA (56·1% and 34·3%, respectively). There were no significant differences in the relative frequencies of T-NHL subtypes. This study provides new insights into differences in the relative frequencies of NHL subtypes in different geographic regions. Epidemiological studies are needed to better characterize and explain these differences.

The clonal relationships between pre-cancer and cancer revealed by ultra-deep sequencing.

The study of the relationships between pre-cancer and cancer and identification of early driver mutations is becoming increasingly important as the value of molecular markers of early disease and personalised drug targets is recognized, especially now the extent of clonal heterogeneity in fully invasive disease is being realized. It has been assumed that pre-cancerous lesions exhibit a fairly passive progression to invasive disease; the degree to which they, too, are heterogeneous is unknown. We performed ultra-deep sequencing of thousands of selected mutations, together with copy number analysis, from multiple, matched pre-invasive lesions, primary tumours and metastases from five patients with oral cancer, some with multiple primary tumours presenting either synchronously or metachronously, totalling 75 samples. This allowed the clonal relationships between the samples to be observed for each patient. We expose for the first time the unexpected variety and complexity of the relationships between this group of oral dysplasias and their associated carcinomas and, ultimately, the diversity of processes by which tumours are initiated, spread and metastasize. Instead of a series of genomic precursors of their adjacent invasive disease, we have shown dysplasia to be a distinct dynamic entity, refuting the belief that pre-cancer and invasive tumours with a close spatial relationship always have linearly related genomes. We show that oral pre-cancer exhibits considerable subclonal heterogeneity in its own right, that mutational changes in pre-cancer do not predict the onset of invasion, and that the genomic pathway to invasion is neither unified nor predictable. Sequence data from this study have been deposited in the European Nucleotide Archive, Accession No. PRJEB6588.

A novel genomic signature reclassifies an oral cancer subtype.

Verrucous carcinoma of the oral cavity (OVC) is considered a subtype of classical oral squamous cell carcinoma (OSCC). Diagnosis is problematic, and additional biomarkers are needed to better stratify patients. To investigate their molecular signature, we performed low-coverage copy number (CN) sequencing on 57 OVC and exome and RNA sequencing on a subset of these and compared the data to the same OSCC parameters. CN results showed that OVC lacked any of the classical OSCC patterns such as gain of 3q and loss of 3p and demonstrated considerably fewer genomic rearrangements compared to the OSCC cohort. OVC and OSCC samples could be clearly differentiated. Exome sequencing showed that OVC samples lacked mutations in genes commonly associated with OSCC (TP53, NOTCH1, NOTCH2, CDKN2A and FAT1). RNA sequencing identified genes that were differentially expressed between the groups. In silico functional analysis showed that the mutated and differentially expressed genes in OVC samples were involved in cell adhesion and keratinocyte proliferation, while those in the OSCC cohort were enriched for cell death and apoptosis pathways. This is the largest and most detailed genomic and transcriptomic analysis yet performed on this tumour type, which, as an example of non-metastatic cancer, may shed light on the nature of metastases. These three independent investigations consistently show substantial differences between the cohorts. Taken together, they lead to the conclusion that OVC is not a subtype of OSCC, but should be classified as a distinct entity.

Classification of non-Hodgkin lymphoma in Algeria according to the World Health Organization classification.

The relative distribution of non-Hodgkin lymphoma (NHL) subtypes differs markedly around the world. The aim of this study was to report this distribution in Algeria. A panel of four hematopathologists classified 197 consecutive cases according to the World Health Organization classification, including 87.3% B-cell and 12.7% T- or natural killer (NK)-cell NHLs. This series was compared with similar cohorts from Western Europe (WEU) and North America (NA). Algeria had a significantly higher frequency of diffuse large B-cell lymphoma (DLBCL: 52.8%) and a lower frequency of follicular lymphoma (FL: 13.2%) compared with WEU (DLBCL: 32.2%; FL: 20.0%) and NA (DLBCL: 29.3%; FL: 33.6%). The frequency of mantle cell lymphoma was lower in Algeria (2.5%) compared with WEU (8.3%). Smaller differences were also found among the NK/T-cell lymphomas. In conclusion, we found important differences between Algeria and Western countries, and further epidemiologic studies are needed to explain these differences.

Next-generation sequencing analysis for detecting human papillomavirus in oral verrucous carcinoma.

OBJECTIVE: The etiology of oral verrucous carcinoma is unknown, and human papillomavirus 'involvement' remains contentious. The uncertainty can be attributed to varied detection procedures and difficulties in defining 'gold-standard' histologic criteria for diagnosing 'verrucous' lesions. Their paucity also hampers investigation. We aimed to analyze oral verrucous lesions for human papillomavirus (HPV) subtype genomes. STUDY DESIGN: We used next-generation sequencing for the detection of papillomavirus sequences, identifying subtypes and computing viral loads. We identified a total of 78 oral verrucous cases (62 carcinomas and 16 hyperplasias). DNA was extracted from all and sequenced at a coverage between 2.5% and 13%. RESULTS: An HPV-16 sequence was detected in 1 carcinoma and 1 hyperplasia, and an HPV-2 sequence was detected in 1 carcinoma out of the 78 cases, with viral loads of 2.24, 8.16, and 0.33 viral genomes per cell, respectively. CONCLUSIONS: Our results indicate no conclusive human papillomavirus involvement in oral verrucous carcinoma or hyperplasia.

Sequential gene targeting to make chimeric tumor models with de novo chromosomal abnormalities.

The discovery of chromosomal translocations in leukemia/lymphoma and sarcomas presaged a widespread discovery in epithelial tumors. With the advent of new-generation whole-genome sequencing, many consistent chromosomal abnormalities have been described together with putative driver and passenger mutations. The multiple genetic changes required in mouse models to assess the interrelationship of abnormalities and other mutations are severe limitations. Here, we show that sequential gene targeting of embryonic stem cells can be used to yield progenitor cells to generate chimeric offspring carrying all the genetic changes needed for cell-specific cancer. Illustrating the technology, we show that MLL-ENL fusion is sufficient for lethal leukocytosis and proof of genome integrity comes from germline transmission of the sequentially targeted alleles. This accelerated technology leads to a reduction in mouse numbers (contributing significantly to the 3Rs), allows fluorescence tagging of cancer-initiating cells, and provides a flexible platform for interrogating the interaction of chromosomal abnormalities with mutations.

Investigation of p16(INK4a) as a prognostic biomarker in oral epithelial dysplasia.

BACKGROUND: Human papilloma virus is a risk factor for oropharyngeal cancer. Evidence for a similar aetiological role in the development of oral dysplasia or its transformation to oral cancer is not as clear. Meta-analyses estimate the prevalence of high-risk human papilloma virus (HPV) serotypes to be three times higher in pre-malignant lesions and cancer than in normal oral mucosa. However, this does not imply a causal relationship. Conflicting results are reported from the few studies examining the prognostic significance of HPV positivity in the development of oral cancer. We aimed to examine the ability of p16(INK4a) protein expression, a surrogate marker of HPV infection, to predict malignant progression in a large cohort of oral dysplasia patients. METHODS: One hundred forty eight oral dysplasia cases underwent immunohistochemical analysis using a monoclonal antibody against p16(INK4a) . Clinical factors were also collated on each case. Slides were double scored independently by two trained observers. Univariate analyses using both logistic and Cox regression models were performed. RESULTS: Thirty nine of 148 cases progressed to cancer. Ten of 148 cases (7%) were p16(INK4a) positive. High grade of dysplasia (P = 0.0002) and lesion morphology (P = 0.03) were found to be prognostic of malignant progression. p16(INK4a) score was not prognostic in this cohort (P = 0.29). This did not change with a time to event analysis (P = 0.24). CONCLUSION: Few studies have assessed the aetiological role of HPV in cancer development from dysplastic lesions. Our study, using one of the largest cohorts of oral dysplasia, demonstrated a low rate of p16(INK4a) positivity and was unable to confirm a prognostic ability for this biomarker.

Classification of non-Hodgkin lymphoma in Central and South America: a review of 1028 cases.

The distribution of non-Hodgkin lymphoma (NHL) subtypes differs around the world but a systematic study of Latin America has not been done. Therefore, we evaluated the relative frequencies of NHL subtypes in Central and South America (CSA). Five expert hematopathologists classified consecutive cases of NHL from 5 CSA countries using the WHO classification and compared them to 400 cases from North America (NA). Among the 1028 CSA cases, the proportions of B- and T-cell NHL and the sex distribution were similar to NA. However, the median age of B-cell NHL in CSA (59 years) was significantly lower than in NA (66 years; P < .0001). The distribution of high-grade (52.9%) and low-grade (47.1%) mature B-cell NHL in CSA was also significantly different from NA (37.5% and 62.5%; P < .0001). Diffuse large B-cell lymphoma was more common in CSA (40%) than in NA (29.2%; P < .0001), whereas the frequency of follicular lymphoma was similar in Argentina (34.1%) and NA (33.8%), and higher than the rest of CSA (17%; P < .001). Extranodal NK/T-cell NHL was also more common in CSA (P < .0001). Our study provides new objective evidence that the distribution of NHL subtypes varies significantly by geographic region and should prompt epidemiologic studies to explain these differences.

Non-Hodgkin lymphoma in Chile: a review of 207 consecutive adult cases by a panel of five expert hematopathologists.

The distribution of subtypes of non-Hodgkin lymphoma (NHL) in Latin America is not well known. This Chilean study included 207 consecutive cases of NHL diagnosed at five cancer centers in the capital, Santiago, and one center in Viña del Mar. All cases were reviewed and classified independently by five expert hematopathologists according to the 2001 World Health Organization classification of NHL. A consensus diagnosis of NHL was reached in 195 of the 207 cases (94%). B-cell lymphomas constituted 88% of NHL, and diffuse large B-cell lymphoma (DLBCL, 38.5%) and follicular lymphoma (25.1%) were the most common subtypes. There was a high frequency of marginal zone B-cell lymphoma (10.3%), as well as of extranodal natural killer (NK)/T-cell lymphoma, nasal type (2.6%) and adult T-cell leukemia/lymphoma (0.5%). Extranodal presentation was seen in 74 of the 195 cases (38%) and the most common extranodal presentation was in the stomach (37.6%). The most common gastric lymphoma was DLBCL (54.5%) followed by mucosa-associated lymphoid tissue (MALT) lymphoma (41%). Overall, the frequency of NHL subtypes in Chile is between that reported in Western and Eastern countries, which is probably a reflection of the admixture of ethnicities as well as the environment and socioeconomic status of its population.

Next-generation sequencing for simultaneous determination of human papillomavirus load, subtype, and associated genomic copy number changes in tumors.

Human papillomavirus (HPV) infection in cases of squamous cell carcinoma of the oropharynx is a powerful predictive and prognostic biomarker. We describe how the use of next-generation sequencing can provide a novel method for the detection of HPV in DNA isolated from formalin-fixed paraffin-embedded tissues. Using this methodology in a cohort of 44 head and neck tumors, we identified the samples that contained HPV sequences, the viral subtype involved, and a direct readout of viral load. Specificity of HPV detection by sequencing compared to traditional detection methods using either PCR or p16 immunohistochemistry was 100%. Sensitivity was 50% when either compared to PCR [confidence interval (CI) = 29% to 71%] or 75% when compared to p16 (CI = 47% to 91%). In addition, we demonstrate the ability of next-generation sequencing to detect other HPV subtypes that would not have been detected by traditional methods, and we demonstrated the ability to apply this method to any tumor and any virus in a panel of eight human cancer cell lines. This methodology also provides a tumor genomic copy number karyogram, and in the samples analyzed here, a lower level of chromosome instability was detected in HPV-positive tumors compared to HPV-negative tumors, as observed in previous studies. Thus, the use of next-generation sequencing for the detection of HPV provides a multiplicity of data with clinical significance in a single test.