—  SPECIALTY CONFERENCE  —

Gastrointestinal Pathology

Case 5 - Proliferative and Dysplastic Lesions of Squamous Mucosa of Hypopharynx and Esophagus Associated with Human Papillomavirus Type 66 Infection

A. Scott Mills
Virginia Commonwealth University


Click on each slide thumbnail image for an enlarged view
Case Presentation:
This patient is a middle-aged male who was referred to our institution for evaluation of abnormalities noted at upper GI endoscopy. Repeat EGD at VCUHS showed multiple mucosal plaques in the hypopharynx and esophagus that had the appearance of those illustrated in Figure 1. The plaques in the esophagus were most numerous in the upper and middle thirds and failed to stain with Lugol's solution. The initial biopsies of these plaques showed the histologic features noted in Figures 2A & 2B. Immunohistochemistry for human papillomavirus (HPV) was non-reactive. PCR was positive for Human papillomavirus Type 66.

During the first year after referral, multiple lesions in the esophagus were ablated by photodynamic therapy (PDT) and/or excised by endoscopic mucosal resection (EMR). Varying degrees of squamous hyperplasia and dysplasia were noted in biopsy material (Figures 3-5). Thirteen months after referral, a lesion involving the right aryepiglottic fold was biopsied by the ENT service (Figures 6A & 6B). This lesion was irradiated.

After completion of radiation therapy to the larynx / hypopharynx, multiple sessions of photodynamic therapy were resumed to ablate lesions in the esophagus. These resulted in strictures that were relieved by multiple dilatations.

About 42 months after referral, an ulcerated 1.4 cm nodule was detected in the middle third of the esophagus (Figure 7). Biopsy findings of this lesion are shown in Figures 8A & 8B. This malignant neoplasm was staged T3, N1 by endoscopic ultrasound (EUS) and was subsequently treated with radiation therapy.


Case 5 - Figure 1
Endoscopic appearance of mucosal plaques.
Case 5 - Figure 2A
Mucosal plaque. (A) Acanthotic squamous mucosa with focal koilocytosis and parakeratosis.
Case 5 - Figure 2B
Mucosal plaque. (B) Higher magnification of koilocytosis and parakeratosis.

Case 5 - Figure 3
Mucosal plaque with slight nuclear atypia and spiked surface.
Case 5 - Figure 4
Mucosal plaque with moderate nuclear atypia.
Case 5 - Figure 5
Squamous cell carcinoma in situ.

Case 5 - Figure 6A
Hypopharyx. (A) Squamous cell carcinoma with sarcomatoid metaplasia.
Case 5 - Figure 6B
Hypopharyx. (B) Higher magnification of same.
Case 5 - Figure 7
Endoscopic appearance of 1.4 cm mass in middle esophagus.

Case 5 - Figure 8A
Mid-esophageal mass. (A) Squamous cell carcinoma with sarcomatoid metaplasia.
Case 5 - Figure 8B
Mid-esophageal mass. (B) Higher magnification of same.

Diagnoses :
  • Proliferative and dysplastic lesions of squamous mucosa of hypopharynx and esophagus associated with Human papillomavirus type 66 infection

  • Poorly differentiated squamous cell carcinoma with sarcomatoid metaplasia of the hypopharyx

  • Poorly differentiated squamous cell carcinoma with sarcomatoid metaplasia of the mid-esophagus.

Key Points:
  • Human papillomavirus (HPV) preferentially infects cutaneous and mucosal squamous epithelium.

  • HPV DNA has been identified in >99% of cervical squamous cell carcinomas.

  • The oncogenic potential of each HPV type is determined by its respective prevalence in cervical cancer. "High-risk" types in descending order of prevalence include HPV 16, 18, 45, 31, 52, 33, 58, 35, 59, 51, 56, 39, 73, 82, and 68. HPV 66 is classified as "Probably high-risk". "Low-risk" types include HPV 6, 11, 40, 42, 43, 44, 54, 61, 70, and 72.

  • HPV DNA has been detected in 23.5% of squamous papillomas of the esophagus worldwide using PCR methods.

  • HPV DNA has been detected in 21.4% of squamous cell carcinomas of the esophagus worldwide using PCR methods. High-risk HPV16 and HPV18 have been the most common types identified.

  • Regional differences in HPV prevalence in esophageal SCC have been attributed to variations in molecular testing methods, but it is also possible that different environmental influences (perhaps acting in synergy) could drive carcinogenesis in diverse geographic areas.

Introduction :
Human papillomavirus (HPV) is a double-stranded DNA virus that preferentially infects cutaneous and mucosal squamous epithelium. It is best recognized clinically for its prominent role in cervical carcinogenesis and its association with anogenital warts. HPV DNA has been identified in >99% of cervical cancers, 94% of women with intraepithelial neoplasia (CIN), and 46% of women with normal cervical cytology. [1] Annually, approximately one-half million new cases of cervical cancer are diagnosed worldwide and one-quarter million women die of the disease. HPV infection of anogenital epithelium is one of the most common sexually–transmitted diseases and is estimated to occur in up to two-thirds of young adults within the first 2 years of sexual activity. [2] The role of HPV infection in the development of intraepithelial and invasive neoplasia in non-genital squamous epithelium is being actively investigated.

HPV Biology:
The circular genome of the Human papillomavirus harbors 8 open reading frames (ORF). [1, 2, 3, 4, 5, 6] The L2 and L1 genes encode minor and major capsid proteins, respectively. The L2 protein aids in the self-assembly of the intact virion and may play a role in the specificity of the virus for keratinocyte surface receptors. There is considerable intertypic variation of the L1 gene sequence. A particular HPV type is defined as a complete genome that has an L1 gene sequence that is at least 10% dissimilar to that of any other HPV type. [7] At last count, 130 HPV types have been identified.

The E1, E2, E4, E5, E6, and E7 genes encode proteins that are transcribed at different times of the viral life cycle. The E1 and E2 genes are highly conserved in all HPV types and are involved with viral DNA replication and genome encapsidation. The E4 gene is also highly conserved. One of the functions of the E4 protein is to interact with cytoskeletal proteins to facilitate viral assembly. This effect may be responsible for the cytopathic koilocytosis noted in HPV infected tissue. The E5 protein upregulates growth factor receptors and has weak transforming activity. It appears to play a role in inducing the epithelial hyperplasia characteristic of HPV infected lesions. [3] The E6 and E7 genes encode proteins that disrupt cell cycle checkpoints, thus ensuring that HPV-infected cells do not exit the cell cycle. The latter is essential because HPV replication requires that DNA synthesis remain intact in keratinocytes throughout all epithelial layers. This is in contrast to normal stratified squamous epithelium, in which the differentiated cells of the upper strata cease to function and exist only as keratin-filled sacs. [1] One of the functions of E6 protein is to bind and degrade p53, thus effectively relieving restrictions on cellular DNA synthesis and promoting viral replication. Similarly, the E7 protein binds and degrades the retinoblastoma (Rb) family of proteins. The E2 protein serves as a major regulator of E6- and E7- mediated cell cycle activities.

During the early vegetative phase of the HPV life cycle, the virus exists as nuclear episomes. The high-risk oncogenic types of HPV are characterized by an ability to integrate their genomes into the host genome. Although the sites of integration are usually random, the E2 gene of the virus is typically disrupted in the process. Loss of E2 regulation of E6- and E7-mediated cell cycle activity results in disregulation of growth control and genomic instability that promotes the development of cancer.

Currently, the oncogenic potential of each HPV type is determined by its respective prevalence in cervical cancer. [8] Using this classification, "high-risk" types in descending order of prevalence include HPV 16, 18, 45, 31, 52, 33, 58, 35, 59, 51, 56, 39, 73, 82, and 68. HPV 16 accounts for 54.5% and HPV 18 for 11% of single infections with cervical carcinoma. HPV16/HPV18 co-infection is noted in 36% of cervical carcinomas, a figure 4 times higher than any other dual co-infection. [9] HPV 26, 53, and 66 are "probably high risk" types. "Low risk" types include HPV 6, 11, 40, 42, 43, 44, 54, 61, 70, and 72. HPV 6 and 11 are present in more than 90% of anogenital warts. HPV 34, 57, and 83 are classified as "undetermined risk". When putting all of these HPV types in perspective, one should note that the current quadrivalent vaccine for HPV provides immunity to the high-risk types 16 and 18 and the low-risk types 6 and 11 only.

Interestingly, most HPV infections, including those secondary to high-risk oncogenic types, resolve within 12 to 18 months. Malignant transformation is rare. This suggests that a number of different factors play a role in carcinogenesis, including variations in the host immune response or the ability of different HPV types to evade the immune system.

HPV Testing:
The detection of HPV in tissues, whether for clinical or research purposes, has usually been done using molecular techniques. These methods include: direct probe ("non-amplified") methods such as Southern blot and in situ hybridization (ISH); the FDA-approved signal amplification method Digene Hybrid Capture ®2; and target amplification methods such as PCR. [10]

ISH has been used in research studies to detect HPV in esophageal squamous papillomas; both Southern blot and ISH have been used to detect HPV in esophageal squamous cell carcinomas. Both techniques lack the analytical sensitivity of amplification methods, and Southern blot is impossible to perform on formalin-fixed tissue.

The Digene Hybrid Capture ®2 Technology (HC2) is the only FDA-approved method for detection of HPV in cervical cytology specimens. Fresh biopsy material can also be assayed, but the technique is not applicable to formalin-fixed tissue. HC2 is a signal amplification, solution-phase hybridization assay that uses two cocktails of RNA probes. One set of RNA probes contains sequences complementary to the DNA sequences of 5 low-risk HPV types (6, 11, 42, 43, and 44). The second RNA probe set has sequences complementary to the DNA sequences of 13 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68). [11] These cocktails must be used in two separate reactions (one to detect low-risk types and the other to detect high-risk types), and neither reaction provides details as to the specific types of HPV detected. To reduce costs and to obviate some cross-reactivity between low- and high-risk probes, most clinical assays used for screening or triage are performed with the high-risk cocktail only. To achieve the best balance between sensitivity and specificity in clinical assays, HC2 uses a positive threshold of 1.0 pg HPV DNA/ml or above (approximately 100,000-120,000 HPV genomes). Signal amplification systems are well-suited for clinical applications because they are semi-automated and do not require highly specialized training or sophisticated laboratories to produce accurate results. There is also less risk for cross-contamination of specimens than with PCR because HC2 does not amplify the target DNA. [11]

Target amplification assays such as PCR are the most sensitive means to detect HPV in tissue. Most research studies that have evaluated the prevalence of HPV infection in squamous cell carcinoma of the esophagus have employed PCR methods. Unfortunately, these research studies have spanned the entire history of PCR, and differences in the methodology have made these studies difficult to compare. Specifically, early investigators used unvalidated "home-brew" PCR methods. Beginning in the late 1980's and continuing to the present time, many investigators used PCR methods that target the L1 gene. Three different designs were developed to achieve broad-spectrum detection of HPV-DNA. [13]

The first design, the GP5+/GP6+ method, used one forward and one reverse primer to replicate a 150bp region of L1. Although this region was highly conserved, the short segment of targeted DNA fully complemented only a few HPV genotypes. To compensate for the mismatches, the PCR was performed at a low annealing temperature.

The second design, the MY09/MY11 degenerate primer system, used a complex mixture of many different oligonucleotide primers to compensate for intertypic sequence variation located at the priming sites of a 450bp region of L1. Because synthesis of these degenerate oligonucleotide primers was poorly reproducible, there was high batch-to-batch variation of primers that made this method difficult to control.

The MY09/MY11 method was replaced by a third design, the PGMY method that targeted the same 450bp region of L1. In the PGMY method, primers with random degeneracies were replaced by distinct forward and reverse primers that contained inosine, a nucleotide that matches with any other nucleotide. This mixture of non-degenerate primers could be synthesized with high reproducibility and the PCR could be performed at optimal annealing temperatures. Another PCR method, the SPF10 primer system, also used inosine-containing nucleotides to target a 65bp region of L1 that overlapped with the 450bp sequence of the MY/PGMY region and the 150bp GP5+/6+ region.

PCR has several advantages over HC2 for research applications: 1) increased sensitivity; 2) the ability to perform the assay on formalin-fixed, paraffin-embedded tissue; and 3) the ability to identify either individual or multiple HPV genotypes after amplification. Researchers have historically employed the following techniques to analyze the amplified product: 1) gel electropheresis with ethidium bromide labeling; 2) restriction fragment length polymorphism (RFLP); 3) hybridization (Southern blot; microtiter plate hybridization); 4) sequencing (sequence analysis after cloning; direct sequence analysis); and 5) reverse hybridization (either in microwell plates or on genotyping strips). In some studies, investigators have evaluated the same specimen using different PCR techniques, including one or more PCR methods targeting the L1 gene and another PCR method using genotype-specific priming of the E6 and/or E7 genes.

Considering the different sensitivities and specificities of these historical PCR research assays, the lack of standardization of reagents and procedures, and the plethora of methods used to analyze the amplicons, it is no surprise that it is exceedingly difficult to compare results from different studies. These concerns do not even begin to address the adoption of new PCR techniques that can be used for clinical and/or research purposes. Roche has recently developed its Amplicor® HPV test with the hope to compete with HC2. This PCR method targets a 170bp fragment of the L1 gene that overlaps the regions of the historical PCR methods. After PCR, reverse hybridization of the denatured amplicon is performed in microwell plates to detect the same 13 high-risk HPV types that are identified in the HC2 assay. [14] Finally, Real-time PCR (RT-PCR) methods such as TaqMan have been used in clinical and research venues to perform HPV viral loads on tissue. The clinical applicability of HPV quantification in tissues is controversial.

HPV and Squamous Papilloma of the Esophagus:
Squamous papillomas are uncommon polyps of the esophagus that are covered by mature stratified squamous epithelium. Most are solitary and measure less than 5 mm in size. A branching core of lamina propria is usually present that confers a frond-like configuration to the polyp. Spiked and endophytic papillomas also occur. [15] Traditionally, it has been the prevailing presumption that most papillomas are reactive lesions that arise in the setting of distal esophagitis, particularly that due to gastroesophageal reflux disease. Not infrequently, however, papillomas arise in the middle or even the upper esophagus, suggesting that longstanding HPV infection or other etiologies can induce their formation. [16, 29]

Both hybridization and PCR methods have been used to detect HPV DNA in esophageal papillomas. [16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29] Four studies that performed ISH only [19, 24, 28, 30] (USA, Korea, Italy, USA, respectively; by citation) detected HPV in 9.5% (2/21) of papillomas. One of the HPV-positive patients was from the USA. [19] This patient had "multiple papillomatosis" that was genotyped as HPV 6 or 11. The second patient was from Korea and had a solitary papilloma that was not typed. [24] A fifth study (Switzerland) [18] reported HPV 31-33-35 in a "small minority" of 33 papillomas.

Ten studies from 9 different countries [16, 17, 20, 21, 22, 23, 25, 26, 27, 29] used PCR (alone, or in combination with, ISH) to analyze squamous papillomas. HPV DNA was detected in 23.5% (40/172) of papillomas. HPV genotype analysis of the 40 positive cases showed the following:

High risk types: HPV+ / % of 40
16 (15 / 35.0%)
16-18 co-infection (03 / 7.5%)
35 (01 / 2.5%)
51-52-55 (01 / 2.5%)
58 (01 / 2.5%)
73 (02 / 5.0%)
Sub-Total (23 / 57.5%)
Low Risk Types: HPV+ / % of 40
6 (07 / 17.5%)
6-11 (06 / 15.0%)
11 (01 / 2.5%)
Sub-Total (14 / 35.0%)
Other Types:
20 (01 / 2.5%)
Cutaneous NOS (02 / 5.0 %)
Sub-Total (03 / 7.5%)
Total (40 /100%)

As noted previously, most squamous papillomas of the esophagus are solitary lesions. One might surmise that squamous papillomas associated with reflux esophagitis would be located in the distal esophagus and those associated with HPV infection might occur more often in the proximal esophagus and be multiple. Only a few HPV studies have noted the location and multiplicity of squamous papillomas.

In the ISH studies, only 3 patients were noted to have multiple squamous papillomas. [19, 28, 30] One of these three patients had detectable HPV in the lesions. [19] The Japanese PCR study of squamous papillomas authored by Takeshita et al [16] examined 38 papillomas in 35 patients. Nine papillomas each were located in the upper and lower esophagus; 20 were located in the middle esophagus. The 4 HPV-positive papillomas in this study were all solitary lesions located in the middle esophagus. The 3 patients with multiple papillomas had lesions located in the upper or middle esophagus and none were positive for HPV. The US-Canadian PCR study of Odze et al [20] analyzed 26 papillomas in 21 patients. Four of the 5 patients with multiple papillomas had HPV-positive lesions. The percentage of HPV-positive papillomas was higher in the upper (1/2; 50%) and middle esophagus (6/8; 75%) than in the lower esophagus (6/16; 37.5%), but there were too few cases for these data to be clinically significant. Previously documented or concurrent esophagitis was present in 61% of the patients in the Odze et al study; it was most often associated with squamous papillomas located in the distal esophagus. All patients with severe ulcerative esophagitis (5) or stricturing esophagitis (2) had papillomas located in the distal esophagus.

In summary, international studies have detected HPV DNA by PCR in 23.5% (40/170) of squamous papillomas. High-risk HPV types accounted for 57.5% of the positive cases. Low risk types 6 and 11 accounted for virtually all of the rest. These data suggest a mulifactorial etiology for esophageal squamous papillomas that includes esophagitis and HPV infection.

HPV and Squamous Cell Carcinoma of the Esophagus :
Carcinoma of the esophagus is the 7th leading cause of cancer deaths worldwide. For the past 20 years, adenocarcinoma of the esophagus has been on the increase in industrialized Western societies, but squamous cell carcinoma continues to predominate in some developing areas of the world. Regions with very high incidence of esophageal squamous cell carcinoma include Iran, China, South Africa, and parts of South America. [31] There is considerable geographic diversity, however, and pockets of high incidence also occur in areas of France, Italy, and Eastern Europe. In addition to genetic susceptibility, many environmental risk factors have been proposed, including: alcohol; tobacco; malnutrition; diet (betel nuts, hot beverages, fermented fish sauce, teas, fungal toxins, nitrosamines, etc); poor oral hygiene; and Human papillomavirus. Numerous ISH and PCR studies have been performed to detect HPV DNA in squamous cell carcinomas, precancers, and normal mucosa of the esophagus. Many of the studies prior to 2001 have been reviewed and tabulated in a 2002 review by KJ Syrjänen [32] and will be cited with that reference. Other pertinent literature is cited separately in the accompanied bibliography.

Twenty-five studies from 7 different countries (1986-2006) used ISH to detect HPV DNA in esophageal squamous cell carcinomas. [24, 32, 35, 38, 46, 70, 71, 72, 73, 74] Worldwide, 25.3% (499/1973) of carcinomas had detectable HPV DNA. The vast majority of the carcinomas examined were from China, where 24.1% (403/1673) were HPV-positive.

The table shown on page 8 lists the data of 58 international studies (1992 to 2007) that used PCR to detect HPV DNA in esophageal squamous cell carcinomas. [32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69] In short, HPV DNA was detected by various PCR methods in 21.4% (757/3542) of squamous cell carcinomas of the esophagus worldwide. High-risk HPV16 was the most common type detected, followed by high-risk HPV18. These data may be somewhat skewed, however, because some of the PCR methods employed HPV16- and HPV18-specific E6/E7 primers that detected these types exclusively. Nevertheless, PCR studies that used consensus primers also showed a similar trend with the exception that additional high-risk, and occasionally low-risk, HPV types were also detected. Interestingly, regional variations in the HPV16/HPV18 ratio were noted. For example, all 15 Chilean patients with SCC in one study [67] were HPV18 positive, and all 10 Native Alaskans with SCC in a separate study [69] were HPV16 positive.

The highest prevalence of HPV-positive cases occurred in Korea (67%), Chile (57.7%), Greece (56.7%), Egypt (54%), Native Alaskans (45.5%), South Africa (36.8%), and Hungary (35.7%). The number of carcinomas in these countries, however, constituted only 7.5% (265-3542) of the worldwide cases examined. Of note, only 14% (97/691) of esophageal squamous cell carcinomas from Europe were HPV-positive. Greece, Hungary, and Germany accounted for 62.9% (61/97) of the HPV-positive cases, but only 23.6% (163/691) of the total cases analyzed. Conversely, Belgium, France, Holland, Italy, Portugal, Slovenia, and the UK combined for 37.1% of the positive cases and 76.4% of the total cases examined.

Five Asian countries accounted for 72.9% (552/757) of the HPV-positive squamous cell carcinomas worldwide and 67.3% (2385/3542) of the total worldwide cases examined. Eighteen PCR studies from China reported 23% (337/1467) HPV-positive carcinomas. [This incidence is remarkably similar to the 24.1% incidence of Chinese carcinomas that were HPV-positive by ISH]. Although most of these PCR studies were performed on patients who lived in Chinese provinces known to have a high incidence of esophageal squamous cell carcinoma, there was striking variation in the HPV-positivity rates from different high-incidence areas that ranged from 2% [45] to 80%. [46]

Although some investigators have attributed these regional differences to variations in HPV testing methods between studies, they could also be explained by the effects of diverse environmental influences (perhaps acting in synergy) that are peculiar to the development of carcinoma in individual geographic areas. A number of studies have tried to answer this question by using one PCR method to analyze squamous cell carcinomas (ESCC) from multiple Chinese communities that have different age-adjusted mortality rates for this disease.

For example, Shumaya et al [48] used SPF10 L1 PCR to analyze 26 ESCC from high-incidence Gansu (age-adjusted mortality 29/10 [5]) and 33 ESCC from low-incidence Shandong (4/10 [5]). In Gansu, 65% (17/26) of the carcinomas were HPV-positive (12 HPV16; 1 HPV16-18 co-infection; 1 HPV16-51 co-infection; 1 HPV18; 2 undetermined type) and in Shandong only 6.1% (2/33) were HPV positive (1HPV6-16 co-infection;1 HPV18). The HPV DNA in all HPV16-positive cancers was integrated. In a separate study, Li et al [75] used balloon cytology of the esophagus to screen volunteers from the high risk Anyang area of China: 138 volunteers from a very high-incidence village (132/10 [5]); 68 volunteers from a high-incidence village (52/10 [5]). Samples of normal, mildly-dysplastic, severely dysplastic, and carcinomatous tissue were analyzed by PCR using HPV16- / HPV18-specific E6 primers and HPV16-specific E7 primers. In the very high-incidence village, 72% (99/138) of the donors had HPV16-positive tissues. In the high-incidence village, 37% (25/68) of the donors had HPV16-positive tissue. One could conclude from these studies that HPV16 might be an important infectious factor in esophageal carcinogenesis in high-incidence areas of Gansu and Anyang, China.

In contrast, Si et al [45] used consensus MY09/11 L1 PCR to analyze 319 carcinomas from 5 separate Chinese provinces with different age-adjusted mortality rates: Linxian (60/10 [5]); Xi-an (47/10 [5]); Sichaun (30/10 [5]); Shangtou (28/10 [5]); and Hong Kong (13/10 [5]). The HPV-positivity rates were surprising: Linxian (1 of 49: 2%); Xi-an (10 of 57: 17.5%); Sichaun (19 of 100: 19%); Shangtou (4 of18: 22%); and Hong Kong (9 of 95: 9.5%). This study supports the hypothesis that environmental factors other than HPV may play a predominant role in esophageal carcinogenesis in Linxian.

Interestingly, Gao et al [12] performed a screening cytology study of 740 adult subjects in Linxian. Digene Hybrid Capture ®2 was used to detect 13 high-risk HPV types in esophageal balloon cytology samples from 702 of the 740 test subjects (475 -- no dysplasia; 102 -- mild dysplasia; 83 -- moderate dysplasia; 38 -- severe dysplasia; 4 ESCC). These authors reported their results using 3 different HPV DNA positivity cutpoints (≥ 1.0 pg/ml; ≥ 2.0 pg/ml; and ≥ 3.0 pg/ml). When using the detection threshold "≥ 1.0 pg/ml" common to cervical cytopathology applications, 47% (327/702) of the cytology specimens were positive for high-risk HPV. This includes 2 of 4 squamous cell carcinomas, neither of which were HPV-positive using the higher cutpoints. These data suggest that many residents of Linxian, China harbor ≥ 1.0 pg/ml / ≤ 2.0 pg/ml of high-risk HPV DNA in their esophageal mucosa. Cross-examination of the results of this HC2 study with the data of the PCR study by Si et al, however, suggests that the association of HPV infection of the esophagus with the subsequent risk of progression to esophageal squamous cell carcinoma in Linxian is debatable.

As confusing as these studies may be, the weight of the molecular evidence supports some role for HPV in esophageal carcinogenesis. Specifically, a significant percentage of squamous cell carcinomas of the esophagus harbor HPV DNA. High-risk HPV16 and HPV18 have been the predominate types detected. Moreover, some of the HPV-positive cancers show integration of the HPV genome. [74] Balloon cytology screening studies for esophageal neoplasia have also detected HPV DNA in normal esophageal mucosa, mild dysplasia, severe dysplasia, and early cancers in high-incidence areas of China. [12, 75] The relative influence that HPV may have in esophageal carcinogenesis with respect to other environmental risk factors, however, remains controversial.

Future Trends
It is difficult to predict the future of any field of research that relies heavily on molecular techniques. I suspect that Hybrid Capture ®2, or some other iteration of signal amplification, will play a major role in HPV detection in fresh clinical specimens. This technology is semi-automated and does not require specialized laboratories or highly-trained personnel to produce accurate results.

Target amplification technologies such as PCR will probably be the tools of choice for HPV research. Before we can easily compare the data from different PCR studies, however, much work needs to done internationally to standardize and validate these methods. Compared to signal amplification methods, PCR has increased sensitivity, can be performed on formalin-fixed paraffin-embedded tissue, and can identify individual or multiple HPV genotypes (co-infections) after amplification. I believe we can expect more RT-PCR studies to determine the relationship, if any, of HPV viral load to the risk of neoplastic transformation. Researchers may also profile gene expression within esophageal carcinomas using microarrays, and then analyze these data separately for HPV-positive and HPV-negative tumors.

Finally, there will be considerable impetus to develop and administer efficacious and inexpensive vaccines worldwide. To be successful, vaccination programs will have to overcome potential political and economic obstacles. Even if effective and widely implemented, the impact of any vaccine will not be fully appreciated for a couple of decades.

HPV PCR of SCC of the Esophagus (1992 to 2007)

HPV+ / n Cases (%)
Africa
Egypt [33] 27/50 (54.0)
S. Africa [32, 34] 21/57 (36.8)
Total 48/107 (44.9)
Asia
China [32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] 337/1467 (23.0)
India [32, 49] 79/242 (32.2)
Iran [50, 51] 47/178 (26.4)
Japan [32, 42, 52, 53, 54, 55] 73/474 (15.4)
Korea [32] 16/24 (67.0)
Total 552/2385 (23.1)
Europe
Belgium [32] 1/21 (04.8)
France [56] 0/75 (00.0)
Germany [57, 58] 24/77 (31.2)
Greece [59] 17/30 (56.7)
Holland [60] 0/63 (00.0)
Hungary [61] 20/56 (35.7)
Italy [32, 62, 63] 8/80 (10.0)
Portugal [32] 9/16 (05.6)
Slovenia [32, 64] 2/141 (01.4)
Sweden [32, 65] 16/110 (14.5)
UK [32] 0/22 (00.0)
Total 97/691 (14.0)
South America
Brazil [66] 26/165 (15.8)
Chile [67] 15/26 (57.7)
Columbia [67] 6/47 (12.8)
Total 47/238 (19.7)
USA
Lower 48 [38, 68] 3/99 (03.0)
Native Alaska [69] 10/22 (45.5)
Total 13/121 (10.7)
Worldwide Total 757/3542 (21.4)

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