“We found that ctHPVDNA alone had excellent diagnostic accuracy. When we added physical exam and imaging findings into an algorithm with ctHPVDNA, this maintained high diagnostic accuracy and increased confidence in a non–tissue-based diagnosis.”—Daniel Faden, MD

Key Points

• A prospective, observational study compared a noninvasive approach using liquid biopsies to detect circulating human papillomavirus (HPV) E7 gene DNA from human papillomavirus–associated head and neck squamous cell carcinoma (HPV-HNSCC) with a conventional diagnosis based on tissue sampling by fine-needle aspiration (FNA) and/or surgical biopsies.
• The noninvasive diagnostic strategy was significantly more accurate, was less costly, and allowed a more timely diagnosis.
• The study authors caution that more data are needed before implementing noninvasive plasma circulating tumor human papillomavirus DNA (ctHPVDNA) testing as a clinical tool either as a diagnostic test or for monitoring during and after treatment.

A new prospective, observational study finds that cell-free HPV DNA tests may diagnose HPV-HNSCC more accurately, at lower cost, and more quickly than tissue-based approaches. The study appears in Clinical Cancer Research (doi:10.1158/1078-0432.ccr-21-3151).

According to the researchers, “HPV-associated cancers make up 5% of all cancers worldwide … HPV-HNSCC is the most common HPV-associated malignancy in the United States and continues to increase in incidence.” The goals of the study, says study author Daniel Faden, MD, an assistant professor in the Department of Otolaryngology at Harvard Medical School Massachusetts Eye and Ear, Massachusetts General Hospital, and the Eli and Edythe L. Broad Institute of MIT and Harvard in Boston, Massachusetts, were to compare the diagnostic accuracy of a ctHPVDNA-based diagnosis, combined with routine cross-sectional imaging and a physical examination, with the standard tissue-based clinical workup for the diagnosis of HPV-HNSCC. Additionally, the researchers investigated whether or not ctHPVDNA produced quicker results and lower costs than tissue biopsies using modeling. “This study is the first to evaluate a liquid biopsy-based noninvasive diagnostic approach for HPV-associated head and neck cancer and shows strong proof of principle to support such an approach, disrupting the existing dogma,” says Dr. Faden.

Study Details

The researchers recruited 70 consecutive patients diagnosed with HPV-associated oropharyngeal squamous cell carcinoma, nasopharyngeal carcinoma, or sinonasal squamous cell carcinoma at the Head and Neck Surgical Oncology Clinic at Massachusetts Eye and Ear between January 21, 2020, and March 30, 2021 (minus 4 months because of the coronavirus disease 2019 pandemic). Each candidate was at least 18 years old and agreed to contribute blood samples during a standard-of-care diagnostic workup. Only 61 of these potential participants qualified for the final analysis after 9 were excluded because they had not undergone direct HPV testing and did not have tissue available for testing or because they had already had at least partial treatment. The study's control group consisted of 45 patients who were diagnosed with HPV-negative HNSCC and 25 healthy individuals without cancer.

For the liquid biopsy procedure, 10 to 20 mL of peripheral blood was processed for the collection of ctHPVDNA in plasma. The researchers designed and validated custom droplet digital polymerase chain reaction assays for DNA from HPV E7 genotypes 16, 18, 33, 35, and 45.

The researchers also used modeling methods to compare liquid biopsy–based and tissue-based standard of care regarding the time to diagnosis (diagnostic interval) and the cost of diagnostic procedures (using fee schedules from the Centers for Medicare and Medicaid Services).

Study Results

In 61% (n = 37) of the patients eventually diagnosed with HPV-HNSCC, FNA of a lymph node was the first procedure for tissue sampling (surgical biopsy was the initial procedure for the remaining 39%). A diagnosis of HPV-HNSCC was successful in only 46% of the FNA specimens, in part because cystic necrosis of HPV-HNSCC nodal metastases can result in aspiration of nondiagnostic/inadequate material to perform confirmatory tests needed to make the diagnosis, such as p16 immunohistochemistry. In these cases, it was necessary to perform repeat FNA or surgical biopsy, which increased the cost and the time to diagnosis. In addition to these pre-analytic/specimen adequacy limitations, the researchers noted in their article that the “interpretation of p16 IHC on FNA specimens lacks consensus and is variable across pathologists and institutions.”

The sensitivity and specificity of the noninvasive liquid biopsy approach were 98.4% and 98.6%, respectively, with positive and negative predictive values of 98.4% and 98.6%, respectively. The sensitivity and specificity of a combined approach using ctHPVDNA and criteria based on imaging plus physical examinations were 95.1% and 98.6%, respectively. The accuracy of this noninvasive approach was much higher than that of the traditional standard of care; the Youden indices (defined as sensitivity + specificity – 1) for the 2 approaches were 0.968 and 0.707, respectively (P < .0001.)

Dr. Faden says that because using a standalone liquid biopsy for diagnosing HPV-HNSCC would lead to eventual treatment of that cancer, 100% specificity is necessary. “False positives would not be tolerated,” he says. “We found that ctHPVDNA alone had excellent diagnostic accuracy. When we added physical exam and imaging findings into an algorithm with ctHPVDNA, this maintained high diagnostic accuracy and increased confidence in a non–tissue-based diagnosis.”

The study found that a large reduction in the time to diagnosis—an average of 26 days per patient—may be possible with liquid biopsies. This improvement is due primarily to the fact that scheduling patients for biopsies can add days or even weeks to the diagnosis in addition to the pathology processing time, whereas bloodwork can be obtained often on the same day or even ahead of time. “Importantly, time lag between presentation and treatment has been shown to be an independent predictor or survival in HNSCC,” they wrote.

The researchers' cost modeling suggested that using a noninvasive approach with liquid biopsies may save more than $6000 on average in comparison with an approach based on FNA and/or surgical biopsies.

Study Interpretation

“This study joins a host of others discussing the possibility of incorporating circulating tumor DNA into the diagnosis and monitoring of response to therapy in patients with head and neck cancer,” says Anurag Singh, MD, a professor of oncology and director of radiation research in the Department of Radiation Medicine at the Roswell Park Comprehensive Cancer Center in Buffalo, New York. In his experience, “delays in treatment due to the inability to make a diagnosis of HPV-positive head and neck cancer with routine procedures are rare. Following patients for recurrence after therapy, however, is fraught with significant costs and false positive tests. In this setting, liquid biopsy technology will certainly progress to allow routine follow-up on our patients' response to therapy with liquid biopsies. Ideally, the additional value to our patients would be to allow less onerous and less costly testing with fewer false positives and false negatives.”

“The take-home message here for cancer researchers and clinicians is that a liquid biopsy-based diagnostic approach for HPV-associated head and neck cancer may have improved accuracy, reduced cost, and a shorter time to diagnosis compared to standard clinical workup,” adds Dr. Faden. “These findings suggest that this approach could be a viable alternative to traditional tissue biopsy in the future. The data for using ctHPVDNA detection in HPV-associated head and neck cancer are certainly growing, and I strongly believe that in the near future, ctHPVDNA will be how we diagnose and monitor all HPV-associated head and neck cancer patients. However, we must first generate more data from large, diverse patient populations and clinical settings, and review this data judiciously as a community, before adopting such an approach the standard of care. Specifically, we need more well-designed, prospective clinical trials testing ctHPVDNA compared to existing approaches.”

中文翻译：

---

无创活检可能更快、更有效地诊断 HPV 相关的头颈癌

“我们发现单独的 ctHPVDNA 具有出色的诊断准确性。当我们将体格检查和影像学检查结果添加到 ctHPVDNA 算法中时，这保持了较高的诊断准确性并增加了对非组织诊断的信心。”——Daniel Faden，医学博士

关键点

• 一项前瞻性观察性研究比较了使用液体活检检测人乳头瘤病毒相关头颈部鳞状细胞癌 (HPV-HNSCC) 中循环人乳头瘤病毒 (HPV) E7 基因 DNA 的非侵入性方法与基于精细组织取样的常规诊断方法。针吸 (FNA) 和/或手术活检。
• 无创诊断策略明显更准确，成本更低，并允许更及时的诊断。
• 该研究的作者警告说，在将无创血浆循环肿瘤人乳头瘤病毒 DNA (ctHPVDNA) 检测作为临床工具作为诊断检测或治疗期间和治疗后的监测之前，需要更多的数据。

一项新的前瞻性观察性研究发现，与基于组织的方法相比，无细胞 HPV DNA 检测可以更准确、成本更低、更快地诊断 HPV-HNSCC。该研究发表在临床癌症研究(doi:10.1158/1078-0432.ccr-21-3151)。

据研究人员称，“HPV 相关癌症占全球所有癌症的 5%……HPV-HNSCC 是美国最常见的 HPV 相关恶性肿瘤，并且发病率继续增加。” 研究的目标，研究作者 Daniel Faden 医学博士说，他是哈佛医学院马萨诸塞州眼耳耳鼻喉科、马萨诸塞州总医院以及波士顿麻省理工学院和哈佛大学的 Eli and Edythe L. Broad 研究所的助理教授。 , 马萨诸塞州，将基于 ctHPVDNA 的诊断结合常规横断面成像和体格检查与基于组织的标准临床检查诊断 HPV-HNSCC 的诊断准确性进行比较。此外，研究人员调查了 ctHPVDNA 是否比使用建模的组织活检产生更快的结果和更低的成本。“这项研究是第一个评估基于液体活检的非侵入性诊断方法用于 HPV 相关头颈癌的研究，并显示了支持这种方法的强有力的原则证明，打破了现有的教条，”Faden 博士说。

学习详情

研究人员在 2020 年 1 月 21 日至 2021 年 3 月 30 日期间在马萨诸塞州眼耳部头颈外科肿瘤诊所连续招募了 70 名被诊断患有 HPV 相关口咽鳞状细胞癌、鼻咽癌或鼻窦鳞状细胞癌的患者（减去4 个月，因为 2019 年冠状病毒病大流行）。每个候选人至少 18 岁，并同意在标准护理诊断检查期间提供血液样本。在 9 人之后，这些潜在参与者中只有 61 人有资格进行最终分析，因为他们没有接受直接的 HPV 检测，也没有可用于检测的组织，或者因为他们已经接受了至少部分治疗。研究'

对于液体活检程序，处理 10 至 20 mL 外周血以收集血浆中的 ctHPVDNA。研究人员设计并验证了针对 HPV E7 基因型 16、18、33、35 和 45 的 DNA 的定制液滴数字聚合酶链反应测定法。

研究人员还使用建模方法来比较基于液体活检和基于组织的护理标准关于诊断时间（诊断间隔）和诊断程序成本（使用医疗保险和医疗补助服务中心的收费表）。

研究结果

在最终诊断为 HPV-HNSCC 的 61% (n = 37) 患者中，淋巴结 FNA 是组织取样的第一个程序（其余 39% 的初始程序是手术活检）。HPV-HNSCC 的诊断仅在 46% 的 FNA 标本中成功，部分原因是 HPV-HNSCC 淋巴结转移的囊性坏死可导致吸入非诊断性/不充分的材料来进行诊断所需的确认性测试，例如 p16免疫组织化学。在这些情况下，有必要进行重复 FNA 或手术活检，这增加了诊断的成本和时间。除了这些分析前/标本充分性限制之外，研究人员在他们的文章中指出，“对 FNA 标本 p16 IHC 的解释缺乏共识，并且在病理学家和机构之间存在差异。”

无创液体活检方法的敏感性和特异性分别为 98.4% 和 98.6%，阳性和阴性预测值分别为 98.4% 和 98.6%。使用 ctHPVDNA 和基于影像学加体格检查的标准的联合方法的敏感性和特异性分别为 95.1% 和 98.6%。这种无创方法的准确性远高于传统护理标准；两种方法的约登指数（定义为敏感性 + 特异性 – 1）分别为 0.968 和 0.707（P < .0001。）

Faden 博士说，因为使用独立的液体活检来诊断 HPV-HNSCC 会导致最终治疗该癌症，因此需要 100% 的特异性。“我们不会容忍误报，”他说。“我们发现单独的 ctHPVDNA 具有出色的诊断准确性。当我们将体格检查和影像学检查结果添加到 ctHPVDNA 算法中时，这保持了高诊断准确性并增加了对非组织诊断的信心。”

研究发现，液体活检可能会大大缩短诊断时间——平均每位患者 26 天。这种改进主要是由于安排患者进行活检除了病理处理时间外，还可能会增加几天甚至几周的诊断时间，而血液检查通常可以在同一天甚至提前获得。“重要的是，就诊和治疗之间的时间差已被证明是 HNSCC 的独立预测因子或生存率，”他们写道。

研究人员的成本模型表明，与基于 FNA 和/或手术活检的方法相比，使用液体活检的无创方法平均可以节省 6000 多美元。

学习解释

“这项研究与许多其他人一起讨论了将循环肿瘤 DNA 纳入头颈癌患者对治疗反应的诊断和监测的可能性，”肿瘤学教授兼放射研究主任 Anurag Singh 医学博士说。纽约布法罗罗斯威尔公园综合癌症中心的放射医学系。根据他的经验，“由于无法通过常规程序诊断 HPV 阳性头颈癌而导致治疗延误的情况很少见。然而，在治疗后跟踪患者复发的代价是巨大的成本和假阳性测试。在这种情况下，液体活检技术肯定会进步，以允许对我们的患者对液体活检治疗的反应进行常规随访。理想情况下，

“癌症研究人员和临床医生的重要信息是，与标准临床检查相比，基于液体活检的 HPV 相关头颈癌诊断方法可能会提高准确性、降低成本并缩短诊断时间，”法登博士补充道。“这些发现表明，这种方法在未来可能成为传统组织活检的可行替代方案。在 HPV 相关头颈癌中使用 ctHPVDNA 检测的数据肯定在增长，我坚信在不久的将来，ctHPVDNA 将成为我们诊断和监测所有 HPV 相关头颈癌患者的方式。然而，我们必须首先从大量、多样化的患者群体和临床环境中生成更多数据，并作为一个社区审慎地审查这些数据，然后才能采用这种方法作为护理标准。