Impact of the Identification of Nonhuman Genetic Signatures in the Diagnosis and Management of Carcinoma of Unknown Primary

Authors:
Arantzazu Barquín García Gynecological, Genitourinary, and Skin Cancer Unit, HM CIOCC Madrid (Centro Integral Oncológico Clara Campal), Hospital Universitario HM Sanchinarro, HM Hospitales, Madrid, Spain
Institute of Applied Molecular Medicine (IMMA), Department of Basic Medical Sciences, Facultad de Medicina, Universidad San Pablo CEU, CEU Universities, Urbanización Montepríncipe, Madrid, Spain
Institute of Applied Molecular Medicine (IMMA), Department of Basic Medical Sciences, Facultad de Medicina, Universidad San Pablo CEU, CEU Universities, Urbanización Montepríncipe, Madrid, Spain

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Sara Palacios-Zambrano Department of Pathology-Therapeutic Target Laboratory, Hospital Universitario HM Sanchinarro, Madrid, Spain

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Felipe Lozano Alarcón Department of Pathology-Therapeutic Target Laboratory, Hospital Universitario HM Sanchinarro, Madrid, Spain

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Beatriz Paumard-Hernández Laboratorio de Genética Molecular AbaCid, Hospital Universitario HM Sanchinarro, Madrid, Spain

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Miguel Quiralte Pulido Gynecological, Genitourinary, and Skin Cancer Unit, HM CIOCC Madrid (Centro Integral Oncológico Clara Campal), Hospital Universitario HM Sanchinarro, HM Hospitales, Madrid, Spain
Institute of Applied Molecular Medicine (IMMA), Department of Basic Medical Sciences, Facultad de Medicina, Universidad San Pablo CEU, CEU Universities, Urbanización Montepríncipe, Madrid, Spain

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Paloma Navarro Gynecological, Genitourinary, and Skin Cancer Unit, HM CIOCC Madrid (Centro Integral Oncológico Clara Campal), Hospital Universitario HM Sanchinarro, HM Hospitales, Madrid, Spain
Institute of Applied Molecular Medicine (IMMA), Department of Basic Medical Sciences, Facultad de Medicina, Universidad San Pablo CEU, CEU Universities, Urbanización Montepríncipe, Madrid, Spain

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Laura Rodríguez Laboratorio de Genética Molecular AbaCid, Hospital Universitario HM Sanchinarro, Madrid, Spain

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Isabel Salas Villar Department of Pathology-Therapeutic Target Laboratory, Hospital Universitario HM Sanchinarro, Madrid, Spain

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Jesús García-Donas Gynecological, Genitourinary, and Skin Cancer Unit, HM CIOCC Madrid (Centro Integral Oncológico Clara Campal), Hospital Universitario HM Sanchinarro, HM Hospitales, Madrid, Spain
Institute of Applied Molecular Medicine (IMMA), Department of Basic Medical Sciences, Facultad de Medicina, Universidad San Pablo CEU, CEU Universities, Urbanización Montepríncipe, Madrid, Spain

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This report presents the case of a 62-year-old woman who was diagnosed in 1999 with stage I cervical carcinoma treated by surgical resection. In 2021, she presented to the emergency department with a complaint of predominantly right-sided lower back pain. A CT scan of the lumbosacral region revealed a bone lesion in the L5 vertebra and retroperitoneal lymphadenopathies suggestive of malignancy. Histology of the L5 vertebra biopsy showed a poorly differentiated carcinoma with an inconclusive immunophenotypic profile. Treatment for carcinoma of unknown primary was started with a combination of carboplatin and paclitaxel every 21 days. A genomic study of the biopsy specimen performed on the FoundationOne CDx platform identified a nonhuman genetic signature compatible with HPV. The presence of HPV 18 DNA in the specimen was confirmed by PCR-reverse dot blot, and the immunophenotypic profile was expanded, revealing strong and diffuse p16 expression, thus corroborating the molecular findings. In view of these findings, the case was reclassified as a recurrence of the cervical adenocarcinoma that had been diagnosed and treated 23 years earlier. Based on the new results, and according to first-line cervical carcinoma protocols, bevacizumab at 15 mg/kg every 21 days was added to her chemotherapy regimen. The identification of HPV DNA sequences by next-generation sequencing facilitated the correct diagnosis and led to a modification of the first-line therapeutic approach.

Sequencing techniques have evolved significantly since the genetic information of an organism (the bacteriophage phi X174) was first sequenced by Frederick Sanger and colleagues in 1974.1 Technological and practical improvements have been introduced, and next-generation sequencing (NGS) techniques are now being incorporated into daily clinical practice.2 One of the factors that has helped the implementation of NGS has been the decline in the cost of sequencing. For example, sequencing a megabase in 2008 cost approximately $8,000, but by 2013, it cost $0.10.2,3

Carcinoma of unknown primary (CUP) is one of the greatest challenges for clinicians.4 Almost all tumors of this type (90%) are initially categorized as adenocarcinomas based on their morphologic characteristics. The remaining 10% of cases are identified as melanomas, sarcomas, squamous cell carcinomas, undifferentiated neoplasms, or lymphomas.5,6 The difficulties in diagnosing this entity and their resistance to treatment compared with other types of tumors mean that therapeutic options are scarce and nonspecific.7

The incorporation of NGS into health care practice has required the creation of multidisciplinary teams that include not only doctors, pathologists, and clinicians but also biologists and genetic experts who can integrate and interpret morphologic and molecular information. The results, although complex, answer diagnostic, prognostic, and predictive questions that help treat patients and offer them a better quality of life.8

Clinical Case

Our patient was a 62-year-old former smoker, multiparous, not using hormonal contraceptives, and had an untreated multinodular goiter. In 1999, she was diagnosed with microinvasive endocervical adenocarcinoma (8 × 1.3 mm) via conization and treated with a vaginal hysterectomy during which an epidermoid carcinoma in situ was also identified. The patient has been monitored since then.

In November 2021, she presented to the emergency department with predominantly right-sided lower back pain, radiating to the posterior and lateral side of the right lower limb and worsening depending on her position. Physical examination found a previously identified goiter, soft abdomen that was not tender to palpation, and difficulty with dorsiflexion of the right foot. A CT scan of the lumbosacral region conducted in the emergency department revealed a bone lesion in the L5 vertebra and retroperitoneal lymphadenopathies suggestive of malignancy. She was referred to the medical oncology department to complete the study, which included lumbosacral MRI, pelvic MRI, and PET/CT. These studies confirmed retroperitoneal lesions consistent with pathologic lymphadenopathies and identified a tumor mass adjacent to the right iliac psoas, in addition to bone metastases in the sacral bone and what appeared to be secondary lung lesions, along with right hydronephrosis. Given these observations, a bone biopsy of the L5 vertebra was performed. The histologic study showed a solid epithelial mass consisting of atypical cells with pleomorphic and hyperchromatic nuclei (Figure 1A), positive for immunohistochemical markers CKAE1/AE3 (Figure 1B), CK7, and GATA3, but negative for the expression of CK20, PAX8, S100, TTF1, calretinin, and estrogen and progesterone receptors. These findings led to a final diagnosis of poorly differentiated carcinoma with inconclusive immunophenotypic profile.

Figure 1.
Figure 1.

(A) Hematoxylin-eosin and (B) immunohistochemistry staining for CKAE1/AE3 (original magnification x100 for both).

Citation: Journal of the National Comprehensive Cancer Network 22, 1D; 10.6004/jnccn.2023.7079

In view of these results, the extension study was completed with bilateral mammography and ultrasound that ruled out malignancy, and cystoscopy with biopsy by transurethral resection; pathology analysis also ruled out malignancy. With these results, breast and urothelial carcinoma were excluded.

After the patient was diagnosed with a stage IV tumor of unknown primary, first-line chemotherapy treatment was started with carboplatin at area under the curve 5 and paclitaxel at 175 mg/m2 every 21 days. The first cycle was administered on March 10, 2022.

A parallel genomic study of the bone tumor biopsy was conducted on the Roche NGS FoundationOne CDx (F1CDx) platform,9 revealing the gene alterations shown in Table 1.

Table 1.

Summary of NGS Results

Table 1.

F1CDx also identified a signature unrelated to the patient genetic background. Bioinformatic algorithms pointed to the presence of DNA sequences of HPV. Because detection of HPV using this procedure is not clinically approved, the suspected result had to be confirmed using another validated technique. We therefore requested PCR-reverse dot blot for the detection and typing of HPV in the patient’s tumor tissue, confirming the presence of HPV genotype 18 DNA sequences (data not shown).

The immunophenotypic profile was also expanded, revealing strong diffuse p16 expression, corroborating and supporting the molecular findings (Figure 2).

Figure 2.
Figure 2.

Immunohistochemical staining for p16 (original magnification x200).

Citation: Journal of the National Comprehensive Cancer Network 22, 1D; 10.6004/jnccn.2023.7079

These findings led to the case being reclassified as a recurrence of the cervical adenocarcinoma that had been diagnosed and treated 23 years earlier.

Based on the new results, bevacizumab was prescribed at a dose of 15 mg/kg every 21 days, in line with standard treatment protocols for advanced cervical carcinoma. At the time of writing, the patient had completed 6 cycles of carboplatin with paclitaxel, 4 of which were administered concomitantly with bevacizumab, and had experienced a partial response. She is currently continuing with bevacizumab monotherapy as maintenance treatment.

Discussion

This report presents the case of a 62-year-old woman with an initial diagnosis of advanced adenocarcinoma of unknown primary. NGS techniques helped identify the presence of genetic material typical of HPV. The diagnostic confirmation by PCR-reverse dot blot and immunohistochemistry for p16 led to the case being reclassified as a recurrence of cervical carcinoma that had been diagnosed and treated 23 years earlier. The treatment regimen was modified accordingly.

CUP is a relatively common clinical entity, accounting for approximately 2% of all invasive cancers. Within this category, tumors from many primary sites with varying biology are represented. This heterogeneity has made the design and interpretation of clinical studies difficult. The tests that are successful in identifying a primary site in most patients with advanced cancer include history, physical examination (including pelvic examination in females), complete blood counts, serum chemistries, urinalysis, tumor markers, CT scans, mammography, and, furthermore, focused evaluation of specific signs and symptoms. These procedures identify a primary site in 10% to 35% of patients.10 Additionally, PET scans may be useful in specific clinical settings, and endoscopic studies should be performed in the presence of abdominal symptoms. Finally, the most important improvements to the evaluation of CUP are diagnostic tests performed on biopsy tissue, including specialized pathologic testing to refine the initial histologic diagnosis through an exhaustive immunohistochemistry staining workup and molecular cancer classifier assays that may use site-specific gene expression profiles to identify the site of origin.

When considering treatment options, it is important to note that approximately 60% of CUPs do not fit into any specific origin.11 For these patients, empirical chemotherapy has been considered the standard treatment. Such treatment options include combinations containing a platinum agent plus other cytotoxic agents (taxanes, gemcitabine, irinotecan), with median survivals of 7 to 10 months and 2-year survival rates of 20% to 25%.12 Moreover, comprehensive molecular profiling may identify molecular alterations for which specific targeted therapy has proven efficacious. In addition, therapies targeting several molecular abnormalities (eg, high levels of microsatellite instability [MSI], tumor mutational burden, TRK fusions) have tumor type–agnostic available treatments. Most importantly, NGS-driven clinical trials can be offered to these patients.

Advances in the knowledge and interpretation of the results of modern NGS platforms have facilitated not only the identification of specific mutations of interest but also the detection of gene fusions/translocations and changes in the number of copies or patterns of DNA damage, such as those caused by defects in repair systems, including mismatch repair genes or homologous recombination.13 Moreover, in 2023, the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Occult Primary were updated to recommend consideration of NGS in the initial workup.10

In our case, the FoundationOne CDx molecular panel was used to analyze tumor DNA and simultaneously detect exogenous DNA from the patient’s specimen.14 When the exogenous DNA is of human origin, any alterations found are filtered until a certain percentage of contamination is reached during the secondary analysis of the sequence. If their genetic characteristics are not of human origin, the molecular panel uses its algorithms and sequences to identify the genetic characteristics of certain viruses, including HPV. HPV is integrated into characteristic regions of the genome and is also detected by analyzing the sample using mutational tumor burden and MSI algorithms.13,15

Cervical carcinoma is the fourth most common tumor in women and the fourth most frequent cause of cancer death among women, with 570,000 cases and 311,000 deaths in 2018 worldwide.16 It is also the most diagnosed tumor in 28 countries and the one that causes the highest mortality in 45 countries.16

HPV causes approximately 5% of all cancers.17 It is the virtually necessary, but not sufficient, cause of cervical carcinoma, with a prevalence of up to 99.7% in samples18; genotypes 16 and 18 cause 70% of this disease type.18,19 Other factors that determine the development of cervical carcinoma include smoking, high parity, continued use of hormonal contraceptives, promiscuity, and coinfection by HIV.20,21 The first 2 are the most important in mediating the progression of early lesions to precancerous and cancerous states.21,22

One of the characteristics of tumors derived from HPV infection is that their gene signatures are different from tumors derived from other causes, such as squamous cell carcinomas of the head and neck. It has been shown that tumors derived from HPV infection mainly present helicase domain mutations of PIK3CA, loss of TRAF3, and amplifications of the E2F1 gene.23 Gene signatures derived from other causes, such as tobacco smoke, show loss of TP53 function, inactivation of CDKN2A, and frequent alterations in the number of copies of 3q26/28 and 11q13/22, including amplifications.23

The incorporation of genetic signatures such as MSI, tumor mutational burden, and homologous repair deficiencies represented a step forward in the development of NGS for clinical use.24 Although currently under evaluation by the FDA, the identification of DNA signatures not related to the patient could also be a relevant innovation. This information could help detect contamination with external DNA or the presence of genetic sequences of oncovirus, such as HPV.25 Thus, maximizing the information obtained by NGS platforms could lead to a wider adoption of this technology and the real implementation of precision medicine in daily practice.

One of the mutations detected in the tumor sample analyzed by NGS was NRAS p.G12D. This activating mutation is frequently found in nonsquamous cell cervical cancer (8.2% vs 2.2% in squamous cell tumors; P<.001) and is significantly associated with HPV18 infection (P<.003).26 Jiang et al26 used multivariate analysis to demonstrate that NRAS p.G12D is a prognostic marker for relapse (P=.019) and found that patients with tumors with the NRAS p.G12D mutation showed a higher percentage of relapses compared with those without the mutation (42.9% vs 20.0%; P=.007).

Another protein detected in the analysis of our patient’s samples was p16. Expression of p16 in cervical carcinoma is a surrogate marker of HPV infection; strong and diffuse staining is consistent with HPV-positive tumors, and negative staining is indicative of tumors with no HPV involvement.27,28 In prognostic terms, p16 expression has been associated with increased survival.27 In contrast, tumors negative for p16 expression are associated with older patients, more advanced disease, and a worse prognosis.27

Results of the NGS panel determined that the tumor was a recurrence of the previously treated tumor, and because of this, the treatment regimen could be modified accordingly. Bevacizumab has become the standard treatment for cervical cancers. In a phase III, randomized, multicenter trial that included 442 patients from 3 countries, a significant improvement in overall survival of 16.8 months was observed in the group treated with the combination of chemotherapy plus this antiangiogenic drug, compared with 13.3 months in the chemotherapy-alone group (P=.007). Furthermore, in patients who had not received pelvic radiation therapy, overall survival in the chemotherapy plus bevacizumab group was 24.5 months, compared with 16.8 months in the non-bevacizumab group (P=.11). Although more fistulas of any grade were observed in the bevacizumab group, they did not result in medical emergency, sepsis, or death.29

Conclusions

The interpretation algorithms of modern NGS platforms offer additional information beyond point mutations. In our case, the identification of HPV DNA sequences led to correct diagnosis and adaptation of the patient’s therapeutic regimen.

Acknowledgments

Medical writing support was provided under the guidance of the authors by Antoni Torres-Collado, PhD, and Javier Arranz-Nicolás, PhD, from Medical Statistics Consulting (MSC), Valencia, Spain, and was funded by Roche Spain, Madrid, Spain, in accordance with Good Publication Practice (GPP3) guidelines.

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Submitted May 11, 2023; final revision received August 31, 2023; accepted for publication August 31, 2023. Published online December 27, 2023.

Disclosures: Dr. García-Donás has disclosed receiving grant/research support from Roche Laboratories, Inc.; and serving on a speakers’ bureau for Roche Laboratories, Inc. The remaining authors have disclosed that they have not received any financial consideration from any person or organization to support the preparation, analysis, results, or discussion of this article.

Correspondence: Jesús García-Donás, PhD, MD, HM CIOCC Madrid, Hospital Universitario HM Sanchinarro, HM Hospitales, Calle de Oña, 10, 28050 Madrid, Spain. Email: jgarciadonas@hmhospitales.com
  • Collapse
  • Expand
  • Figure 1.

    (A) Hematoxylin-eosin and (B) immunohistochemistry staining for CKAE1/AE3 (original magnification x100 for both).

  • Figure 2.

    Immunohistochemical staining for p16 (original magnification x200).

  • 1.

    Sanger F, Air GM, Barrell BG, et al. Nucleotide sequence of bacteriophage phi X174 DNA. Nature 1977;265:687695.

  • 2.

    Gagan J, Van Allen EM. Next-generation sequencing to guide cancer therapy. Genome Med 2015;7:80.

  • 3.

    Barba M, Czosnek H, Hadidi A. Historical perspective, development and applications of next-generation sequencing in plant virology. Viruses 2014;6:106136.

  • 4.

    Subbiah IM, Tsimberidou A, Subbiah V, et al. Next generation sequencing of carcinoma of unknown primary reveals novel combinatorial strategies in a heterogeneous mutational landscape. Oncoscience 2017;4:4756.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5.

    Pavlidis N, Fizazi K. Carcinoma of unknown primary (CUP). Crit Rev Oncol Hematol 2009;69:271278.

  • 6.

    Hainsworth JD, Fizazi K. Treatment for patients with unknown primary cancer and favorable prognostic factors. Semin Oncol 2009;36:4451.

  • 7.

    Qaseem A, Usman N, Jayaraj JS, et al. Cancer of unknown primary: a review on clinical guidelines in the development and targeted management of patients with the unknown primary site. Cureus 2019;11:e5552.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 8.

    Angerilli V, Galuppini F, Pagni F, et al. The role of the pathologist in the next-generation era of tumor molecular characterization. Diagnostics (Basel) 2021;11:339.

  • 9.

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    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10.

    Ettinger DS, Stevenson MM, Ahn D, et al. NCCN Clinical Practice Guidelines in Oncology: Occult Primary. Version 2.2023. Accessed November 1, 2022. To view the most recent version, visit https://www.nccn.org

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11.

    Blaszyk H, Hartmann A, Bjornsson J. Cancer of unknown primary: clinicopathologic correlations. APMIS 2003;111:10891094.

  • 12.

    Lee J, Hahn S, Kim DW, et al. Evaluation of survival benefits by platinums and taxanes for an unfavourable subset of carcinoma of unknown primary: a systematic review and meta-analysis. Br J Cancer 2013;108:3948.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13.

    Chung CH, Guthrie VB, Masica DL, et al. Genomic alterations in head and neck squamous cell carcinoma determined by cancer gene-targeted sequencing. Ann Oncol 2015;26:12161223.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14.

    Lechner M, Frampton GM, Fenton T, et al. Targeted next-generation sequencing of head and neck squamous cell carcinoma identifies novel genetic alterations in HPV1 and HPV– tumors. Genome Med 2013;5:49.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15.

    Oyervides-Muñoz MA, Pérez-Maya AA, Rodríguez-Gutiérrez HF, et al. Understanding the HPV integration and its progression to cervical cancer. Infect Genet Evol 2018;61:134144.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16.

    Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394424.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17.

    de Martel C, Plummer M, Vignat J, et al. Worldwide burden of cancer attributable to HPV by site, country and HPV type. Int J Cancer 2017;141:664670.

  • 18.

    Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:1219.

  • 19.

    WHO. Cervical cancer. Accessed September 20, 2023. Available at: https://www.who.int/news-room/fact-sheets/detail/cervical-cancer

  • 20.

    Cohen PA, Jhingran A, Oaknin A, et al. Cervical cancer. Lancet 2019;393:169182.

  • 21.

    Castellsagué X, Muñoz N. Chapter 3: cofactors in human papillomavirus carcinogenesis—role of parity, oral contraceptives, and tobacco smoking. J Natl Cancer Inst Monogr 2003:2028.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 22.

    Pista A, de Oliveira CF, Cunha MJ, et al. Risk factors for human papillomavirus infection among women in Portugal: the CLEOPATRE Portugal Study. Int J Gynaecol Obstet 2012;118:112116.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 23.

    Lawrence MS, Sougnez C, Lichtenstein L, et al. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature 2015;517:576582.

  • 24.

    Hill BL, Graf RP, Shah K, et al. Mismatch repair deficiency, next-generation sequencing-based microsatellite instability, and tumor mutational burden as predictive biomarkers for immune checkpoint inhibitor effectiveness in frontline treatment of advanced stage endometrial cancer. Int J Gynecol Cancer 2023;33:504513.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 25.

    Mühr LSA, Guerendiain D, Cuschieri K, et al. Human papillomavirus detection by whole-genome next-generation sequencing: importance of validation and quality assurance procedures. Viruses 2021;13:1323.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 26.

    Jiang W, Xiang L, Pei X, et al. Mutational analysis of KRAS and its clinical implications in cervical cancer patients. J Gynecol Oncol 2018;29:e4.

  • 27.

    da Mata S, Ferreira J, Nicolás I, et al. P16 and HPV genotype significance in HPV-associated cervical cancer—a large cohort of two tertiary referral centers. Int J Mol Sci 2021;22:2294.

    • PubMed
    • Search Google Scholar
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