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IN THE SPOTLIGHT

Predictive Potential of Head and Neck Squamous Cell Carcinoma Organoids Sarah J. Hill 1 , 2 and Alan D. D’Andrea 1 , 2 , 3

1 Department of Radiation Oncology, Dana-Farber Cancer Institute, Har-vard Medical School, Boston, Massachusetts. 2 Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massa-chusetts. 3 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts. Corresponding Author: Alan D. D’Andrea, Dana-Farber Cancer Institute, 450 Brookline Avenue, HIM 243, Boston, MA 02215. Phone: 617-632-2112; Fax: 617-632-6069; E-mail: alan_dandrea@dfci.harvard.edu Cancer Discov 2019;9:828–30 doi: 10.1158/2159-8290.CD-19-0527 ©2019 American Association for Cancer Research.

Summary: In this issue of Cancer Discovery , Driehuis and colleagues establish culture conditions for the rapid, effi -cient, and long-term expansion of healthy human oral mucosa and head and neck squamous cell carcinoma (HNSCC) tumor organoids. The HNSCC tumor organoids provide a functional platform for analyzing tumor cell phenotype, tumorigenic potential, and drug and radiotherapy response, and they have a potential role in clinical decision-making.

See related article by Driehuis et al., p. 852 (8).

Head and neck squamous cell carcinomas (HNSCC), which arise from the mucosal lining of the oral cavity, pharynx, and larynx, are increasing in incidence worldwide ( 1 ). Risk fac-tors for HNSCC include smoking and alcohol consumption, as well as exposure to human papillomavirus (HPV; ref. 2 ). Analysis of the mutational landscape of HNSCCs ( 3 ) has not revealed druggable targets for the cancer, and current therapy entails, in many cases, a complex combination of surgery, radiation, and chemotherapy. Aggressive treatment, some-times entailing induction chemotherapy ( 4 ), can be curative, but relapse rates may exceed 50%. Immunotherapy has also recently emerged as another modality for HNSCC, and some successful studies with immune checkpoint therapy have been reported.

Given the heterogeneity of HNSCCs, in terms of their drug responsiveness and aggressive potential, there is a vexing need for reliable model systems, which can identify treatment strategies for individual patients. Through the successful development of three-dimensional culture techniques and advanced growth factor supplementation, organoid models have been established recently for a wide range of human epithelial cancers ( 5, 6 ). Organoids are generated from fresh, viable tissue samples from human tumors. These models are faster, easier, and less expensive to generate than patient-derived xenograft (PDX) mouse models. They often uncover clonal heterogeneity of tumors and can be generated without long periods of ex vivo selection. Importantly, organoid cul-tures can be subjected to a wide battery of functional assays, and they can be rapidly tested for their sensitivity to novel

drugs, drug combinations, and drugs given in defi ned tem-poral sequences. In some cases, organoids contain immune cells representative of the microenvironment of the primary tumor, setting the stage for the development and in vitro test-ing of new immunotherapies ( 7 ).

In this issue, Driehuis and colleagues provide the fi rst detailed description of culture conditions for organoids from normal human stratifi ed mucosal lining epithelial cells or from HNSCC tumor cells ( 8 ). The organoid culturing was rapid, with successful passage within 10–14 days of ex vivogrowth, and it exceeded 65% effi ciency. This high success rate for HNSCC, compared with the lower effi ciency of organoid generation from other tumor types such as prostate and pan-creatic cancers, may result in part from the relatively easier access of larger tumor biopsies from head and neck tumors. The rapid outgrowth and high effi ciency of HNSCC organoids, which is also observed for high-grade serous ovarian tumor organoids ( 9 ), may result, at least in part, from their underlying p53 mutations (the most common genetic mutation in these cancers). This intrinsic rapid cellular growth rate for HNSCC or ovarian cancer organoids may allow immune therapy testing in the organoid cultures with the tumor-infi ltrating lympho-cytes from the tumor. Importantly, Driehuis and colleagues were able to subject these organoids to a battery of novel functional assays not used previously in other organoid pub-lications. Among the assays employed, the investigators used mutation and transcriptional profi ling, chromosome stability, xenotransplantation, and novel drug and conventional chemo-therapy/radiation (chemo/RT) testing.

A previous study from another laboratory group ( 10 ) described culture conditions for HNSCC organoids and par-tially characterized the organoids with respect to ex vivo drug sensitivity and tumorigenicity. The new study by Driehuis and colleagues signifi cantly extends this earlier work. It describes robust long-term culture conditions for HNSCC cells in a well-defi ned medium, and shows, by histologic and genetic comparison, that the organoid cells are indeed tumor-derived and not overgrown wild-type cells. Accordingly, the organoids described by Driehuis and colleagues are amenable to a wider range of functional analyses, including but not limited to drug screening, and offer the possibility of predicting drug response of individual patients with HNSCC in the clinic.

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The new study by Driehuis and colleagues describes a bat-tery of novel functional assays as well as novel applications of organoid models. First, the organoids derived from normal mucosal lining cells were used to model herpes simplex virus and HPV infection, providing a clear advantage over previous studies employing immortalized epithelial cell lines. Second, the HNSCC cells from individual patients could be expanded long-term and cryopreserved without loss of cell viability. Third, tumor organoids were exposed to a wide range of clini-cally relevant drugs or radiation treatments, thereby revealing the heterogeneous sensitivities of HNSCC tumor cells from individual patients. In some cases, drug sensitivities corre-lated with specific tumor-derived mutations, thus highlight-ing the potential application of the model system in clinical decision-making. Fourth, in anecdotal cases, the organoid drug responses correlated with the clinical response of 7 patients with HNSCC.

Perhaps the most interesting and novel part of the new work is the observed correlation between the radiotherapy sensitivity of the HNSCC organoids and the clinical response of the corresponding patient with HNSCC. Previous stud-ies have demonstrated that organoids can partially predict the clinical response of metastatic gastrointestinal cancers to chemotherapy or to targeted therapy (11). But this new work by Driehuis and colleagues extends the use of tumor organoids to the prediction of clinical radiotherapy response.

The results also confirm the ability of some drugs such as cisplatin to sensitize tumor cells to radiation.

The work also underscores the importance of testing whole panels of drugs in the organoids, including the conventional drugs for HNSCC such as cisplatin, docetaxel, and fluoroura-cil, as well as experimental targeted agents. Indeed, all of these drugs should be tested, even if there are no obvious genetic markers in the tumor that might indicate whether a targeted drug will be effective. For instance, in one case an organoid responded strongly to a PI3K inhibitor, even though there was no clear activating mutation in the PI3K gene in the orga-noid. Accordingly, in some cases the functional studies may be more informative than the genetic survey.

The authors stress that their study does not claim to prove the predictive potential of HSNCC-derived organoids in guiding therapy. Their study was aimed instead to establish the experimental basis for a future prospective clinical trial, which in itself will be a large endeavor. The mucosal lining organoid culture platform described in the study will allow the derivation and propagation of wild-type and cancer cells from individual patients with HNSCC. Matched, isogenic organoids can also be established from patients in the post-treatment setting, providing there is sufficient viable residual tumor tissue after treatment available for biopsy.

Ideally, the organoid cultures will be adapted as a correla-tive biomarker platform for patients with HNSCC enrolled

Figure 1.  Combination of genomic profiling and a functional organoid platform for HNSCC. As shown, primary or relapsed tumor tissue from patients with HNSCC can be analyzed genetically through DNA sequencing and RNA sequencing. Simultaneously, organoid cultures can be generated for the functional tests and drug screening described in the text. Combining these data may yield an improved predictor of clinical response of individual patients.

Sequencing

Tumor organoids Drug screening

Predictive potential

Sequencing report

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in a large prospective coclinical trial. For instance, patients could be enrolled in a trial using a conventional chemo/RT regimen in which wild-type tissue organoids, pretreatment tumor organoids, and post-treatment tumor organoids are established from their corresponding biopsy sites. Careful dissection of the fresh tissue by a trained pathologist is required to separate the normal tissue from the tumor tissue prior to establishing the organoids. Such a trial would offer several advantages. First, if the trial is successful, it could establish whether the chemo/RT sensitivity of the organoids indeed matches the chemo/RT sensitivity of the patient in the clinic. A scenario in which the pretreatment tumor orga-noid is resistant to the chemo/RT, and the patient exhibits a corresponding clinical resistance to the same combination, could lead to an appropriate change in therapy. Second, the establishment of post-treatment tumor organoids may allow the identification of molecular mechanisms of tumor resist-ance. Depending on the mechanism, these organoids may have an acquired sensitivity to another drug. In this case, post-treatment tumor organoids could provide the rationale for an alternative therapy. The drug-resistant organoids have added value because they will accelerate the development of new drugs and drug combinations.

In summary, Driehuis and colleagues have established the starting point for a prospective trial in which orga-noid chemo/RT response and patient clinical response can be directly compared. Establishing the predictive value of HNSCC organoid testing will require large numbers of patients. Moreover, such a trial must take into account a large number of clinical variables. First, the site of the primary tumor (say, oral cavity, pharynx, or larynx) or stage is likely to influence the functional activity of the corresponding orga-noids. Second, the tumors will have wide-ranging differences in genetic constitution. Accordingly, it is essential for each organoid culture to have genetic profiling, including muta-tional screening and transcriptional analysis. Combining the genetic profile of an organoid with its cellular phenotype (Fig. 1) may allow patients to be divided into smaller subsets which are more or less likely to respond to a specific therapy. Third, many relapsed tumors and even primary tumors are likely to exhibit clonal heterogeneity. Accordingly, multiple tumor biopsies from the same patient may yield organoid cultures with different functional attributes and different drug responses. The likelihood of clonal heterogeneity would mandate the use of combination therapy, which again can

be easily tested with the tumor organoid platform. Despite these caveats, the establishment of an organoid platform for HNSCC is likely to lead to important advances in HNSCC diagnostics and treatment.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsWe are grateful to Dr. Geoffrey Shapiro for critical reading and

helpful discussion.

Published online July 1, 2019.

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2019;9:828-830. Cancer Discov   Sarah J. Hill and Alan D. D'Andrea  Carcinoma OrganoidsPredictive Potential of Head and Neck Squamous Cell

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