Immunotherapy Combined With Chemotherapy and Radiotherapy for a Patient With Cancer of Unknown Primary: A Case Report


 Background: Cancer of unknown primary (CUP) is metastatic at diagnosis with unknown primary site, indicating a high degree of malignancy with poor prognosis. The development and application of targeted therapy and Immunotherapy is the current research hotspot, and provide more treatment options for CUP.Case Presentations: A 36-year-old freeworking male presented with pain on the right hip in April 2018. After various examinations, he was diagnosed as CUP. He received chemotherapy, immunotherapy and local radiotherapy in our department. However, the use of radiotherapy after immunotherapy resulted in severe pneumonia. Conclusions: Compared with traditional treatments, immunotherapy is effective treatment with fewer side effects and better patient tolerance. However, treating physicians should continue to pay special attention to the occurrence of side effects, when radiotherapy combined with immunotherapy.


Background
Cancer of unknown primary (CUP) is a mysterious type of cancer. It is metastatic at diagnosis with unknown primary site, indicating a high degree of malignancy with poor prognosis. 1 In a previous study, CUP was diagnosed as a malignant tumor and the metastasis was con rmed through histology. However, despite a comprehensive diagnostic work-up, the primary site was not determined. 2 The development and application of targeted therapy and immunotherapy is the current research hotspot, and provide more treatment options for CUP. Recently, a patient with CUP and multiple systemic metastases was treated using immunotherapy combined with chemotherapy and radiotherapy in our department.

Case Presentations
A 36-year-old freeworking male presented with pain on the right hip in April 2018. Positron emission computed tomography (CT) was performed, demonstrating probable bone metastasis and surrounding soft tissue masses in the pelvis ( Figure 1A) and the upper lobe of the right lung ( Figure 1B). Emission CT of bone was conducted, which revealed bone metastasis. Pelvic puncture of the soft tissue masses in the posterior superior acetabulum and the iliac crest indicated poorly differentiated mucinous adenocarcinoma. Immunohistochemistry con rmed the presence of mucinous adenocarcinoma, with some cells being signet-ringoid, cytokeratin 7 (CK7) (+), CK19 (+), thyroid transcription factor-1 (−), anaplastic lymphoma kinase-CST (−), CK20 (−), caudal-related homeodomain transcription 2 (CDX2) (−), and Napsin A (−). Pathological consultation at the Fudan University Shanghai Cancer Center indicated that the tumors were derived from the digestive tract. However, following gastroscopic and colonoscopic examination, the primary site remained unknown. The patient was subjected to immune-related gene testing. The results showed programmed cell death 1 ligand 1 (PD-L1) positivity (25%), the tumor mutational burden (10.48%), and mutation abundance of exon 5 in TP53 (49.49%). Therefore, the patient was diagnosed with CUP.
In May 2018, the patient received palliative care to alleviate the pain and special conformal radiotherapy with DT40GY/20F for the bone metastasis and surrounding soft tissue mass ( Figure 1A). Subsequently (June 2018), he received chemotherapy with "XELOX" regimen (i.e., capecitabine 1,500 mg in the morning and 2,000 mg in the evening per os d1-14+oxaliplatin 130 mg/m 2 intravenous d1). The selection of the regimen was based on recommendations pertaining to rst-line chemotherapy for the treatment of advanced tumors of the digestive tract.
During the treatment period, the pain in the left ilium intensi ed. Conformal radiotherapy with DT40GY/20F to the site was initiated on June 15, 2018. The size of the mass on the chest wall was 15×15 mm ( Figure 1C), and the mass in the lung was enlarged ( Figure 1D). These ndings indicted progressive disease. A biopsy for the mass of the chest wall was performed to identify the primary lesion. According to the follow-ups performed between October 2018 and April 2019, the patient had been able to conduct normal daily life activities, with a markedly improved quality of life and absence of additional abnormalities.
However, the patient presented with recurrent cough and hemoptysis on April 18, 2019. Lung CT imaging revealed the presence of new tissues ( Figure 3A). These new tissues detected in the right main bronchus were removed using an endoscope ( Figure 3B). The size of the largest new tissue was 3×1 cm, and the pathological diagnosis suggested hemorrhage and brous exudation ( Figure 3C). The new tissues detected through a second respiratory endoscopy suggested the presence of malignant epithelial tumors of the salivary gland type, based on the morphology with H&E staining, ×40 (Figures 4A) Figure 3D). These ndings indicated progressive disease. After consultation with the Fudan University Shanghai Cancer Center, the use of local radiotherapy was suggested after discontinuation of immunotherapy. In June 2019, the patient received radiotherapy with DT45GY/15F for the lesions in the right lung, and mediastinal and hilar lymph nodes. The treatment alleviated the patient's cough and hemoptysis did not recur. Two weeks later, it was observed that the mass in the lung had decreased in size ( Figure 3E). However, interstitial in ammation occurred after DT36Gy/12F radiotherapy ( Figure 3F). Radiotherapy was discontinued and anti-infective and antiin ammatory treatments were administered. The in ammation in the lung was resolved ( Figure 3G). In the end, this patient died from Recurrent pneumonia in October 2019.

Discussion And Conclusion
The treatment strategy for CUP is challenging. Poorly differentiated neuroendocrine tumors are invariably characterized by aggressive clinical behaviors, and usually associated with a poor prognosis. Typically, CUP responds to chemotherapy (cisplatin and etoposide) with 70−80% overall response rates. 3 Ohta et al. found that patients with CUP who received chemotherapy showed a relatively good prognosis. 4 However, chemotherapy has no impact on overall survival. Notably, gene detection can identify the source of CUP tissues, while another promising strategy involves liquid biopsies that detect circulating tumor DNA in patients with CUP. The detection of gene mutations (e.g., mismatch repair and tumor mutational burden) may help with immunotherapy, and plays an important role in the development of treatment strategies against CUP. 4 Immunosuppressive agents carry considerable potential for the treatment of patients with cancer, such as advanced non-small cell lung cancer (NSCLC) and small cell lung cancer. 5 Programmed cell death protein 1 (PD1) is a member of the immunoglobulin family B7-CD28. It is expressed on monocytes, B cells, activated T cells, and dendritic cells as an immunosuppressive receptor. 6 In healthy organisms, PD1 is mainly expressed in activated immune cells, promoting the maturation of T cells and regulating excessive immune responses through a negative regulatory mechanism. PD-L1 and PD-L2 are ligands for PD1. Over-activation of the PD1/PD-L1 pathway leads to inhibition of the immune response, 7 thereby preventing excessive in ammatory responses. PD-L1 is the major ligand, and its expression in tumor tissues (e.g., NSCLC, breast cancer, melanoma, gastric cancer, renal cancer, etc.) is signi cantly higher than that recorded in normal tissues. [8][9][10][11][12] Blockage of PD1/PD-L1 inhibits the proliferation and viability of T and B cells. It plays an important role in tumor immune escape, and promotes the progression of tumors. Overexpression of PD1 ligands in tumor cells cause weakening or even death of T cells. 13 Therefore, blockage of PD1/PD-L1 enhances the effect of immune cells on tumor cells, and that has got bene ts from clinical trials.
Nivolumab is one of the drugs which block PD1/PD-L1. In 2015, it was reviewed by the Food and Drug Administration (USA) for the treatment of previously treated advanced NSCLC. The Food and Drug Administration (USA) approved nivolumab as the immune checkpoint inhibitor for the treatment of this disease. In a phase 3 clinical trial (CheckMate-017), 51,272 patients with lung squamous cell carcinoma were randomized to receive either nivolumab or platinum and docetaxel. The results showed that nivolumab was signi cantly better than the docetaxel regimen in terms of objective response rate (20% vs. 9%, respectively; P=0.008), and signi cantly prolonged progression-free survival and overall survival.
Clinically, approximately 60% of patients with NSCLC have abnormal PD-L1 expression. 14 However, the correlation between the expression of PD-L1 and prognosis remains unclear. It is recommended to determine the expression of PD-L1 in NSCLC patients without a driver gene mutation. Immunosuppressive therapy or combined chemotherapy with PD-1 is recommended for patients with high PD-1 expression, demonstrating the new direction toward individualized therapy for NSCLC based on gene detection. Therefore, it is emphasized that gene testing should be performed to determine the presence of gene mutations and PD-1 expression in patients prior to the administration of lung cancertargeted drugs and immune drugs. Another phase II study found that PD-L1 expression in patients with lung squamous cell carcinoma was not related to treatment e cacy. After treatment with an anti-PD-L1 agent, partial remission was achieved in 24% of PD-L1-positive patients and 14% of PD-L1-negative patients. Currently, there are many challenges in clinical practice that require urgent attention. For example, the PD-1/PD-L1 monoclonal antibody is not effective in all patients. Therefore, it is especially important to select the population that would bene t the most from this therapy. The CheckMate-227 study showed that the tumor mutational burden may be helpful in predicting the e cacy of immunotherapy. 15 PD-1 immunotherapy may be appropriate for patients carrying TP53 and KRAS mutations. 16 Currently, there is no gold standard for the detection of PD-L1, and it remains unclear whether PD-L1 could become an ideal molecular marker. Nivolumab was listed in China for second-line treatment of NSCLC in 2018. Therefore, there is less data in clinical practice in China. It's a new trail or challenge for this patient in our case report to accept immunotherapy.
Local radiotherapy was also used in this case, resulting in a certain therapeutic effect against CUP. In a case of primary retroperitoneal serous adenocarcinoma, the combination of immunotherapy with radiotherapy decreased the tumor size, and subsequently led to durable disease control. 17 Another research study showed that radiotherapy may improve systemic responses to anti-PD1/PD-L1-directed immune therapy in Merkel cell carcinoma, as well as the immunotherapy has the clarity on abscopal regression. 18 Coincidentally, a case report also suggested that immunotherapy increases the chance of an abscopal effect occurring after radiation therapy in a patient with lung adenocarcinoma treated with atezolizumab and subsequently combined with brain irradiation for the treatment of metastasis. 19 However, unexpectedly, serious interstitial pneumonia in the contralateral lung eld was noted in the present case, which may have been related to the long-term use of immunotherapy. Traditional radiation pneumonitis is usually associated with the range of exposure and dose distribution. However, in this case, the range of interstitial pneumonia was mainly observed in the contralateral lung eld. This suggests that low-dose radiation may be the trigger for the activation of immune pneumonia. Alternatively, the paracrine signaling of the tumor may trigger this immune pneumonia. Radiation induces a bystander response and abscopal effects through a series of biological effects in irradiated cells, or cellular communication with non-irradiated local neighbors or distant cells. 20 This has been adopted by an increasing number of scholars. However, distinguishing between radiation pneumonitis and immune checkpoint blockade-related pneumonitis remains important and challenging. 21 Therefore, the combination of immunotherapy with radiotherapy requires caution, due to the risk of immune in ammation. Further research is warranted to identify optimal approaches for incorporating radiotherapy in combination treatments with respect to dosage, as well as the fractionation and sequencing of therapies. 22 We reported a unique case of CUP with multiple systemic metastases in a 36-year-old male. In patients in whom the primary tumor site cannot be identi ed, apart from conventional chemotherapy and radiotherapy, immunotherapy can also be an effective treatment with fewer side effects and better patient tolerance. Besides, the use of radiotherapy after immunotherapy requires caution, due to the risk of immune in ammation. The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed as a potential con ict of interest.

Funding Not Applicable
Authors' Contributions PW and SD provided this case. QN, KL, and CP wrote, reviewed, and/or modi ed the manuscript. All authors have read and approved the manuscript Ethics Approval and Consent to Participate The study was approved by the Human Ethics Review Committee of Jiangsu Taizhou People's Hospital.

Consent to Publish
Written informed consent to publish this report and the associated medical images was provided by the patient's wife.

Availability of Data and Materials
All datasets generated for this study are included in the manuscript and/or the supplementary les.