At the beginning of the diagnostic process there is a basic diagnostic program, see Table 5.

With this basic diagnosis, a search for common primary tumors, an orienting staging and - if not clinically obvious - the determination of the optimal localization for tissue sampling can be achieved in a short time. Examination of asymptomatic regions beyond the basic program is not useful. Diagnostics repeated during the course do not contribute to primary tumor identification. Only rarely is the primary tumor symptomatic during the course of the disease and thereby identified (<10%).

PET-CT at the beginning of the diagnosis is desirable, but - except for cervical lymph node metastases with unknown primary tumor - not accepted by the health care authorities (GBA) in Germany and also not part of international guidelines [10, 19, 20]. PET-CT allows rapid staging, additional cross-sectional CT and/or MRI examinations can often be omitted, and further diagnostics can be more focused. A recent meta-analysis concluded that therapy-relevant findings are found by PET-CT in one third of patients [25]. Identification of the primary tumor is successful in 18-28%, previously unknown metastases are found in 10-19%.

The ESMO guideline recommends the use of PET-CT additionally in singular metastases or oligometastatic disease, if there is a prospect of local ablative therapy in curative intention with surgery and / or radio(chemo)therapy [20]. This is to exclude possible further metastases before local treatment. Before PET-CT is performed in Germany, an explanatory tumor board decision and, if necessary, an application for cost coverage to the health insurance company is recommended, which should be made with reference to the ESMO guideline and the Onkopedia guideline available here. In most cases the time spent for awaiting the results of this approach is not worthwhile the possible additional information.

Supplementary examinations are performed specifically according to the patient's history, findings, and working diagnosis. Above all, a search should be made for therapeutically relevant, prognostically favorable groups, see Table 7. Further diagnostics in the first stage are based on the medical history, the localization of the tumor manifestations, and histology. Recommendations are summarized in Table 6.

The first diagnostic program is followed by a second level according to tumor presentation and histology including immunohistochemistry. The following flow charts (Figure 1 - 7) are partial results of the CUPISCO study - the first international large prospective study with central pathology review and central expert review of each individual patient before inclusion in the study [28]. These algorithms use the criteria of immunohistochemistry, local lymph node stations, and distant metastases to provide guidance as to whether a malignant mass should be interpreted as a primary tumor or as a metastasis, thus facilitating the differential diagnosis between CUP syndrome and known tumor entities. The result is a much clearer definition of 'true' CUP patients. The algorithms have already been included in the current ESMO guidelines [20]. They are of essential practical importance.

• In women, this represents a defined special case of the CUP syndrome.

• For differentiation of 'true' CUP vs. breast cancer, see Figure 4.

• As a rule, in women diagnosis and therapy as for nodal-positive breast cancer.

Frequency and primary tumor

• In women almost always (>75%), in men occasionally breast cancer as primary tumor (in men often BRCA-associated).

• Otherwise frequently lung cancer, occasionally amelanotic melanoma, malignant lymphoma, soft tissue sarcoma (immunohistochemistry!).

• 5-year survival 50-88%.

Therapy for adeno- or undifferentiated carcinoma [20, 40, 41]

• Axillary lymphadenectomy (level I + II)

  • Mastectomy or lumpectomy is not required.

• (Neo)adjuvant chemotherapy as in nodal-positive breast cancer, see Onkopedia Female Breast Cancer

  • followed by radiotherapy (including the ipsilateral mamma) as after breast-conserving surgery

  • followed by anti-hormonal therapy for receptor-positive tumor

  • additional anti-HER2 therapy for HER2-positive tumor.

Therapy for squamous cell carcinoma

• Axillary lymphadenectomy

• followed by irradiation including the infra- and supraclavicular lymph node regions.

Frequency and primary tumor

• This manifestation is rare; frequently, the primary tumor is identifiable (see Figures 1, 2, and 4).

• Primary tumors are often pulmonary, but thymic carcinoma, breast cancer, or primary mediastinal germ cell tumor should also be considered.

• In some cases, long-term survival after surgery or combined radiochemotherapy has been described.

• By means of immunohistochemistry, a closer assignment of the possible primary tumor is often possible.

• Always search for driver mutations and immunogenicity.

Complementary diagnostics

• PET-CT and bronchoscopy, if required EBUS (endobronchial ultrasound) or transesophageal ultrasound, see Onkopedia Non-Small-Cell Lung Cancer.

Therapy

• Primary radiochemotherapy as for NSCLC, surgical resection including suspicious lung portions, if indicated; followed by radiotherapy/radiochemotherapy.

• If a significant driver mutation or immunogenicity is detected: see chapter 6.1.2.6.

Frequency and primary tumor

• This manifestation is rare, then often due to a locoregional primary tumor (vulva, vagina, cervix, penis, urethra, urinary bladder, anus, lower extremity skin).

Complementary diagnostics

• Dermatologic examination, gynecologic examination, urologic examination, proctoscopy, cystoscopy; histology/immunohistochemistry (exclusion of amelanotic melanoma or soft tissue sarcoma), HPV status in tumor tissue.

Therapy

• Surgical resection

• followed by radiotherapy including the iliac lymphatic drainage pathways; if indicated, as radiochemotherapy with fluoropyrimidine-based chemotherapy.

Frequency and primary tumor

• Identification of the primary tumor is successful in 50-75% of cases. In most cases it is a lung carcinoma, less frequently a breast carcinoma.

Complementary diagnostics

• Craniofacial MRI, PET-CT; biopsy for histology and immunohistochemistry (molecular pathology) mandatory.

Therapy [42, 43, 44]

• Surgical resection, if neurosurgically possible, postoperative radiotherapy of resection bed.

• Gamma knife or stereotactic radiotherapy for unresectable lesions <5cm.

• Whole-brain irradiation only in cases of incomplete resection or numerous foci or according to histologic findings (small-cell neuroendocrine carcinoma).

Frequency and primary tumor

• It is crucial to distinguish this from primary lung cancer, see Figures 1 and Figure 2.

• Solitary lung metastases as the only tumor manifestation are extremely rare, usually multiple lesions are detected.

Therapy

• Atypical resection, frozen section, lobectomy if indicated, systematic lymphadenectomy if required.

• If indicated, radiotherapy or adjuvant chemotherapy, depending on histology and individual case.

Frequency and primary tumor

• Rare, usually multiple lesions.

• In the case of osteolysis, a search should be made in particular for lung and renal cell carcinoma, and in the case of (partially) osteoplastic characteristics, particularly for prostate or breast carcinoma, but also gastric cancer.

Complementary diagnostics

• Histological confirmation! (DD plasmacytoma, Paget's disease, primary bone tumor, etc.)

Therapy

• Surgical resection with curative intention

• followed, if indicated, by radiotherapy.

• In case of unfavorable localization, primary definitive (not palliative!) radiotherapy.

• Depending on histology/histochemistry, immune checkpoint blockade or targeted therapy can be considered [45].

Frequency and primary tumor

• Rare, usually multiple lesions present [35].

• Well-differentiated neuroendocrine tumors may have a favorable prognosis.

Therapy [36]

• Surgical resection or local ablative therapy.

• Palliative chemotherapy, if locoregional therapy is not feasible, see below, Chapter 6.1.3 (disseminated disease).

Is no longer considered as CUP syndrome.

Frequency and primary tumor

• Solitary lymph node metastases of malignant melanoma have a better prognosis with unknown primary tumor than with known primary tumor in several studies [46, 47], but not unequivocally [48].

• A recent comparative genomic analysis shows no significant differences between melanomas with unknown versus known primary [49].

Therapy

• As for malignant melanoma with known primary tumor.

Frequency and primary tumor

• Rare; lung or breast carcinoma most common primary tumor [50].

• Merkel cell carcinoma has received special attention because of its specific and promising treatment options [51, 52]

• This can be clearly diagnosed histologically: neuroendocrine carcinoma with evidence of Merkel cell polyomavirus.

Therapy

• Surgical resection, followed by radiotherapy, if indicated.

• For Merkel cell tumor, immune checkpoint blockade with avelumab or pembrolizumab, if curative resection/radiotherapy is not possible.

Are no longer considered as CUP syndrome.

• Can be reliably differentiated from other carcinomas by immunohistochemistry, see Table 1

• Consultation with the pathologist is essential!

• Attention is required in men, patients < 50 years, poorly differentiated carcinoma, retroperitoneal/mediastinal/pulmonary, rapid progression.

For details, see Onkopedia Germ-cell Cancer (German Version only).

Female serous high-grade carcinoma does no longer count as CUP syndrome.

Criteria

• Female sex, peritoneal carcinomatosis, no other metastases ± malignant pleural/pericardial effusion ± retroperitoneal lymph node metastases.

• Patients with typical histology (serous papillary adenocarcinoma) should be classified as "primary peritoneal adenocarcinoma" rather than CUP syndrome.

• The prognosis is somewhat less favorable than for primary ovarian carcinoma [59].

Therapy:

• As for ovarian cancer, with debulking surgery (if applicable), carboplatin/paclitaxel-based adjuvant or preoperative chemotherapy supplemented with bevacizumab.

In case of histology not compatible with ovarian cancer, especially adenocarcinoma compatible with pancreato-biliary or gastric profile ('upper GI') may present as a 'prognostically unfavorable CUP syndrome'. In some cases, the primary tumor is found in the appendix [60]. For therapy, see chapter 6.1.3 and Figure 8.

Other criteria

• Liver metastases and/or peritoneal carcinomatosis.

Therapy

• As for metastatic colorectal carcinoma, see Onkopedia Colon Cancer and Onkopedia Rectal Cancer, respectively

Other criteria

• For differential diagnosis, refer to algorithm in Figure 6. Pulmonary or skeletal metastases, retroperitoneal lymph node metastases may be present [20].

Therapy

• As for metastatic renal cell carcinoma see Onkopedia Renal Carcinoma.

The therapeutic goal is palliative, i.e., prolongation of life / preservation or improvement of quality of life. The most important criterion for the choice of therapy is the characterization of the tumor biology.

In all patients with a possible therapeutic consequence, molecular testing by NGS (next generation sequencing) for possible driver mutations as well as for immunogenic tumor biology should be performed [20]. In any case, this includes diagnostics for microsatellite instability (MSI-H) or defects in mismatch repair enzymes (dMMR). These defects lead to a very high somatic mutation burden in a wide variety of primary tumors with good response to therapy with immune checkpoint inhibitors such as pembrolizumab. For this substance, a tumor-agnostic approval for first-line therapy is available by both the FDA and the EMA; thus, also for CUP syndrome.

Studies in CUP with regard to other predictors of a possible response to immune checkpoint blockade show a high TMB - tumor mutational burden - as a relevant criterion, the cutoff seems to be 10-12 mutations/megabase [14, 61].

In CUP, small phase II studies with pembrolizumab or with nivolumab as monotherapy [22, 23] show an objective remission rate (ORR) of approximately 20%, PFS of 4 months, and OS of 11-14 months. A phase II study with ipilimumab plus nivolumab as combination therapy shows an ORR of 16% in the overall group. This includes a 60% response with a small fraction of long-term survivors with high TMB (compared to only 7.7% at low TMB). In contrast, PDL1 status (pos. vs. neg.) has no prognostic significance in this study [61].

PDL1 expression (TPS and CPS) can provide an important tool for treatment with chemotherapy plus immune checkpoint blockade, especially in the presence of an immunogenic tumor (lung, gastroesophageal, cholangiocellular, urinary bladder, squamous cell carcinoma, and many others). A cut-off for TPS or CPS has not been determined in CUP. In 2nd- or further-line therapy, the option of immune checkpoint blockade in case of PDL1 expression has to be considered individually.

Therapy

• In case of MSI-H or dMMR: therapy with pembrolizumab

• In case of high tumor mutation burden (TMB-high, >12 mut./Megabase): PD1/PDL1 checkpoint inhibitor +/- CTLA4 inhibitor (note: off-label).

• In case of immunogenic tumor biology with PDL1 expression: consider PD1/PDL1 checkpoint inhibitor in the 2nd line of therapy (note: off-label).

Probably the most exciting current treatment approach is molecular targeted therapy based on genetic alterations of so-called 'druggable targets' on the tumor cells. In this process, molecular profiles of the tumor cells are created - optimally by means of NGS (next generation sequencing) - with the aim of identifying suitable targeted substances (e.g., tyrosine kinase inhibitors). The interpretation of the (often complex) findings takes place in a Molecular Tumor Board (MTB). Based on the recommendation of the MTB, an application for reimbursement of the proposed treatment must usually be written in Germany, which in turn is often approved by the Medical Service.

Genetic alterations are found in more than 85% of cases, while changes that are amenable to currently approved substances are found in about 30-35% [62, 63]. The subset of 'unfavorable CUP' could therefore become significantly smaller than before with consistent NGS testing. Alterations in KRAS, PIK3CA, FGFR (≥ 8% each) are frequent, followed by BRAF and C-ERBB2; alterations in EGFR, MET, KRAS p.G12C, ROS1, NTRK and ALK are less frequent. Case reports and initial studies confirm the efficacy of this targeted approach [77]. The CUPISCO trial with 630 enrolled patients is investigating whether molecular characterization achieves better results than empirical chemotherapy in 'unfavorable CUP'; publication of first results in this regard is expected in the 2nd half of 2023 [21].

Therapy

• As for known primary tumor with corresponding driver mutation.

If no indicators for molecular targeted therapy or immune checkpoint blockade are found, empirical chemotherapy is recommended. The choice of chemotherapy, e.g., combination therapy or monotherapy, should be based on age, comorbidity, LDH, functional status, and patient preferences, analogous to a geriatric assessment.

An overview of the therapy structure is given in Figure 8.

First-line therapy

Currently, empirical chemotherapy continues to be the best-proven treatment. Data are available from several hundred patients treated in trials comparing different chemotherapy regimens. However, a randomized comparison to a best supportive care approach has never been studied in clinical trials. Treatment with carboplatin and paclitaxel has been best documented. Adding an EGFR antibody or a histone deacetylase inhibitor to chemotherapy without a biological marker as a selection criterion has shown no benefit [64, 65].

Empirical chemotherapy options supported by studies include:

• Carboplatin-paclitaxel-based chemotherapy [66, 67].

• Cisplatin - gemcitabine ('French regimen') (cave toxicity) [68]

• Gemcitabine - irinotecan (cave toxicity) [69, 70]

In many cases, treatment analogous to the presumed primary tumor is carried out in practice; but this approach is not evidence-based. It can be useful in individual cases, but it can also reduce the chances of therapeutic success: namely, if not the most therapy-sensitive, but rather the presumed most probable primary tumor is treated and no good treatment options exist for it. For example, the supposedly broadly effective combination capecitabine-oxaliplatin is not effective in first-line CUP even if criteria for a GI primary tumor are present, if the group of colon-typical CUP has been thoroughly excluded (see Chapter 6.1.2.5), PFS was only 2.5 months, OS only 7.5 months in a well-documented prospective study [75].

In two randomized trials and a meta-analysis, aligning therapy with results by genetic profiling using a 92-gene test (CancerType ID) with the aim of better determining the possible primary tumor showed no advantage for 'personalized' therapy oriented to the presumed primary tumor over empirical chemotherapy [18, 71, 72]. Therefore, as a general rule, empirical chemotherapy is preferable due to the better evidence.

Details of the regimens can be found in the German Treatment Protocols Appendix.

Second-line therapy

There are no convincing studies for second-line therapy. The few available studies included small numbers of selected patients. If a driver mutation is detected, the corresponding agent, e.g., a TKI, should be given at the latest in the second line. In case of high tumor mutation burden (TMB-high) or pronounced PDL1 expression, immune checkpoint blockade is reasonable if not already given in the first line. In other cases, an alternative chemotherapy regimen from the first-line treatment options may be reasonable for patents in good general performance. In addition, in the second line, empiric therapy should always be considered based on the working diagnosis.

In squamous cell carcinomas, histology does not allow reliable conclusions with respect to the location of the primary tumor. HPV detection in metastases may point at primary gynecologic malignancies (cervical, vulvar/vaginal cancer), anal or penile cancer or oropharyngeal tumors. In the case of cervical lymph node metastases, a primary tumor in the oropharynx is likely, if HPV is detected, see Chapter 6.1.1.1. In numerous studies on CUP syndrome, squamous cell carcinomas are treated like adeno- and undifferentiated carcinomas, although a statement on the success of this therapy in the case of squamous cell histology is not possible due to its rarity.

Therapy

• Combined radiochemotherapy, e.g., cisplatin/fluoropyrimidine or carboplatin AUC=2 / paclitaxel 50mg/m² weekly, plus immune checkpoint blockade if necessary for PDL1 expression.

• In second-line therapy, an immune checkpoint inhibitor is useful (if not already given in first-line), especially if there is evidence of immunogenic tumor biology.

• The administration of an EGFR antibody should be discussed on a case-by-case basis.

For the therapy structure, see Figure 8.