Management of Germ Cell Tumours of the Testes in Adult Patients: German Clinical Practice Guideline, PART II – Recommendations for the Treatment of Advanced, Recurrent, and Refractory Disease and Extragonadal and Sex Cord/Stromal Tumours and for the Management of Follow-Up, Toxicity, Quality of Life, Palliative Care, and Supportive Therapy Open Access

We are presenting the main content of the new German clinical practice guideline in a publication series; part I covers the following chapters: epidemiology, classification systems, diagnostics, prognosis, and treatment recommendations for the localized stages [1]. Here, part II covers the recommendations for the metastatic stages, for recurrent and refractory disease, and for follow-up, toxicity management, quality of life, palliative care, and supportive therapy.

The online supplementary German S3 guideline (see www.karger.com/doi/10.1159/000511245 for all online suppl. material) is freely available for download in the German language in both a short and a long version (http://www.awmf.org/leitlinien/detail/ll/043-044.html).

The objectives of this guideline have been described previously in part I [1].

The methods of this guideline have been described previously in part I [1].

See part I for the previous recommendations [1]. See also Tables 1-3 for the dosages and applications.

19In patients with cSIIA seminoma either radiotherapy or chemotherapy with 3 cycles of BEP (bleomycin, etoposide, and cisplatin; alternatively, 4 cycles of EP [etoposide and cisplatin] if bleomycin is contraindicated) is recommended. The lack of prospective randomized controlled trials for the direct comparison of radiotherapy versus chemotherapy for seminoma cSIIA/B does not allow a valid statement regarding the superiority or equivalence in oncological efficacy of either therapy. We recommend informing the patient about the advantages and disadvantages of both treatment options (grade A: very low quality of the evidence) [e2–e6].

20It is recommended that patients with cSIIB seminoma receive chemotherapy with 3 cycles of BEP or, in case of contraindications to bleomycin, 4 cycles of EP. Alternatively, radiotherapy can be performed. The lack of prospective randomized controlled trials for the direct comparison of radiotherapy versus chemotherapy for seminoma cSIIB does not allow a valid statement regarding the superiority or equivalence in oncological efficacy of either therapy. We recommend informing the patient about the advantages and disadvantages of both treatment options (grade A: very low quality of the evidence) [e2, e4, e6].

21Radiotherapy with 30 Gy (total dose) is recommended for patients with cSIIA seminoma and radiotherapy with 36 Gy (total dose) is recommended in stage cSIIB seminoma (A, 2b).

22We recommend performing a control imaging with an abdominal/pelvic CT scan 2–3 months after radiotherapy. It is recommended to proceed equally after chemotherapy. The result of this examination is also the starting point for follow-up care (expert consensus).

23In non-seminomatous cSIIA/B GCT we recommend treatment according to the International Germ Cell Cancer Collaborative Group (IGCCCG) with chemotherapy (3–4 cycles of BEP), and in case of a residual tumour we recommend treatment with residual tumour resection (RTR). Exception: in GCT initially classified as 100% teratoma in the orchiectomy specimen without a marker increase, retroperitoneal lymph node dissection (RPLND) is recommended (A, 2b) [e7, e8].

24Patients with serum tumour marker-negative non-seminomatous cSIIA GCT should be monitored first (serum tumour marker monitoring, repeat imaging after 6–8 weeks) or, alternatively, a diagnostic RPLND should be performed (B, 2b) [e7, e8].

25In patients with serum tumour marker-negative cSIIA non-seminomatous GCT and after primary RPLND with pN1 findings (PS IIA) in the R0 status, active surveillance is recommended (A, 1b). Alternatively, 2 cycles of EP can be administered if there is no pure teratoma (0) [e9–e12, e7].

26Patients after primary RPLND in serum tumour marker-negative cSIIA non-seminomatous GCT with pN2 findings (PS IIB) should be monitored in the R0 status (B, 1b). Alternatively, if there is no pure teratoma, 2 cycles of BEP or EP can be administered (0) [e13, e9, e10, e12, e7].

27The standard regimen of polychemotherapy for metastatic disease is BEP (statement, 1b) [e9].

28Growth factors should be used in BEP as a secondary prophylaxis in case of neutropenic fever in the previous cycle (B, 1b) [e9, e14, e15].

29When applying the VIP regimen (etoposide, ifosfamide, cisplatin), we recommend using a myeloid growth factor (G-CSF) as a primary prophylaxis (A, 1b) [e14, e16].

30In patients with metastatic IIC/III stage GCT of the good prognosis group according to the IGCCCG, we recommend polychemotherapy with 3 cycles of BEP with application of cisplatin and etoposide for 5 days (A, 1b) [e17–e19, e15].

31In patients with acute life-threatening metastatic gonadal GCT, we recommend primarily administering chemotherapy without a prior orchiectomy (expert consensus).

32After completion of chemotherapy in patients with gonadal GCT, we recommend performing an orchiectomy of the initially affected testis if this has been postponed due to acute life-threatening advanced metastatic disease at diagnosis (A, 4) [e20–e23].

33In patients with stage IIC/III metastatic seminoma and a good prognosis we recommend 3 cycles of BEP chemotherapy (A, 1a) [e24].

34In patients with stage IIC/III metastatic seminoma and a good prognosis we recommend 4 cycles of EP if bleomycin is contraindicated (A, 4) [e25, e26].

35In patients with metastatic seminoma and an intermediate prognosis, we recommend 4 cycles of BEP (A, 1b).

36In patients with metastatic seminoma and an intermediate prognosis, we recommend 4 cycles of VIP if bleomycin is contraindicated (A, 1b) [e27].

37In patients with metastatic non-seminomatous GCT stage IIC/III and a good prognosis, we recommend 3 cycles of BEP (A, 1b) [e18].

38Patients with metastatic non-seminomatous GCT stage IIC/III and a good prognosis should receive 4 cycles of EP if contraindications to bleomycin exist (B, 1b) [e18, e28].

39In patients with metastatic non-seminomatous GCT and an intermediate prognosis we recommend 4 cycles of BEP (A, 1b) [e27].

40In patients with metastatic non-seminomatous GCT and an intermediate prognosis, we recommend 4 cycles of VIP if contraindications to bleomycin exist (A, 1b) [e27].

41In patients with metastatic non-seminomatous GCT and a poor prognosis, we recommend 4 cycles of BEP (A, 1b) [e9].

42In patients with metastatic non-seminomatous GCT and a poor prognosis, we recommend 4 cycles of VIP if contraindications to bleomycin exist (A, 1b) [e29].

43In patients with primary mediastinal disease, bone and/or brain metastases, and/or an inadequate serum tumour marker decline, we recommend considering treatment intensification (A, 1b–4) [e30–e34].

44It is recommended that the evaluation of treatment intensification be performed at a centre with proven experience (expert consensus).

45We do not recommend general intensification of first-line therapy in favour of a high-dose chemotherapy with autologous stem cell transplantation for the unselected group of all GCT patients with a poor prognosis (A, 1b) [e35, e36].

46To assess the response to therapy, we recommend analysis of the serum tumour marker decline after 1, or at the latest after 2, cycles of standard-dose chemotherapy (A, 1b) [e35].

47In patients with an inadequate serum tumour marker decline after 1 or 2 cycles of cisplatin-based first-line therapy, intensification of chemotherapy should be considered on an individual basis (grade B: moderate quality of the evidence)[e37].

48When chemotherapy is intensified by means of primary high-dose therapy, sequential therapy should be performed with 3 cycles of high-dose VIP, each followed by autologous stem cell transplantation (B, 1b) [e38, e36].

See also Table 4 [e36].

49In patients with brain metastases at the initial diagnosis, we recommend either referral of patients to a GCT centre with proven experience or consultation at a centre with proven expertise prior to therapy (expert consensus).

50In patients with brain metastases at the initial diagnosis, we recommend 4 cycles of chemotherapy (BEP, VIP) according to the IGCCCG poor prognosis group (A, 2b). Alternatively, primary therapy intensification can be performed (0) [e30, e39, e40, e33].

51Due to insufficient data, no reliable statement regarding the benefit of post-chemotherapeutic radiotherapy and/or resection (in case of residual brain metastasis*). Any additional treatment should be decided upon based on interdisciplinary discussion on an individual basis (statement, 2b) [e30, e39, e40, e15, e33].

52Due to documented long-term toxicity of whole brain radiotherapy of the neurocranium, stereotactic radiotherapy should be sought if possible (B, 2b) [e39, e40, e33].

53In patients with bone metastases at the initial diagnosis, we recommend 4 cycles of chemotherapy (BEP, VIP) according to the IGCCCG intermediate prognosis (seminoma) and poor prognosis (non-seminomatous GCT) group (A, 4) [e34].

54We recommend that patients with bone metastases at the initial diagnosis be referred to a centre with proven experience, or that such a centre be consulted prior to therapy (expert consensus).

55Primary high-dose chemotherapy is no standard approach (in patients with bone metastases*), but it should be evaluated on an individual basis and considered where appropriate (B, 2b) [e32].

56We recommend considering subsequent local therapy of bone metastases following chemotherapy on an individual basis (resection or radiation if technically possible). Due to insufficient data, a reliable statement regarding the benefit of post-chemotherapeutic radiotherapy and/or resection of residual bone lesions cannot be provided (A, 4) [e34, e41].

57We recommend performing decompression of tumour-induced hydronephrosis before initiating chemotherapy (expert consensus).

58In patients with renal impairment, we do not recommend general replacement of cisplatin by carboplatin. The individual treatment decision should be made after consulting a centre with proven experience or a second-opinion online portal such as eKonsil Urologie (Hodentumor) in Germany (https://hodentumor.zweitmeinung-online.de/) (expert consensus).

59Carboplatin-based chemotherapy is associated with increased recurrence rates and increased tumour-related mortality and it should therefore be restricted to justifiable individual cases (statement, 1b) [e42–e44].

60We recommend that patients with active HIV disease receive sufficient antiretroviral therapy before initiating chemotherapy. It is recommended that the choice of antiretroviral therapy consider possible interactions with the cytostatic agents applied (expert consensus).

61Active HIV disease is not a contraindication to stage-matched chemotherapy. Primary prophylactic administration of G-CSF (unless it is with VIP therapy) and antibiotic prophylaxis are not required (expert consensus).

62In patients with active HIV disease*, we recommend determining the CD4 cell count before each chemotherapy cycle. With CD4 counts <200/μL, antibiotic prophylaxis with cotrimoxazole should be administered (expert consensus).

63Patients with advanced/metastatic GCT disease should preferably be treated by multidisciplinary teams at centres with proven experience in order to maximize the chance of cure and prevent treatment-associated adverse events by avoiding overtreatment (expert consensus).

* Additional information due to the abbreviated presentation of the text.

64Immediate chemotherapy without a prior orchiectomy should only be initiated in patients with life-threatening symptoms and high serum tumour markers for AFP and β-hCG (B, 4) [e45].

65FDG-PET/CT can be applied in seminoma patients with normal or normalized serum tumour markers who have residual disease larger than 3 cm in diameter after completion of therapy (0, 1a). The CT as part of the FDG-PET/CT scan should preferably be performed as a contrast-enhanced CT (expert consensus) [e46, e47].

66We recommend performing FDG-PET/CT for the evaluation of serum tumour marker-negative residual tumours in seminoma patients not earlier than 6 weeks after completion of chemotherapy (expert consensus).

67In case of positive metabolic activity in FDG-PET/CT, intensified follow-up (CT and serum tumour marker control) should be performed (expert consensus).

68After completion of first-line therapy of a metastatic seminoma, RTR should not be performed (B, 1b) [e48].

69Restaging after first-line chemotherapy should not include an FDG-PET/CT scan in patients with non-seminomatous GCT (B, 2b) [e49, e50].

70In patients with non-seminomatous GCT, we recommend resection of residual retroperitoneal and pulmonary tumours >1 cm that persist after completion of first-line chemotherapy and achievement of serum tumour marker normalization. It is recommended that the management of residual tumours at other sites be decided upon individually (A, 2b) [e51].

71Surgical removal of residual pulmonary metastases >1 cm is recommended. It is recommended that bilateral thoracic RTR be decided on interdisciplinarily when the histology of the resection of the first side is available. It is recommended that the resection be carried out using a parenchyma-saving approach with the opportunity for digital palpation (A, 4) [e52, e53].

72It is recommended that RTR be performed in centres with proven experience with the opportunity for interdisciplinary surgical care (e.g., liver resections, vascular grafting, spinal neurosurgery) (A, 2b) [e54–e56].

73Retroperitoneal RTR should preferably be performed using a nerve-sparing technique (B, 4). It is recommended that the template of RTR be defined depending on the initial tumour site (A) [e57–e59].

74Tumour resection only (so-called lumpectomy) should not be performed (B, 2b) [e57, e60–e63].

75In patients with R0 resection upon RTR, follow-up is recommended. In case of vital tumour cells, an individual approach is recommended, depending on the histology, the postoperative serum tumour markers, and morbidity, since a benefit for survival after adjuvant chemotherapy is currently unclear (expert consensus).

76In patients after salvage chemotherapy of a retroperitoneal non-seminomatous GCT relapse, retroperitoneal RTR should be performed even in tumours <1 cm if tumour markers have normalized (B, 4) [e64].

1In patients with recurrent seminoma after initial cSI, we recommend classification and treatment according to the extent of disease based on IGCCCG criteria (expert consensus).

2In patients with a recurrent non-seminomatous GCT after the initial stage I, we recommend classification and treatment based on IGCCCG criteria. Only in patients with recurrence earlier than 6 months after adjuvant therapy with 2 cycles of BEP should alternative chemotherapy regimens be considered (expert consensus).

3We recommend classification of patients with recurrence after first-line chemotherapy for metastatic disease according to the risk classification of the International Prognostic Factors Study Group (IPFSG) (expert consensus).

4Patients with recurrence after cisplatin-based combination chemotherapy due to primary metastatic disease can receive conventional-dose chemotherapy according to the TIP (paclitaxel, ifosfamide, cisplatin) or VIP regimen (0, 2b) [e65].

5Patients after cisplatin-based combination chemotherapy for primary metastatic disease should receive primary salvage chemotherapy using sequential high-dose chemotherapy with 3 cycles of HD-CE (carboplatin + etoposide) and autologous stem cell support (B, 1b). Alternatively, patients with relapse following cisplatin-based combination chemotherapy for primary metastatic disease can receive conventional-dose chemotherapy according to the TIP or VIP regimen (0) [e66–e68].

6Patients (with recurrence*) after cisplatin-based combination chemotherapy for primary metastatic disease should be included in clinical trials if possible (expert consensus).

7In non-seminomatous GCT patients undergoing salvage chemotherapy for a retroperitoneal relapse, RTR should be performed even in residual tumours <1 cm after normalization of tumour markers (B, 4) [e64].

* Additional information due to the abbreviated presentation of the text.

8In patients with recurrent bone metastases, we recommend performing a systemic combination chemotherapy (expert consensus).

9Since bone metastases at recurrence represent a high-risk situation, the patient should preferentially be treated with high-dose chemotherapy (expert consensus).

10After systemic therapy of recurrent bone metastases, additional local therapy (resection or radiotherapy) should always be evaluated. Comparative prospective data are not available for these two therapy options (expert consensus).

11For vitality assessment after systemic chemotherapy, a biopsy can be considered prior to local therapy in patients with extensive bone metastases (expert consensus).

12In patients with cerebral metastases at relapse, high-dose chemotherapy should be administered (B, 2b). Retrospective studies indicate a survival benefit from consolidation radiotherapy. In case of incomplete remission, it is recommended that radiotherapy be performed additionally (A, 2b). In case of complete remission of cerebral metastases in imaging after salvage chemotherapy, the situation is unclear and additive radiotherapy should be considered individually (B, 2b) [e33].

13In patients with solitary residuals after salvage chemotherapy, we recommend performing stereotactic radiation and/or neurosurgical resection of brain metastases (expert consensus).

14In patients with multiple residuals after salvage chemotherapy, it is recommended that radiotherapy of the neurocranium be performed (expert consensus).

15In patients with marker-negative late recurrence and in whom complete resectability seems possible, we recommend performing a primary surgical resection (A, 2b) [e69, e70].

16Patients with a non-resectable late recurrence have a poor prognosis. These patients should primarily receive chemotherapy (either conventional or high-dose chemotherapy) and secondary resection should be considered (B, 4) [e71].

17In patients with recurrence or progressive relapse after multiple chemotherapy regimens, we recommend offering a combination chemotherapy with gemcitabine and oxaliplatin +/– paclitaxel (GOP). Following a response to therapy with GOP, the resection of all residual tumours is recommended (A, 4) [e72–e77].

18In patients with growing teratoma syndrome, we recommend complete surgical resection (A, 4) [e78–e80].

19In cSI patients with malignant somatic transformation, we recommend RPLND (A, 4) [e81, e82].

20In patients with metastatic disease and components of malignant somatic transformation, we recommend performing a GCT-based chemotherapy first, followed by RPLND (expert consensus).

21In patients with malignant somatic transformation relapsing after GCT therapy, we recommend choosing a chemotherapy regimen based on the histology of the somatic malignancy (A, 4) [e83, e81, e82].

1The spermatocytic tumour – formerly spermatocytic seminoma – is a very rare tumour that requires differentiation from classical seminoma. The diagnostic measures do not differ from those applied in gonadal GCT (expert consensus).

2The pure spermatocytic tumour limited to the testes is a tumour with a good prognosis, which is sufficiently treated by orchiectomy and surveillance (expert consensus).

3The presence of sarcomatous components in a spermatocytic tumour indicates a highly malignant transformation of the tumour with a poor prognosis and requires a multimodal therapy concept with chemotherapy, RTR, and possibly radiation therapy. The sequence of the individual steps needs to be determined individually for each patient (expert consensus).

4It is recommended that sarcomatous components of the spermatocytic tumour be ruled out upon histological evaluation (expert consensus).

5Ninety-five percent of all male adult GCT originate in the testicles; 5% are of primarily extragonadal origin (expert consensus).

6We recommend confirming the diagnosis of extragonadal GCT histologically only in case of an ambiguous constellation of serum tumour markers (expert consensus).

7The determination of the serum tumour markers (AFP, β-hCG, LDH) in extragonadal GCT is recommended for confirmation of the diagnosis, for correct classification according to the IGCCCG, and for monitoring of the treatment responses (expert consensus).

8All patients with mediastinal non-seminomatous extragonadal GCT are classified as belonging to the poor prognosis group (stage III) according to the IGCCCG. Due to their very poor prognosis, high-dose chemotherapy should be chosen primarily. Performance of a radical RTR should yield a good response (B, 2b). We recommend the involvement of centres with proven experience in the therapy (A, 2b) [e84].

9We recommend to classify patients with retroperitoneal non-seminomatous extragonadal GCT depending on their serum tumour marker levels according to the IGCCCG classification (A, 2b) [e84].

10We recommend performing therapy of extragonadal GCT in analogy to metastatic gonadal GCT according to the histology and the corresponding IGCCCG classification. Centres with proven experience should be involved in the therapy (expert consensus).

11In patients suspected of having a Leydig cell tumour or a Sertoli cell tumour, we recommend determining the serum tumour markers (AFP, β-hCG, LDH) as well as testosterone, LH, and FSH for endocrinological evaluation (expert consensus).

12We recommend establishing intraoperatively the histological diagnosis by frozen section. We recommend performing organ-preserving surgery in small tumours (<2 cm) if no malignancy criteria are present (A, 3) [e85].

13Malignant gonadal stroma tumours of Leydig cell or Sertoli cell origin should be treated with radical orchiectomy (expert consensus).

14If histological examination of Leydig cell or Sertoli cell tumours reveals criteria for the malignancy, RPLND should be performed in stage I patients (B, 2b) [e54, e86, e87].

15In malignant Leydig cell or Sertoli cell tumours we recommend performing an RPLND in stage II patients (expert consensus).

There is no international consensus on follow-up recommendations for patients with GCT in full remission after curative therapy. A number of publications address follow-up schedules.

1MRI of the abdomen/pelvis should replace CT of the abdomen/pelvis in the follow-up care of patients with GCT if performed at centres with proven experience to reduce radiation exposure. Experienced radiologists are required for the interpretation of results (expert consensus).

The recommendations given in Tables 5-11 are based on the following publications and are separately illustrated for the different patient groups [e88–e90].

1Acute toxicity occurring during therapy should be recorded systematically and appropriate therapeutic measures should be taken (expert consensus).

2In the follow-up care, the examination and, if necessary, the treatment of therapy-related late sequelae and long-term toxicity play a central role in addition to the search for disease recurrence (expert consensus).

3After radiation or chemotherapy, fertility impairment is common in GCT patients compared to the normal male population and can persist as a long-term sequela after completion of treatment (expert consensus).

4GCT patients are at risk of clinically relevant hypogonadism. Therefore, we recommend asking the patient about related symptoms and determining serum levels of testosterone and LH as part of the diagnostic measures during therapy and follow-up (expert consensus).

5In patients with hypogonadism, therapy follows the recommendations for testosterone replacement therapy (expert consensus).

6During cisplatin-containing chemotherapy for metastatic GCT, thromboembolism prophylaxis with low-molecular-weight heparin should be used to reduce the risk of thromboembolic events in patients (B, 2b) [e91].

1In patients with GCT, quality-of-life data should be collected before and during treatment (expert consensus).

2The EORTC QLQ-TC 26 questionnaire for quality-of-life assessment, which was specifically developed for patients with GCT, is available in the German language (statement, 3b) [e92].

3Depending on individual needs, it is recommended that all patients eligible for rehabilitation be offered post-treatment rehabilitation (expert consensus).

4It is recommended that the rehabilitation of patients with GCT be carried out in rehabilitation clinics offering age-specific services with oncological competence and by using multimodal therapy concepts (expert consensus).

1If incurable disease becomes obvious, we recommend informing the patient and, when required, his or her relatives, at an early stage about palliative care options. Access to these should be facilitated (expert consensus).

2Low-threshold psychosocial counselling, support, and treatment should be available to all affected persons and their relatives in every phase of the disease, as well as in the long term (expert consensus).

• The lack of prospective randomized controlled trials for the direct comparison of radiotherapy versus chemotherapy for seminoma cSIIA/B does not allow a valid statement regarding the superiority or equivalence of either therapy with respect to oncological efficacy.

• In patients with acute life-threatening disease with metastatic gonadal GCT in stages IIC/III, we recommended administerimng chemotherapy upfront and postponing orchiectomy of the initially affected testis until completion of the chemotherapy.

• We recommend analysis of the serum tumour markers after 1, or at the latest after 2, cycles of standard-dose chemotherapy.

• Particular attention and careful consideration are required in patients with contraindications to bleomycin, with brain and bone metastases, with renal impairment, or with active HIV infection.

• In patients with primary mediastinal disease, bone and/or brain metastases, and/or an inadequate serum tumour marker decline, we recommend evaluating treatment intensification.

• We recommend classifying and treating patients with relapsing seminoma or non-seminomatous GCT after the initial stage I based on their recurrent stage by analogy to primary metastatic seminoma/non-seminomatous GCT according to IGCCCG criteria.

• There is no international consensus on follow-up recommendations for patients with GCT in full remission after curative therapy.

• In patients with a serum tumour marker-negative late recurrence, we recommend performing a primary surgical resection in cases where complete resectability is technically feasible.

• In patients with relapse or progression after multiple chemotherapy, we recommend offering a combination chemotherapy with gemcitabine and oxaliplatin +/– paclitaxel (GOP). Following a response to therapy with GOP, resection of all residual tumours is recommended.

• Acute toxicity occurring during therapy should be recorded systematically and appropriate therapeutic measures should be taken.

• In the follow-up care, the examination, and, if necessary, the treatment of therapy-related late sequelae and long-term toxicity play a central role in addition to the search for disease recurrence.

• In patients with GCT, quality-of-life data should be collected before and during treatment.

• Research gaps in the context of this guideline were particularly evident for healthcare research and with respect to risk factors for disease incidence. In addition, the statements related to the poor prognosis group are based on limited data. The evaluation of follow-up schedules by long-term studies is also still pending.

The German Guideline Program in Oncology of the Association of the Medical Scientific Societies (AWMF), the German Cancer Society (DKG), and the German Cancer Aid Foundation (DKH) supervised this project and gave methodological support. Administrative support by Ingrid Rambow (Münster, Germany) was greatly appreciated. Uwe Hölzel (Dresden, Germany) also participated in this project.

The evaluation of conflicts of interests showed only minor relevance on the topic and can be obtained via request.

Support was received from the DKH (reference No. 70112789).

All of the authors have made substantial contributions to the conception or design of this work and the acquisition, analysis, and interpretation of data for this work. They drafted this work or revised it critically for important intellectual content and approved the final version to be published. They have agreed to be accountable for all aspects of this work in ensuring that questions related to the accuracy or integrity of any part of this work are appropriately investigated and resolved.

S. Kliesch and S. Schmidt contributed equally to this study and should be considered co-first authors.