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Targeting VEGF/VEGFR to Modulate Antitumor Immunity

In addition to the crucial role in promoting the growth of tumor vessels, vascular endothelial growth factor (VEGF) is also immunosuppressive. VEGF can inhibit the function of T cells, increase the recruitment of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), and hinder the differentiation and activation of dendritic cells (DCs). Recent studies have investigated the role of antiangiogenic agents in antitumor immunity, especially in recent 3 years. Therefore, it is necessary to update the role of targeting VEGF/VEGFR in antitumor immunity. In this review, we focus on the latest clinical and preclinical findings on the modulatory role of antiangiogenic agents targeting VEGF/VEGFR in immune cells, including effector T cells, Tregs, MDSCs, DCs, tumor-associated macrophages, and mast cells. Our review will be potentially helpful for the development of combinations of angiogenesis inhibitors with immunological modulators.

Meanwhile, VEGF is also immunosuppressive. The effects of VEGF on immune cells are summarized in Figure 1 and are reviewed in detail in the main text. Given the immunosuppressive role of VEGF, scientists have recently tried to restore the antitumor immunity by targeting VEGF/VEGFR. In this review, we focus on the latest clinical and preclinical findings on the modulatory role of antiangiogenic agents targeting VEGF/VEGFR in immune cells, such as effector T cells, regulatory T cells (Tregs), myeloidderived suppressor cells (MDSCs), and dendritic cells (DCs).

eFFeCTOR T CeLLS effects of veGF on effector T Cells
Zhang et al. found that strong expression of VEGF was detected in ovarian carcinoma tissues without T cells, while low expression of VEGF was detected in ovarian carcinoma tissues with T Cells (15). Ohm et al. 's study indicated VEGF impeded the development of T cells from early hematopoietic progenitor cells, indicating the potential immunosuppressive role of VEGF in tumors (16). But the direct effects of VEGF on T-cell function were not investigated in these two studies. Basu et al. found that VEGFRs were expressed on recently activated and memory subsets of human CD4 + T cells (17). VEGF-VEGR interactions resulted in the activation of the MAPK and PI3K-Akt signaling pathways in human CD4 + T cells (17), similar to in endothelial cells (18). VEGF could also induce the production of IFN-γ and IL-2 and mediate migratory responses in human CD4 + CD45RO + memory T cells (17). However, mounting evidence supports the suppressive role of VEGF/VEGFR in T cells (19). Ziogas et al. found that VEGF significantly reduced the cytotoxic activity of T cells derived from peripheral blood samples, and that activated T cells expressed increased VEGFR2. Anti-VEGFR2 reversed the VEGF-induced suppression of T cells (20). Similar results were also observed in T cells from ascites secondary to ovarian cancer (21). In addition to the direct effects of VEGF on T cells, VEGF could also suppress T-cell function through combination with cyclooxygenase by upregulating FasL on the endothelium (22). Kaur  were upregulated in CD47-deficient murine CD4 + T cells, and the resulting autocrine VEGFR2 signaling enhanced proliferation and some TCR responses in the absence of CD47 (23). This may explain the conflicting findings regarding whether VEGF was an inhibitor or stimulator in T-cell function. Because CD47 is ubiquitously expressed in human cells (24), it is possible that VEGF suppresses the function of T cells in most circumstances.
enhancing T-Cell Function by Antiangiogenic Agents Targeting veGF/veGFR Regarding the immunosuppressive role of VEGF in T-cell function, it is biologically reasonable that interfering with VEGF/ VEGFR can enhance antitumor immunity by improving T-cell function. There are some clinical and preclinical findings supportive of this hypothesis. Manzoni et al. found that bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody, could increase B-cell and T-cell compartments in patients treated with a bevacizumab-based first-line therapy for metastatic colorectal cancer (25). Bevacizumab also improved cytotoxic T-lymphocytes response in patients with metastatic non-small cell lung cancer (NSCLC) (26). Sunitinib is a multi-target tyrosine kinase inhibitor that can block VEGFR1, VEGFR 2, and VEGFR3, plateletderived growth factor receptors α and β, stem cell factor receptor, and Flt3. Sunitinib was approved by the FDA for the treatment of renal cell carcinoma (RCC) and imatinib-resistant gastrointestinal stromal tumor (GIST) in 2006 (27,28). Sunitinib was found to reduce the expression of IL-10, Foxp3, PD-1, CTLA4, and BRAF, but increased Th1 cytokine (IFN-γ) in isolated tumorinfiltrating lymphocytes (TILs) in an MCA26 (colon cancer cells) bearing mouse model. An increase in the proportion of CD4 + and CD8 + T cells was also observed in TILs in sunitinib-treated mice, whereas expressions of the inhibitory molecules PD-1 and CTLA4 were obviously reduced after sunitinib treatment. T cells from sunitinib-treated mice exhibited stronger cytotoxic activity against MCA26 tumor cells. These results indicate that sunitinib can modify the tumor microenvironment, resulting in a shift of cytokine and costimulatory molecule expression profiles that could favor T-cell activation and Th1 responses (29). Likewise, Schmittnaegel et al. 's study suggested that dual angiopoietin-2 and VEGFA inhibition elicited antitumor immunity by increasing the proportion of CD8 + T cells that expressed an activated, IFN-γ or CD69 + phenotype in both transgenic and transplanted mammary tumor models (30). Voron et al. found that VEGFA produced in the tumor microenvironment enhanced the expression of PD-1 and other inhibitory checkpoints involved in CD8 + T-cell exhaustion, including PD-1, CTLA-4, Tim-3, and Lag-3 (28). This effect could be reversed by antibodies targeting VEGFR2 (28), which is similar to Bamias et al. 's findings (21). Voron et al. 's study also indicated that VEGFA enhanced the expression of inhibitory checkpoints involved in T-cell exhaustion via the activation of the VEGFR2-PLCγ-calcineurin-NFAT pathway (28). In addition to increasing T-cell activity, targeting VEGF/VEGFR also can promote T-cell infiltration in the tumor microenvironment. Targeting VEGF/VEGFR not only hinders the sprouting of new vessels (31,32) but can also normalize the vasculature.
High endothelial venules (45) may be another mechanism by which T-cell infiltration can be promoted by targeting VEGFR2. HEVs are located in all lymphoid organs except the spleen and specialized postcapillary venules with portals through which blood-borne lymphocytes enter into secondary lymphoid organs (36,46,47). Recent studies have suggested that various human tumors could develop areas of HEVs and their presence was associated with a decreased tumor size and improved patient outcome (48,49). A recent study by Allen et al. demonstrated that a combination of anti-VEGFR2 and anti-PD-L1 antibodies could induce HEVs in murine models. These HEVs enhanced lymphocyte infiltration and activity through activation of lymphotoxin β receptor (LTβR) signaling, and eventually improved the treatment efficacy (36).

TARGeTinG veGF/veGFR TO DeCReASe THe nUMBeR OF Tregs
Regulatory T cells are immunosuppressive and can suppress or downregulate induction and proliferation of effector T cells (50). Tregs express the biomarkers CD4, FOXP3, and CD25 (51). The expression of VEGF has been shown to be positively associated with intratumoral Tregs, which are prognostic markers for the poor outcomes of various malignancies (52-54). Suzuki et al. showed for the first time that VEGFR2 is selectively expressed by FOXP3 high but not FOXP3 low Tregs (55). Neuropilins acted as co-receptors, increasing the binding affinity of VEGF for VEGFRs (56,57). Promoted VEGF signaling through conjunction with neuropilin-1 may enhance Treg activation and create a tolerogenic environment (57). It is therefore reasonable that targeting VEGFA/VEGFR can modulate antitumor immunity by interfering with inhibitory Tregs.
Sunitinib has been reported to reduce the number of Tregs in tumor-bearing mice and in patients with metastatic renal carcinoma (29,(58)(59)(60). Sunitinib could target various receptors as mentioned above, and these studies (29, 58-60) did not investigate through which receptor sunitinib decreased the number of Tregs or the direct effects of VEGF on Tregs. Then, Terme et al. investigated patients receiving bevacizumab, a monoclonal antibody targeting VEGF, for metastatic colorectal cancer and treated colon cancer-bearing mice (CT26) with drugs targeting the VEGF/VEGFR axis. This study suggested that VEGF could promote the proliferation of Tregs and VEGF/VEGFR antibodies or inhibitors could decrease the number of Tregs in both patients with mCRC and the mouse models. This proliferation was inhibited by VEGF/VEGFR2 blockade (61), similar to the findings of Suzuki et al (55). In addition, sunitinib (55,61), the anti-VEGFR2 antibody DC101 (62) and a chimeric receptor blocking VEGFR1/ R2 (63) could also reduce the number of Tregs in tumors.

TARGeTinG veGF/veGFR TO inHiBiT THe ACCUMULATiOn AnD THe ACTiviTY OF MDSCs
Myeloid-derived suppressor cells were initially identified in tumor-bearing mice as cells co-expressing CD11b and Gr1 (64).  (66). The accumulation of MDSCs was shown to be associated with an increase in intratumoral VEGF concentration during disease progression in pancreatic-ductal adenocarcinoma-bearing mice (45).
Huang et al. found that VEGF could induce the accumulation of Gr1 + CD11b + cells by VEGFR2 and activation of JAK2 and STAT3 (67), but not VEGFR1 in tumor-bearing mice (68). Besides, MDSC enhanced by VEGF could induce the development of other immunosuppressive cells, including FOXP3 + Tregs, through a TGFβ-dependent and/or independent pathway (69)(70)(71). It is, therefore, reasonable to decrease the accumulation of MDSC by interfering with VEGF/VEGFR axis.
A decrease in the absolute number of MDSC in the spleen, bone marrow, and tumor in various tumor models has been observed after treatment with sunitinib (29,58). The potential mechanisms included the following: sunitinib could act on MDSC by inhibiting STAT3; sunitinib could constrain the M-MDSC and lead to the apoptosis of granulocytic MDSCs (29,58). Sunitinib also resulted in a favorable microenvironment depleted of MDSCs and synergize with HPV vaccine leading to enhanced levels of active tumor-antigen specific CTLs in a tumor-bearing mice model (72). Not only the quantity of MDSC but also the suppressive capacity was affected. In a melanoma-bearing mouse model, Axitinib, a small molecule against VEGFR1, R2, and R3, could induce a reduced suppressive capacity of MDSCs isolated from the spleen or tumor of Axitinib-treated mice compared to vehicle-treated mice. Moreover, treatment with Axitinib induced differentiation of MDSC toward an antigen-presenting phenotype (73). Clinically, sunitinib could result in a reduction of MDSC in RCC patients. The reduction of MDSC was correlated with reversal of T-cell suppression (74). A recent study demonstrated that bevacizumabcontaining regimens significantly reduce the percentage of the granulocytic-MDSCs compared with non-bevacizumab-based regimens in patients with unresectable NSCLC (75).

DenDRiTiC CeLLS effects of veGF on the Differentiation, Maturation, and Activation of DCs
Dendritic cells are antigen-presenting cells of the immune system, which act as messengers between the innate and the adaptive immune systems. Immature DCs are derived from hematopoietic bone-marrow progenitor cells. Immature DCs are highly endocytic. They express relatively low levels of surface MHC-I, MHC-II, and costimulatory molecules such as CD80 and CD68. Hence, immature DCs are unable to process and present them efficiently to T cells (76,77). Mature DCs are characterized by an increased capacity for antigen processing, increased the half-life of surface MHC-peptide complexes, and reduced antigen uptake (76,78). Activated DCs can be distinguished from resting, mature DCs by expression of higher levels of MHC and costimulatory molecules or production of cytokines. Maturation and activation can occur simultaneously (76,79). Thus, factors that interfere with the differentiation, maturation, and activation of DCs can lead to the dysfunction of DCs.
Clinical and preclinical studies indicated that VEGF could impair the differentiation and maturation of DCs. Almand et al. found that an increased plasma level of VEGF was associated with the presence of immature DCs in the peripheral blood of cancer patients. Surgical removal of the tumor could result in partial reversal of the observed effects (80). For patients with colorectal cancer, peripheral DCs were inversely correlated with VEGF serum levels (81). Various studies have indicated that VEGF binding to VEGFR1 blocked the activation of the transcriptional factor NF-κB and resulted in the inhibition of DC maturation in murine models (71,82,83

TARGeTinG veGF/veGFR TO MODiFY THe DiFFeRenTiATiOn, MATURATiOn, AnD ACTivATiOn OF DCs
Scientists have tried to modify the function of DCs by targeting VEGF/VEGFR. Sorafenib is a multikinase inhibitor and can inhibit RAF/MEK/ERK pathway, VEGFR2, VEGFR3, PDGFRβ, Flt-3, and c-KIT (85). Though various studies have investigated the associations between sorafenib and DCs, the results are inconsistent and the role of sorafenib in DCs remains controversial. Hipp et al. found that sorafenib impeded the maturation of DCs, characterized by reduced expression of CD1a, major histocompatibility complex, and costimulatory molecules in response to TLR ligands as well as by their impaired ability to migrate and stimulate T-cell response (85). However, Alfaro et al. 's study indicated that sorafenib could restore the differentiation of DCs assessed by the alloreactive mixed T-lymphocyte reaction (MLR) in the presence of VEGF and supernatants of RCC cells (86). The seemingly paradoxical results from the studies of Hipp (85) (88). Discrepancies among different studies may lie in the intricate effects of sorafenib or distinct experimental designs, the complicated development of DCs, or the use of different animal models. Discrepancies also exist for sunitinib. Though sunitinib was found to increase the frequency of myeloid DCs in patients with renal cancer experiencing tumor regression (89), other studies indicated that the function of DCs was not affected by the use of sunitinib (85,86). Generally, the exact roles of sorafenib and sunitinib in DCs are debatable and further studies are warranted. A preclinical study indicated that bevacizumab could reverse the inhibitory effects of VEGF in the differentiation of monocytes into DCs in vitro (86). Various clinical studies suggested improvements in both quantity and function of DCs after the use of bevacizumab. Bevacizumab was found to increase the number of DCs in peripheral blood of cancer patients and enhance the allostimulatory capacity of DCs against recall antigens (90). In patients with metastatic NSCLC, bevacizumab was found to promote DC activation (26). Significant trafficking of CD163 + DCs across the tumor vasculature was observed in bevacizumab plus ipilimumab post-treatment biopsies in patients with metastatic melanoma, compared to ipilimumab alone (91).

TUMOR-ASSOCiATeD MACROPHAGeS (TAMs) effects of veGF on TAMs
Vascular endothelial growth factor can recruit macrophages to the tumor and promote TAM development (53,92). TAMs have a poor antigen-presenting capacity and a decreased cytotoxic capacity due to the weak NO production (53,93). TAMs can also hinder T-cell activation and proliferation by releasing IL-10, TGFβ, and prostaglandins (53,94).

TAM Might Be involved in the Anti-veGF Resistance
A recent study suggested macrophages could be actively recruited to the tumor microenvironment and were responsible for the anti-VEGF resistance in a mouse model bearing ovarian cancer. The resistance to anti-VEGF failed to occur in a macrophagedeficient mouse model (95). Zoledronic is a bisphosphonate drug and clinically approved for the treatment of bone metastases and osteoporosis. Bisphosphonates can also result in robust macrophage depletion (95)(96)(97). The addition of zoledronic acid at the emergence of resistance to anti-VEGF therapy halted tumor growth and obviously prolonged the survival of mice bearing ovarian cancer (95).
In addition to ovarian cancer, increased TAMs have been observed in the specimens of glioblastomas which progress during bevacizumab treatment and associated with a poor outcome in preclinical and clinical studies (98,99). The accumulation of immune-suppressive cells was induced by increased expressions of stromal-derived factor (SDF)-1α (CXCL12) and C-X-C motif chemokine receptor 4 (CXCR4) (100,101). SDF-1αpromoted the recruitment of macrophages by targeting CXCR4. Decreasing the recruitment of TAMs is another strategy to reverse the anti-VEGF resistance. A preclinical study by Deng et al. found that inhibition of SDF-1α inhibited the recruitment of TAMs induced by VEGF blockade and potentiated its antitumor efficacy in glioblastoma (102). Combination of VEGFR and CXCR4 inhibitors slows progression of GBM xenografts (100).
AMD3100 against CXCR4 was applied with a combination of bevacizumab in patients with recurrent high-grade glioma (HGG) in a phase I clinical trial NCT01339039. In ASCO annual meeting 2014, the preliminary data demonstrated that the combination treatment with bevacizumab and AMD3100 was well tolerated in HGG patients (103). Macrophage migration inhibitory factor (MIF) is another ligand for CXCR4. A recent study by Castro et al. described that MIF was also a mediator of increased macrophages and associated with bevacizumab-resistance in patients with glioblastoma and xenograft models by causing proliferative expansion of M2 macrophages (104).

AnTiAnGiOGeniC THeRAPY AnD MAST CeLLS
Kessler et al. found that mast cells accumulated in tumors before the onset of angiogenesis and resided in close proximity to blood vessels (105,106). Mast cells can participate in the tumor rejection by producing molecules such as IL-1, IL-4, IL-6, and TNF-α. By contrast, mast cells can promote the tumor by enhancing its vascular supply, degradation of the tumor extracellular matrix and immunosuppression (107). Mast cells can synthesize and release angiogenic cytokines, including VEGF, FGF-2, the serine proteases tryptase and chymase, IL-8, TGFβ, TNF-α, and nerve growth factor (NGF) (107).
Similar to the findings in MDSC and TAM, antiangiogenic agents could reverse the tumor-induced immunosuppression by decreasing the tumor-promoting mast cells, and mast cells also played a role in antiangiogenic resistance. Axitinib is a multireceptor inhibitor, which does not only inhibit VEGF receptors but also kinases including fms-like tyrosine kinase 3 (FLT-3), PDGF receptors, and CD117 (cKIT) (108). The latest study found that inhibition of mast cells by axitinib as well as their experimental depletion led to a decreased tumor growth. Treatment with axitinib also resulted in an improved T-cell response, which was pivotal for the therapeutic efficacy (109). A recent study demonstrated that mast cells could decrease the efficacy of antiangiogenic therapy (anti-VEGFR2 antibody DC101) (106). The potential mechanisms included: the degranulation-independent secretion of granzyme B, which liberates alternative pro-angiogenic factors including FGF-1 and GM-CSF from the ECM and the degranulationdependent secretion of FGF-2 (106). Therefore, tumor-promoting mast cells might be a promising therapeutic target to improve the antitumor immunity and reverse the antiangiogenic resistance. Targeting VEGF/VEGFR to Modulate Antitumor Immunity Frontiers in Immunology | www.frontiersin.org May 2018 | Volume 9 | Article 978

COnCLUSiOn
In this review, we summarized the effects of VEGF and antiangiogenic agents on the immune cells, e.g., effector T cells, Tregs, MDSCs, DCs, TAMs, and mast cells. Agents targeting VEGF/ VEGFR can restore the function and enhance the infiltration of effector T cells, decrease the number of immunosuppressive Tregs, TAMs, and mast cells, and inhibit the accumulation and immunosuppressive activity of MDSCs. But the effects of antiangiogenic agents on DCs are inconsistent among different studies and further studies are still needed. MDSC, TAMs, and mast cells also participate in the resistance of antiangiogenic therapy. Our review will be potentially helpful for the development of combinations of angiogenesis inhibitors with immunological modulators.

AUTHOR COnTRiBUTiOnS
JY, JY, and BL conceived the theme of this review and wrote the manuscript.

ACKnOwLeDGMenTS
Many thanks for the great help from the colleagues in the Comprehensive Cancer Centre of Drum Tower Hospital.


PSMA Theranostics: Current Status and Future Directions

Prostate-specific membrane antigen (PSMA) is a promising target for imaging diagnostics and targeted radionuclide therapy (theranostics) of prostate cancer and its metastases. There is increasing evidence of encouraging response rates and a low toxicity profile of radioligand therapy (RLT) of metastatic castration-resistant prostate cancer using 177Lu-labeled PSMA ligands. In this article, we review the current status of diagnostics and therapy using radiolabeled PSMA ligands. We also suggest protocols for patient selection criteria and conduct of PSMA-based RLT. Challenges and opportunities of PSMA theranostics are discussed.

Introduction
Most deaths related to prostate cancer (PC) are due to advanced disease, which results from any combination of lymphatic, blood, or contiguous local spread. Targeted radionuclide therapy is an attractive and quickly developing therapy option for a variety of cancers, such as lymphoma, melanoma, and neuroendocrine tumor. [1][2][3][4] Prostate-specific membrane antigen (PSMA), also known as folate hydrolase I or glutamate carboxypeptidase II, is a type II, 750-amino acid transmembrane protein (100-120 kDa), which is anchored in the cell membrane of prostate epithelial cells. The PSMA is highly expressed on prostate epithelial cells and strongly upregulated in PC. The PSMA expression seems correlated to androgen independence, the presence of metastases, and PC progression. 5 Despite its name, PSMA is not specific to the prostate gland and is expressed in other normal (eg, salivary glands, duodenal mucosa, subset of proximal renal tubular cells, and subpopulation of neuroendocrine cells in the colonic crypts) and neoplastic (eg, subtypes of transitional cell carcinoma, renal cell carcinoma, colon carcinoma, and peritumoral and endotumoral endothelial cell of neovasculature) tissues. [6][7][8] There is no known natural ligand for PSMA, and the reasons for its upregulation in PC remains unknown. The PSMA ligands undergo constitutive cell internalization by PSMA, which, therefore, represents an appealing molecular target for theranostics in metastatic PC. 9 PSMA-Based Imaging

Anti-PSMA Antibodies
The first commercialized anti-PSMA antibody was 111 Incapromab-pendetide (ProstaScint, AYTU Bioscience, Englewood, USA), 10 which was approved by the US Food and Drug Administration in 1996. However, ProstaScint was only able to bind to the intracellular epitope of PSMA and was therefore not capable of visualizing viable PC cells leading to poor clinical performance. 11 In contrast to 111 In-capromab-pendetide, J591 binds to the extracellular domain of PSMA. Labeled with 111 In, J591 can be used for scintigraphy 12 and when labeled with 89 Zr for positron emission tomography (PET). 13,14 Recently, 89 Zr-Df-IAB2M was introduced as an anti-PSMA minibody. 15 Despite good tumor detection rates and contrast in scintigraphy, 16 J591 as well as other radiolabeled antibodies present with limitations such as low tumor penetrability, long delay between injection and imaging, nonspecific accumulation associated with inflammation, and high radiation exposure due to the necessity of radionuclides with long half-lives due to the long plasma half-life of antibodies.

Small-Molecule PSMA Inhibitors
Small-molecule inhibitors have been developed over the past 2 decades. [17][18][19][20][21][22][23][24][25][26][27] The basic chemical structure of the majority of small-molecule PSMA ligands consists of glutamateurea-lysine dimers. This structure derives from hydroxyphosphinyl derivatives described by Jackson et al in 1996. 17 Another significant step forward was the development of urea-based inhibitors by Kozikowski et al 21,23 as well as the inhibition of PSMA by phosphonamidothionate derivatives of glutamic acid. 28 One attribute of small-molecule inhibitors that has a significant impact on imaging and therapy is the feature that these ligands are internalized into the cells after binding to PSMA.

Small-Molecule PSMA Inhibitors for Scintigraphy
123 I-(MIP)-1072 and 123 I-MIP-1095 were the first smallmolecule PSMA inhibitors that were introduced into the clinic in 2008 by Molecular Insight Pharmaceuticals, Inc (MIP, now a subsidiary of Progenics Pharmaceuticals Inc, Cambridge, Massachusetts, USA). 24,29 These ligands demonstrated for the first time the enormous potential of PSMA ligands for imaging and therapy of PC. 25,30 The subsequent replacement of 123 I by 131 I led to the first human therapy with PSMA ligands. 31,32 However, the widespread availability and use of 99m Tc made the development of 99m Tc-labeled PSMA ligands a desirable pursuit. MIP-1404 and MIP-1405 were the first 99m Tc-labeled PSMA ligands, which were introduced into the clinic in 2010. 26,33 Although both ligands presented with promising results, 25 99m Tc-MIP-1404 was chosen to enter further clinical studies due to its lower urinary excretion. 34,35 Currently, 99m Tc-MIP-1404 is the subject of a phase 3 clinical trial designed to evaluate its sensitivity and specificity to detect PC compared to histopathology (ClinicalTrials.gov Identifier: NCT02615067).

PSMA Ligands for PET Imaging
The development of PSMA ligands for PET imaging dates back to as early as 2002. The first tracer published was 11 C-MCG which was used in animal studies. 22 The clinical breakthrough of PET imaging with PSMA ligands was achieved with the invention of 68 Ga-PSMA-11 (also called HBED-CC, HBED, PSMA-HBED, or Prostamedix), which demonstrated excellent characteristics. [36][37][38] Since its clinical introduction in May 2011, PET imaging with 68 Ga-PSMA-11 has spread rapidly worldwide and is regarded as a significant step forward in the diagnosis of recurrent PC. [38][39][40][41][42][43][44] The first publications indicated that this novel method is significantly superior compared to alternative methods used for the detection of recurrent PC. 36,38 Later publications confirmed the high sensitivity and specificity of 68 Ga-PSMA-11 PET/computed tomography (CT). 38-44 68 Ga-PSMA-11 PET/CT is usually conducted at 1 hour post injection (p.i.) according to its first described clinical setup. 37 However, experience showed that most PC lesions present with increased uptake and contrast at later imaging timings (3 hours p.i.), despite the relatively short half-life of 68 Ga. 37 Later publications confirmed these findings. [45][46][47] Alternative PSMA ligands such as 18 F-DCFPyL, 68 Ga-PSMA-617, 68 Ga-PSMA I&T, or 124 I-MIP-1095 demonstrated similar characteristics. 31,[48][49][50] Several studies with large patient cohorts have analyzed the sensitivity of 68 Ga-PSMA-11 PET/CT as well as possibly interacting factors such as prostate specific antigen (PSA), injected amount of tracer, androgen deprivation therapy (ADT), or Gleason Score (GS). [39][40][41] As expected, the probability to detect tumor lesions (sensitivity) increased with the PSA value. At PSA values less than 0.5 ng/mL, the reported detection probability was about 50%. This rate is assumed to be the highest among all methods for imaging recurrent PC. However, as shown in Figure 1, a continuous increase in PSA level does not always correlate with an increase in tumor detection. Even in the subcohort of patients with PSA levels above 10 ng/mL, not all patients presented with a pathologic 68 Ga-PSMA-11 PET/CT. Absent or low PSMA expression as well as small tumor size could be possible reasons for this observation. No association was found between the probability of a pathologic scan and either of age, injected tracer activity, faster PSA doubling times, or faster PSA velocity. No significant association but only a tendency was found between higher GS and a higher probability of a pathologic scan. 39,40 In contrast to GS, an ongoing ADT at the time of the scans was significantly associated with a pathologic 68 Ga-PSMA-11 PET/CT 39,40 although such a therapy can cause a reduction in tumor size as well as PSA values. Both of these effects usually entail a negative impact on tumor detection. It is known from preclinical literature that ADT can increase the PSMA expression in PC cells. 51,52 On the other hand, another reason for the fact that patients with ADT more often showed pathological findings in the 68 Ga-PSMA-11-PET/CT could be that ADT is often started in cases of advanced tumor stages. According to the current knowledge, there is no need to pause an ADT prior to a PSMA ligand scan.
The PSMA is not specific for PC. Since the late 1990s, it is also well-known that the neovasculature of many solid tumors may express PSMA as well. 53 This is reflected by multiple case reports describing PET-positive nonprostatic tissues. 8 However, in case of recurrent PC, 68 Ga-PSMA-11 PET/CT has showed an excellent specificity. 39,54,55 Recently, 18 F-labeled PSMA-ligands were clinically introduced with promising results such as DCFBC, DCFPyL, or PSMA-1007. 48,[56][57][58] They all show promising first results. In theory, 18 F-labeled tracers present with advantages such as higher physical resolution and high amounts of 18 F deriving from cyclotrons. However, future studies will show whether these theoretical advantages can outweigh the faster pharmacokinetics and excellent tumor contrast of 68 Ga-PSMA-11.

PSMA-Targeted Therapy
After rather unsuccessful therapy with 90 Y-CYT-356 monoclonal antibody (mAb) that binds to the intracellular domain of PSMA, 59  Current Status of PSMA-Targeted Radioligand Therapy Table 1 shows a list of current therapy molecules already administered in humans. In the first cohort of 10 patients, mild side effects and considerable rates of PSA response were reported after 1 cycle of radioligand therapy (RLT) with 177 Lu PSMA-617. 64 A decline in PSA was observed in 70% of patients, with more than 50% decline in a few patients. Another recently published study showed the safety and favorable therapy response in a larger cohort of 24 patients who received up to 2 cycles of 177 Lu PSMA-617. Twenty-two patients received 2 cycles of RLT. Eight weeks after the second cycle of 177 Lu PSMA-617 therapy, 15 (68.2%) of 22 patients experienced a PSA decline compared to the baseline PSA value, of who 15 experienced a decline of more than 30% and 13 a decline of more than 50%. 65 Relevant hematotoxicity (grade 3) occurred during the observation period (within 2 months after the last cycle) in 2 patients only. Apart from minor grade 1 or 2 hematotoxicity, the majority of patients did not have any hematotoxicity during the observation period. No relevant nephrotoxicity or hepatotoxicity was observed. The overall survival benefit of RLT in comparison to a historical control group could be demonstrated in another investigation. 66 The estimated median survival was 29.4 weeks in the group treated with 177 Lu PSMA-617, which was noticeably longer than survival in a historical control group with 19.7 weeks (hazard ratio: 0.44; 95% confidence interval: 0.20-0.95; P ¼ .031).
Baum et al presented similar results using 177 Lu PSMA-I&T 67 treating 56 patients with metastasized PC at an early stage of castration resistance. Any PSA decline and a decline of !50% were reported in 80% and a median overall survival of 13.7 months. In this cohort, prior chemotherapy was performed in 25% of patients. Prior therapy with abiraterone was performed in 21% and prior enzalutamide was given in 11% of patients, whereas in the prior study mentioned earlier, pretreatments with chemotherapy, abiraterone, and enzalutamide were administered in 78%, 75%, and 86% of patients, respectively. 66 Results of the abovementioned studies were confirmed by the findings of the German multicenter study including 145 patients with metastatic castration-resistant PC. Biochemical response was defined by the PC work group as a PSA decline !50%. 68 A PSA decline !50% and any PSA decline were reported in 45% and 60% of the patients. 69 The presence of visceral metastases and an alkaline phosphatase !220 U/L were negative predictors of therapy response. Adverse events were analyzed according to common toxicity criteria. Grade 3 to 4 toxicities such as anemia, leukopenia, and thrombocytopenia were reported in 10%, 3%, and 4% of patients treated with 177 Lu PSMA-617. Mild or transient xerostomia were reported in 8% of the patients. Repeated cycles of 177 Lu PSMA-617 RLT can be performed after radionuclide therapy using 223 Ra dichloride, without a higher probability of hematotoxicity. 69 These results about the safety and toxicity were confirmed by another study including 49 patients who were treated with 3 cycles of 177 Lu-PSMA-617. The patients were separated into 2 groups subjected to a history of prior therapy with 223 Ra. Group 1 included 20 patients who had received therapy with 223 Ra prior to 177 Lu-PSMA-617 therapy. Group 2, which was the control group, comprised 29 patients without any history of a bone-targeted radionuclide therapy. No grade 4 hematotoxicity was observed in the entire study population. There were no significant differences between the 2 groups regarding leukopenia and thrombocytopenia. 70 Figure 2A and B shows a case of a 66-year-old patient with metastasized castration-resistant PC with an excellent response to 177 Lu PSMA-617 RLT with history of prior 223 Ra dichloride therapy.
Although there is a specific renal binding of PSMA-ligands, no grade 3 to 4 toxicities were reported after the RLT. Only low-grade renal toxicity has been reported so far. 71 Table 2 presents reported toxicities of 177 Lu-PSMA RLT.
Yordanova et al analyzed changes in creatinine, cyctatin C, glomerular filtration rate, and tubular function assessed by 99m Tc-MAG3 renal scintigraphy in 55 patients receiving at least 3 (range 3-6) consecutive cycles of 177 Lu PSMA-617 every 8 weeks. Significant negative predictor for renal function were age (>65 years), arterial hypertension, and prior renal failure.
Two recently published studies 75,76 analyzed overall survival in patients receiving up to 8 cycles of 177 Lu PSMA-617 RLT. Any PSA decline after the first cycle was a significant prognosticator of survival in both studies (68 vs 33 weeks reported by the first mentioned study and 59 vs 28 weeks reported by the second-listed study). These results could be confirmed by a larger 2-center study in patients pretreated with at least 1 line of chemotherapy and abiraterone and/or enzalutamide. 77 The PSA decline of at least 20.87% was the optimal parameter in predicting improved overall survival in the multivariate analysis. The same study group also showed that in patients who do not respond to the first cycle, further therapy cycles should be performed, because nearly one-third of the patients showed a delayed response after additional therapy cycles. 78 According to currently available results, 177 Lu PSMA-617 is a new and promising therapy option for patients with metastatic PC, but it has not yet reached clinical approval. It should therefore mostly be offered to patients as a salvage therapy; however, a more upfront role of RLT with PSMA ligands leading to regulatory approval would seem prudent.
In the following, we summarize the current therapy protocol most widely used.

Indications for RLT With 177 Lu-PSMA-617
metastatic castrate-resistant PC with PSMA-positive metastatic disease upon PSMA-PET or singlephoton emission computed tomography (SPECT) imaging; after initial hormone therapy (luteinizing hormonereleasing hormone agonists/antagonists): progressive disease, despite newly developed hormone therapies (abiraterone / enzalutamide) progressive disease, despite (first line or second line) chemotherapy (docetaxel and cabazitaxel) or patient not suitable for chemotherapy not suitable for 223 Ra-dichloride due to visceral metastases or diffuse bone marrow metastases; and decision for salvage PSMA therapy at the institutional interdisciplinary tumor board.

Preparation Prior to RLT
PSMA-PET or SPECT imaging to verify PSMA positive lesions. Renal scintigraphy to evaluate renal function and rule out obstructive dysfunction, which should be treated prior to the RLT

Prerequisites for RLT
White blood cells ! 2 tsd/mL. Hb ! 8 g/dL, in the case of symptomatic anemia a red blood cell transfusion should precede the therapy. The RLT with 177 Lu-PSMA-617 may have a positive effect on bone marrow depression because of tumor regression in bone marrow. 79 Platelets ! 75 Â 10 9 /L. Creatinine < 2 mg/dL. Eastern Cooperative Oncology Group 2.

Proposed Protocol
Cooling the salivary glands from 30 minutes before and up to 4 hours after the 177 Lu-PSMA-617 injection for reducing the risk of salivary glands radiation injuries. Using urinary catheter in incontinent patients in the first 48 hours for avoiding any contamination. A dose of 6 to 7.4 GBq 177 Lu-PSMA-617. Injection intravenously as a slow bolus (over about 30 seconds) followed by 1000 mL Ringer or NaCl. Four to 6 cycles of the RLT every 6 to 8 weeks.

Follow-Up After RLT
The PSMA-PET or SPECT imaging before first cycle to confirm PSMA-positive metastatic disease and after the last cycle (interim imaging should also be performed if needed). Laboratory test after RLT should be performed every 2 weeks: blood cell counts, creatinine, and liver panel. Decision to pursue the RLT depending on the imaging and clinical situation.  64 10 10 0 0 0 mild nausea, fatigue Rahbar et al. 66 28 11 0 0 14 mild nausea Ahmadzadehfar et al. 65 24 9 0 0 8.7 mild nausea Baum et al. 67 56 0 0 0 3.5 na Kratochwil et al. 72

Future Perspectives and Remaining Issues
The PSMA-based imaging and RLT is a theranostic approach, which is currently applied in metastatic castration-resistant PC. Radioligand therapy with 177 Lu-PSMA-617 seems to be a promising treatment for metastatic patients with PC. Despite very encouraging early results, several issues will need to be resolved to clarify and establish the exact role of PSMAbased theranostics in PC. With regard to imaging, currently a number of different agents are available, and the most suitable agent from the point of view of optimal imaging characteristics, availability, access, and cost will need to be identified. Similar notion holds for RLT with bor a-particles and the specific clinical situations, which may be best amenable for either type of particles or both. Using this therapy, as a combination with newer antiandrogenic drugs such as abiraterone and enzalutamide, at earlier stages of the therapy must be evaluated in future studies. Larger prospective multicenter studies are needed to evaluate the potential of RLT among and with other available therapy options. Moreover, the long-term results and toxicities must be analyzed after longer follow-up periods. We have to point out that while preliminary protocols have been devised, both the imaging and the therapy protocols will evolve in time as experience with PSMA theranostics grows. An expanded understanding of the effects of various treatments on PSMA expression will be needed to facilitate therapy response assessment. It has been recognized that ADT increases PSMA expression which is not only relevant in recognition of posttreatment PSMA PET imaging changes but also suggests the possibility of synergistic therapeutic effect of androgen deprivation and PSMA-based RLT. Determination of the impact of PSMA theranostics on various outcome measures and comparative cost-benefit analysis will need investigations.

Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The University of Muenster received consulting fees from ABX Advanced Biochemical Compounds, Radeberg, Germany for K.R.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.


s2fieldsofstudy: Biology Medicine

text:
Chorea–Acanthocytosis and the Huntington Disease Allele in an Irish Family

The presence of peripheral blood film acanthocytes can help narrow the differential diagnosis of a familial choreiform disorder. Acanthocytosis is associated with chorea–acanthocytosis (ChAc), McLeod syndrome, pantothenate kinase-associated neurodegeneration (PKAN), and Huntington’s disease-like 2 (HDL-2).1 Huntington disease (HD) can present at a similar age with a similar phenotype, but without acanthocytosis. We report the cases of three adult siblings with genetically confirmed ChAc, and discuss the unusual finding of a co-existing abnormal HD allele (CAG repeat expansion in the range of reduced penetrance) in two of these siblings.

Introduction
The presence of peripheral blood film acanthocytes can help narrow the differential diagnosis of a familial choreiform disorder. Acanthocytosis is associated with chorea-acanthocytosis (ChAc), McLeod syndrome, pantothenate kinase-associated neurodegeneration (PKAN), and Huntington's disease-like 2 (HDL-2). 1 Huntington disease (HD) can present at a similar age with a similar phenotype, but without acanthocytosis. We report the cases of three adult siblings with genetically confirmed ChAc, and discuss the unusual finding of a co-existing abnormal HD allele (CAG repeat expansion in the range of reduced penetrance) in two of these siblings.

Case series
Three siblings developed progressive neurological syndromes beginning in their third or fourth decade (Table 1). Psychiatric symptoms were an early feature in all siblings, including anxiety and obsessive-compulsive features. Over the years, all siblings developed dysarthria and orofacial features, including self-injurious biting, feeding dystonia, and orofacial chorea. Siblings 1 and 3 developed epilepsy (complex partial seizures in sibling 1 and generalized seizures in sibling 3). Limb chorea, and frontal and temporal cognitive deficits occurred further into the disease process, and were present in the elder siblings (1 and 2) by the time of assessment. Sibling 1 died suddenly during follow-up and autopsy suggested cardiorespiratory collapse as the cause of death.

Family history
There are seven siblings in this Irish family, three of whom are unaffected. The final sibling reportedly has some neurological symptoms but has not been fully investigated. There is no other family history of neurological disorder, and no known consanguinity.

Investigations
Laboratory and imaging studies screening for biochemical, infective, or autoimmune causes were unremarkable in each case. Sibling 1 (the eldest) presented first and genetic testing for HD demonstrated that he carried a single normal allele and a HD allele with 37 CAG repeats, in the reduced penetrance range (also subsequently identified in sibling 2). Investigation for ChAc was prompted a short time later after siblings 2 and 3 were also evaluated, because of recognition of the prominent orofacial features, symptom onset in the 20s or 30s, and presence of acanthocytes in peripheral blood. Genetic analysis of the VPS13A (ChAc) gene (NM_033305.2) demonstrated that the three affected siblings are compound heterozygotes, with one copy of the Arg3143fs*5 (c.9427_ 9428delAG in exon 72) mutation that was previously reported in a homozygous state in another (unrelated) Irish family from the same region, 2 and one copy of the novel Pro322Alafs*19 (c.962dupT in exon 12) mutation. Both mutations cause a frameshift resulting in a truncated protein. Genetic analysis of the unaffected mother and one unaffected brother showed they each carry a single mutation (Pro322Alafs*19). We assume that the Arg3143fs*5 mutation was inherited from the siblings' father, who had no history of neurological or psychiatric symptoms and died in his 40s of nonneurological disease. Sibling 3 underwent HD testing at a later date and was found to have 2 normal alleles.

Freely available online
Tremor and Other Hyperkinetic Movements http://www.tremorjournal.org Columbia University Libraries Post-mortem neuropathological findings in sibling 1 Findings in the brain were interpreted as consistent with ChAc. There was prominent loss of neurons and gliosis in the caudate and to a lesser extent the putamen and pallidum, with some early similar changes within the substantia nigra. Furthermore, there was evidence of chronic cerebrovascular disease with vascular lipohyalinosis, and there were acute established changes of preterminal hypoxia-ischemia. The spinal cord and sural nerve were normal. In the deltoid and quadriceps muscles there were myopathic features with some fiber size variability, increased number of fibers containing internal nuclei, type I fiber predominance with grouping, and small type II fibers.

Discussion
ChAc is a rare autosomal recessive disorder caused by mutations of the VPS13A gene on chromosome 9, which encodes the chorein protein.
The function of this protein is not well understood, but it is thought to be involved in architecture of the cytoskeleton and cell survival. 3 Cytoskeletal abnormalities may explain the unusual erythrocyte morphology seen in this condition, but the degree of acanthocytosis is variable and does not seem to correlate with clinical severity. 4,5 Furthermore, while routine laboratory techniques actually demonstrate limited sensitivity in detecting acanthocytosis, the use of isotonically diluted blood and unfixed wet blood preparation improves sensitivity and specificity. 6 In addition, muscle creatine kinase is another useful blood marker that is almost ubiquitously elevated in affected individuals. 7 Symptoms of ChAc typically begin in the third decade (or less frequently in the fourth decade) with dysphagia, dysarthria, chorea, and unsteady or ''rubber man''' gait. 7 Involuntary movements of the orofacial region, with feeding dystonia and self-mutilation from teeth grinding and lip biting are characteristic of the disorder. Involuntary vocalizations are frequent. Psychiatric symptoms, cognitive impairment, epilepsy, and neuropathy are recognized non-motor symptoms. 7 The phenotype of our patients is strongly suggestive of ChAc, with orofacial dystonia, self-injurious behavior, and epilepsy early in the disease. Their clinical features are certainly within the range of severity and age of onset as that described in typical ChAc cases. However, the co-existing VPS13A and HTT gene abnormalities were an interesting finding in siblings 1 and 2. We propose that our patients have clear clinical manifestations of ChAc and the finding of an abnormal HD allele in the reduced penetrance range could simply be an incidental finding. Indeed, post-mortem neuropathological findings in the eldest sibling were consistent with the clinical diagnosis of ChAc, demonstrating marked gliosis and extensive neuronal loss in the striatum (less gliosis would be expected in HD, and neuronal loss should only be extensive in advanced HD). 8 Further support for the hypothesis that the abnormal HD allele is an incidental finding includes evidence that the incidence of the HD allele in the general population is almost certainly higher than previously suspected. A recent large study reported 18 of 7,315 individuals from Western populations had >36 CAG repeats (15 of these were in the reduced penetrance range of 36-39 CAG repeats), suggesting an incidence of HD gene abnormalities of approximately one in 400 in the general population. 9 While penetrance rates of up to 65% have been reported with 36-39 CAG repeats in a cohort attending for diagnostic/predictive testing for HD (i.e., in those with a known family history), 10 the penetrance rate with 36-39 CAG repeats among the general population is closer to 0.2% (although under-ascertainment of clinical cases or capturing individuals at a pre-symptomatic age is possible in a cross-sectional population study such as this). 9 On the other hand, an alternative hypothesis that should be considered is that, should the remaining affected sibling with the CAG repeat expansion live long enough, she could experience a ''double-hit'' from both genetic abnormalities, resulting in compound neurodegenerative changes in affected regions. CAG repeat length demonstrates an inverse correlation with age of onset; 11 therefore, HD alleles in the reduced penetrance range that become symptomatic typically cause late-onset disease, sometimes with no family history of the disorder. 12 However, accurately determining age of onset is challenging in HD, 13 and we expect that the co-existence of another choreiform disorder seen in our patients could make this almost impossible to define. This being said, we will certainly monitor the surviving siblings closely for evolution of phenotype over time, and it will be particularly useful to compare the clinical course of the sibling with the abnormal HD allele to the sibling without this abnormality. A final important issue raised in this family is the implications of detection of the HD allele in the reduced penetrance range for subsequent generations. Regardless of whether an individual carrying this abnormal allele becomes symptomatic in their lifetime, there is a possibility of anticipation with expansion of CAG repeats between parent and offspring, a phenomenon that occurs far more commonly with paternal transmission of the gene. 14 The deceased male sibling 1 has one son who has received genetic counseling and will be followed by clinical genetics over the long term.

Conclusion
We report an Irish kindred with classic presenting features of ChAc, who are compound heterozygotes for this rare neurodegenerative disorder, but also carry a reduced penetrance range expansion of the HTT gene. This case series emphasizes the importance of a detailed family history and clinical examination in the assessment of chorea, and the diagnostic utility of identifying acanthocytes on a peripheral blood film. With recent studies elucidating potential pathophysiologic pathways in ChAc, [15][16][17] potential therapeutic targets may be identified in future.


Using Evidence of Mechanisms to Evaluate Efﬁcacy and External Validity

Previous chapters in Part III develop accounts of how to gather and evaluate evidence of claims about mechanisms. This chapter explains how this evaluation can be combined with an evaluation of evidence for relevant correlations in order to produce an overall evaluation of a causal claim. The procedure is broken down to address efﬁcacy, external validity, and then the overall presentation of the claim. In this chapter, we move from claims about mechanisms to causal claims, i.e., claims of efﬁcacy and external validity. As we have seen in Chap. 6, in order to establish efﬁcacy, one needs to establish both the claim that there is a correlation between putative effect and putative cause and the claim that there is a mechanism connecting the putative effect and cause that can account for the size of the observed correlation. populations question

1. Is clinical study evidence strong enough to make it plausible that there is a mechanism that can account for the size of the correlation? 2. Is there a specific mechanism hypothesis and is the existence of the crucial features of that mechanism hypothesis established?
Correlation claim. The correlation claim is the claim that there is a correlation between the putative cause and effect, conditional on plausible confounders. Note that mechanistic evidence and results from previous clinical studies may rule in some variables as plausible confounders. Mechanistic evidence may also speak to the question whether a certain clinical study is well-conducted and properly controlled for these confounding variables. Given that one has settled on both a set of potential confounders and an assessment of the quality of the design of the relevant studies, deciding whether the putative cause and effect are correlated is a purely statistical question. A meta-analysis, for instance, of relevant studies yields an estimate for the size of the correlation and corresponding confidence interval and p-value. The status of the correlation claim then depends on the width of the confidence interval, the size of the p-value, and the heterogeneity of the studies evaluated. A low p-value may, for instance, lead to a high status of the correlation claim.
Efficacy claim. To obtain the status of an efficacy claim, we combine the status of the corresponding general mechanistic claim with the status of the corresponding correlation claim. Efficacy is established just when it is established that there is a correlation and that there is some mechanism which can account for this correlation (Russo and Williamson 2007;Illari 2011;Clarke et al. 2013Clarke et al. , 2014. More generally, the status of the causal claim can be taken to be the minimum of two statuses: the status of the correlation claim and the status of the general mechanistic claim: Status of an efficacy claim. The status of the claim A is a cause of B is the minimum of: 1. the status of the claim that A and B are appropriately correlated, and 2. the status of the claim that there is an appropriate mechanism linking A and B that can account for this correlation. Hence, a causal claim cannot have a higher status than both the correlation claim and the general mechanistic claim (see discussions in (Russo and Williamson 2007, 2012Russo 2011;Russo 2016, 2017)). To give an example, efficacy is provisionally established if the existence of a correlation is established or provisionally established and the existence of a mechanism that can account for the correlation is provisionally established. Equally, efficacy is provisionally ruled out if a correlation is provisionally ruled out and if the existence of a mechanism that can account for the correlation is provisionally ruled out or of higher status.
Before turning to external validity, we discuss a potential source of confusion: Digression: reinforced concrete. In the framework set out above, there are two separate distinctions in play. First, there is the distinction between evidence of correlation and evidence of mechanisms (Illari 2011). This distinction is core to the approach taken in this handbook: the claim that A is a cause of B is evaluated according to how strongly evidence of correlation supports the claim that A and B are appropriately correlated, and how strongly evidence of mechanisms supports the general mechanistic claim that there is a mechanism linking A to B that can account for the correlation. Second, there is a distinction between clinical studies (which repeatedly measure A and B together) and mechanistic studies (which investigate the details of a putative mechanism linking A and B). It is important to note that these two distinctions do not align. Both clinical and mechanistic studies can provide evidence of correlation (though clinical studies often provide better evidence of correlation than mechanistic studies). Similarly, both clinical and mechanistic studies can provide evidence of mechanisms (although mechanistic studies often provide better evidence). See Clinical studies and mechanistic studies can be mutually reinforcing. Consider an analogy to reinforced concrete, which is formed by placing steel grids into concrete (Clarke et al. 2014). Concrete has high resistance to compressive stresses but fractures under tension. Steel, however, has high strength in tension. So, if steel is placed in concrete to produce reinforced concrete, we get a composite material where the concrete resists the compression and the steel resists the tension. The combination of two different materials produces a material that is much stronger than either of its components. In the same way, combining clinical studies with mechanistic studies produces much stronger overall evidence of efficacy than would either type of evidence on its own, because they compensate for each other's weaknesses. For instance, clinical studies can rule out masking: masking occurs when one or more counteracting mechanisms cancel out the effect of the mechanism of action. On the other hand, mechanistic studies can rule out confounding.
The following scenarios illustrate the idea of reinforced concrete. Scenario 1. Suppose, for instance, that many well conducted RCTs consistently show a correlation between the putative cause and effect and that bench research provides only very low quality evidence for the general mechanistic claim that there exists a mechanism that can account for the size of the correlation. In this case, it might seem that the correlation is established and the existence of the mechanism is speculative. In which case, efficacy is only speculative. However, this misrepresents the evidence for the general mechanistic claim. It confuses evidence obtained only by bench research with total evidence of mechanisms from all sources. Recall from Sect. 6.3 that clinical studies may also yield evidence relevant to the general mechanistic claim that there exists a mechanism-see Joffe (2011) and Williamson (2018, Sect. 2.1). In the above example, the RCTs, when combined with the bench research, can yield a status for the general mechanistic claim that is higher than speculativean application of the reinforced concrete metaphor. Accordingly, the efficacy claim will have a status higher than speculative.
Scenario 2. Suppose low quality clinical studies suggest that there is a correlation. Suppose too that high quality mechanistic studies support key aspects of a specific mechanism hypothesis, but that the possibility of a counteracting mechanism cannot be ruled out. In this case, it is not clear that the proposed mechanism of action can account for the observed correlation, and the general mechanistic claim will not be established. Subsequently, high quality clinical studies are carried out and determine that the net correlation is indeed positive. These studies provide evidence that any counteracting mechanism fails to totally mask the effect of the mechanism of action. The total body of evidence may now suffice to establish the general mechanistic claim (see Sect. 6.3). In this scenario, clinical studies reinforce mechanistic studies when evaluating the general mechanistic claim.
Scenario 3. Suppose certain clinical studies provide low quality evidence of a correlation. One might think that the key concern is confounding, so that when there is high quality evidence of mechanisms that rules out confounding, efficacy is established. However, confounding is not the only problem that arises with low quality evidence of correlation. There is also the problem that the observed correlation may not correspond to a correlation in the underlying data-generating probability distribution. In order to establish efficacy, one needs to establish that there is a genuine correlation in the underlying distribution. Hence, without high quality evidence of correlation, efficacy cannot be established.
Scenario 4. Suppose that initially, certain clinical studies provide low quality evidence of a correlation. Suppose that in this case, it is clear that the studies identify a genuine correlation conditional on certain potential confounders, but that not all plausible confounders have been controlled for. The key concern here, then, is confounding. For instance, there might be a large number of epidemiological studies all showing a correlation between putative cause and effect, but where each study fails to control for some particular variable which may be a confounding variable. Now, if there is also high quality evidence of mechanisms that rules out this variable as a confounder, efficacy is established. In this case, the mechanistic studies boost the status of the correlation claim, to established. In this case, then, the overall status is established.

External Validity
When mechanisms within a study population and the target population are sufficiently similar, one can extrapolate an efficacy claim from the study population to the target population. In this section, we show how to combine evidence of efficacy obtained directly on the target population with evidence obtained by extrapolation from a study population.
Three assessments feed into the evaluation of effectiveness: 1. Efficacy in the target population. Although studies performed directly on the target population will normally be less conclusive than those performed on the study population, they can form the basis of a preliminary evaluation of efficacy in the target population. The preliminary status of the causal claim can be determined as set out in Sect. 7.1. 2. Efficacy in the study population. The status of efficacy in the study population can also be determined by considering the procedure of Sect. 7.1. 3. Similarity of mechanisms in the study and target populations. The status of the general mechanistic claim relevant to external validity (i.e., the claim that the mechanisms of action are sufficiently similar in study and target) can be determined as indicated in Sect. 6.3.
To obtain a final status for efficacy in the target, one can combine the preliminary status in the target population with the status of efficacy in a study population, provided that study and target population share similar mechanisms of action. The status of the causal claim about the target population may be increased (respectively, decreased) by observing that efficacy does (respectively, does not) hold in a study population that is similar to the target population. In this case, causal claims are extrapolated from the study population to the target population. Table 7.1 shows how the status of the causal claim in the target population can be determined from the above three assessments. To change the preliminary status of an efficacy claim given by studies directly on the target population, all evidence of causation in the study population and of similarity of mechanisms needs to be of at least moderate quality, and one or other needs to be high quality. Other quality levels do not change the initial status.
Some remarks help to explain the table and relate it to other approaches that address external validity.
1. If studies on the target population would on their own establish causality in the target population, this is strong, but not infallible, evidence for causation in the target. If there is a study population for which similarity to the target has been established but causation has been ruled out in the study population, then causation in the target population is downgraded to provisionally established.
(Note that this situation is not covered by the protocol for evaluating external validity advocated by the International Agency for Research on Cancer (IARC); see Sect. 8.1 for further discussion of this point.) 2. Changing the preliminary status of a causal claim obtained from evidence gathered on the target population is more common when that evidence is of lower quality. For instance, a provisionally established status may be changed to established only in case of established efficacy in the study and established similarity between study and target. The status arguable, however, may be changed in case of established efficacy in the study and provisionally established similarity. 3. The GRADE working group also evaluates whether evidence from a study population can be used to draw inferences about the target population. In particular, the GRADE working group considers the case where no evidence directly obtained on the target population is available: In general, one should not rate down for population differences unless one has compelling reason to think that the biology in the population of interest is so different from that of the population tested that the magnitude of effect will differ substantially. Most often, this will not be the case.
[...] The above discussion refers to different human populations, but sometimes the only evidence will be from animal studies, such as rats or primates. In general, we would rate such evidence down two levels for indirectness (Guyatt et al. 2011(Guyatt et al. , pp. 1304(Guyatt et al. -1305 Hence, the GRADE working group takes similarity of mechanisms to be established by default when study and target populations are both human populations. This is problematic because it sets the standard of evidence required for extrapolation too low. In the case of animal studies, one can interpret the default assumption of the GRADE working group as being that the causal claim is arguable solely on the basis of causation in animals having been established. Again this is problematic. In our approach, in the absence of evidence of similarity of mechanisms, efficacy in the study population cannot be extrapolated to the target. Hence, even if many high quality RCTs in animals establish efficacy in animals, in the absence of evidence of similarity, nothing can be concluded about efficacy in humans. There is thus a sense in which the approach presented here is more cautious than the GRADE approach to external validity. 4. Causation can be established or ruled out even where no clinical studies on the target are available. This is the case when causation has been established in a study population for which it has been established that it is mechanistically similar to the target population. (This case is captured by the fourth row of Table 7.1, where causation in the target is speculative.) 5. Note that, by similarity of mechanisms we mean that any mechanisms in the target population which counteract this mechanism do not mask the effect of the mechanism of action to such an extent that a net correlation in the target population could not be explained mechanistically (see Sect. 2.3). Consequently, with a mechanism established and some counteracting mechanisms established in the study, a small correlation may be good evidence for causation in the target even if it is not the case that the whole mechanistic structure is similar. After all, this counteracting mechanism would only make the existent correlation smaller in the study than in the target.

Presenting the Status of a Causal Claim
In presenting the status of a causal claim the following questions need to be addressed, and the status of the causal claim presented after the evaluation of evidence.
Presenting the status of the efficacy claim. The following questions should be addressed: 1. What is the population to which the status applies? 2. What is the intervention or exposure level? 3. What is the outcome and how is it measured? 4. What is the status of the correlation claim? How is this status obtained? 5. What is the status of the general mechanistic claim? How is this status obtained? (See Chap. 6.) 6. What is the status of the efficacy claim?
The following box considers the case where efficacy is extrapolated from one to another population Presenting the status of the effectiveness claim. The following questions should be addressed: Standard evidence appraisal systems can be extended to take these considerations into account. For an example of how to incorporate certain aspects of this procedure into a GRADE-style evidence profile, see Sect. 4.6.
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extfieldsofstudy: Medicine Biology

text:
Rhythm and synchrony in animal movement and communication

Animal communication and motoric behavior develop over time. Often, this temporal dimension has communicative relevance and is organized according to structural patterns. In other words, time is a crucial dimension for rhythm and synchrony in animal movement and communication. Rhythm is defined as temporal structure at a second-millisecond time scale (Kotz et al. 2018). Synchrony is defined as precise co-occurrence of 2 behaviors in time (Ravignani 2017). Rhythm, synchrony, and other forms of temporal interaction are taking center stage in animal behavior and communication. Several critical questions include, among others: what species show which rhythmic predispositions? How does a species’ sensitivity for, or proclivity towards, rhythm arise? What are the species-specific functions of rhythm and synchrony, and are there functional trends across species? How did similar or different rhythmic behaviors evolved in different species? This Special Column aims at collecting and contrasting research from different species, perceptual modalities, and empirical methods. The focus is on timing, rhythm and synchrony in the second-millisecond range. Three main approaches are commonly adopted to study animal rhythms, with a focus on: 1) spontaneous individual rhythm production, 2) group rhythms, or 3) synchronization experiments. I concisely introduce them below (see also Kotz et al. 2018; Ravignani et al. 2018).

Introduction
Animal communication and motoric behavior develop over time. Often, this temporal dimension has communicative relevance and is organized according to structural patterns. In other words, time is a crucial dimension for rhythm and synchrony in animal movement and communication. Rhythm is defined as temporal structure at a second-millisecond time scale . Synchrony is defined as precise co-occurrence of 2 behaviors in time .
Rhythm, synchrony, and other forms of temporal interaction are taking center stage in animal behavior and communication. Several critical questions include, among others: what species show which rhythmic predispositions? How does a species' sensitivity for, or proclivity towards, rhythm arise? What are the species-specific functions of rhythm and synchrony, and are there functional trends across species? How did similar or different rhythmic behaviors evolved in different species? This Special Column aims at collecting and contrasting research from different species, perceptual modalities, and empirical methods. The focus is on timing, rhythm and synchrony in the second-millisecond range.
Three main approaches are commonly adopted to study animal rhythms, with a focus on: 1) spontaneous individual rhythm production, 2) group rhythms, or 3) synchronization experiments. I concisely introduce them below (see also Kotz et al. 2018;Ravignani et al. 2018).

Spontaneous Individual Rhythm Production
Spontaneous individual rhythms deal with the temporal structure of individual behaviors, mostly in a non-interactive context. The adjective "spontaneous" here denotes a general methodological trend in observing or recording how an animal behaves spontaneously rather than in response to experimental manipulations. These individual rhythms have been studied in several taxonomic groups. African apes and songbirds are good examples of this (e.g., Arcadi et al. 2004;Trejos-Araya and Barrantes 2018).
Both chimpanzees and bonobos show forms of individual rhythmic behaviors. Bonobos perform "staccato hooting" displays, consisting of series of vocalizations which seem to repeat metronomically twice a second (de Waal 1988;Bermejo and Omedes 1999). In chimpanzees, instead, rhythmic sound production is mostly non-vocal, consisting of sequences of percussive sounds. This so-called "buttress drumming" can be observed in wild chimpanzees, who drum on hollow trees (Arcadi et al. 2004), and their captive conspecifics, who will employ any resonant object to produce loud sequences of sounds (Dufour et al. 2015;Ravignani et al. 2013aRavignani et al. , 2013b. Zebra finches are songbirds whose songs have been thoroughly studied. Classical work on zebra finches has explored the complex ways in which syllables (i.e., elements of a song) are organized, learnt, and reused to produce complex vocal displays (Scharff and Nottebohm 1991;Feher et al. 2009;Lipkind et al. 2013). Recent work, however, has also explored the rhythmic dimension of zebra finches' songs (Saar and Mitra 2008;Benichov et al. 2016;Norton and Scharff 2016;Spierings and ten Cate 2016). For instance, when metronomically-occurring (i.e. isochronous) sounds are overlaid to recordings of zebra finch songs, the syllables' onsets occur-more often than not-on the metronome clicks (Norton and Scharff 2016). Hence, these songbirds' songs appear rhythmically structured so that syllables occur at specific points in time.

Group Rhythms
Beyond individual rhythms, animals can produce vocal or behavioral rhythms in an interactive, coordinated manner (e.g., Couzin 2018). The classical framework of chorusing traditionally studies this kind of phenomena, even in non-auditory modalities (Ravignani et al. 2014). Duets are the simplest form of group chorusing, at least in terms of participants. Examples of duetting include those performed by birds, gibbons, lemurs and anurans. In the study of the precise rhythmic structure of duetting, mammals seem to be historically neglected. For instance, mated gibbons have been reported to sing in pairs, to enhance pair-bonding and defend a territory (Geissmann 2002). From an observational perspective, these duets show good temporal coordination, with gibbon pairs alternating synchronous and antiphonal parts. Quantitative studies, comparable to those in other taxonomic groups, to test the precise mechanisms of vocal rhythmicity in gibbons have only recently appeared (Terleph et al. 2018a(Terleph et al. , 2018b From duets, when we increase the number of participants in a group rhythm, we find for instance "quartets." Neotropical plain-tailed wrens Thryothorus euophrys are known for their 4-parts choruses (Mann et al. 2006). Males and females alternate in their performance of complementary parts, whereas birds of the same sex synchronize.
A particularly fruitful strand of research over the last century has examined chorusing in orthopterans (crickets, bush crickets, etc.) and anurans (Ravignani et al. 2014). One orthopteran species, Neoconocephalus spiza, displays group choruses of hundreds of insects (Greenfield and Roizen 1993). All insects stridulate isochronously (i.e., metronomically) with a constant and small delay between pairs of individuals. More elaborate group rhythms can be seen in the Indo-Malayan katydid of the genus Mecopoda. As in Neoconocephalus, all Mecopoda individuals produce isochronous sounds, with potentially individual-specific periods (Sismondo 1990). When neighbors have similar sound production periods, synchrony, or antiphony ensues. When neighbors have different sound production periods, they slightly adapt to each other and end up being related by small integer ratios (e.g., 2:3). Finally, other species perform rhythmic group displays in non-auditory modalities. Fireflies (e.g., Pteroptyx malaccae) perform visual displays, competing to be the most conspicuous signaler (i.e., the first one to signal) and hence to attract potential mates (Greenfield 2005). This competition for conspicuousness, however, leads to the opposite of individual conspicuousness (as in the Neoconocephalus spiza example above), with hundreds of animals all flashing in perfect synchrony.

Synchronization experiments
A third strand of animal rhythm research stems more from comparative psychology, rather than zoology and bioacoustics. Focusing on the concept of synchrony and prediction, synchronization experiments somehow combine the 2 categories above. In other words, the capacities to partake in group rhythms are tested in isolation. In synchronization experiments, isolated individuals are usually exposed to sounds (or visual stimuli, Takeya et al. 2017;Takeya et al. 2018), which can be as simple as metronomic clicks or much more structured (Patel et al. 2009a;Cook et al. 2013). The typical task consists in 1) extracting a periodic structure from the sound stream, 2) building expectations about future incoming events, and 3) adjusting future behaviors predictively so that they occur in synchrony with the external periodicity extracted from the sound stream ). This work has been extensively reviewed elsewhere (Ravignani et al. 2013b;Patel 2014;Ravignani and Cook 2016;Wilson and Cook 2016;Kotz et al. 2018). However, it is important to notice that a trademark of synchronization experiments is cross-modality. In other words, the modality involved in perceiving a rhythmic stimulus is often (required to be) different from the modality used to produce a synchronous rhythmic behavior (Patel et al. 2009a(Patel et al. , 2009b.

Contributions to This Special Column
Each paper in this Special Column belongs to one of the 3 macroareas described above, or their intersection. Contributed papers are beautifully diverse along several dimensions. Here, instead of rehashing the abstract of each individual paper, I will attempt to point out similarities and differences across papers along a few key dimensions.
Animals discussed include fiddler crabs (Backwell 2019), lemurs (De Gregorio et al. 2019), macaques (Katsu et al. 2019), seals (Ravignani et al. 2019), parrots, and humans (Seki and Tomyta 2019). This is a diverse group of animals, and species' diversity was one of the aims of this Special Column. This diversity is needed if one wishes to understand why animals have rhythm, especially the phylogeny and function of rhythmic behaviors.
Which aspects of temporal structure are tackled in each paper? Two papers investigate the classical issue of synchrony (Backwell 2019;Seki and Tomyta 2019), each however with a fresh spin. Backwell (2019) notices how, across species, synchrony is the norm in the auditory modality, but quite an exception in the visual modality. In addition, by investigating different species of fiddler crabs, one could aim at reconstructing "rhythmic phylogenies." Seki and Tomyta (2019) also focus on synchrony, but do so using a paradigm uncommon in primate and bird experiments. Instead of reinforcing the animals to synchronize, they show how an isochronous metronome sound pushes bird pecking and human tapping behavior to be more synchronous with the metronome. Two more papers tackle the reciprocal influence of individual timing in primate duets (Katsu et al. 2019) and choruses (De Gregorio et al. 2019). Even the one paper where rhythm is studied at the within-individual level and in isolation shows some signatures of interactivity (Ravignani et al. 2019). In fact, harbor seal pups, even when vocalizing in isolation, produce call sequences with a rhythmic structure quantitatively closer to non-isochronous, interactive behaviors than to monologue-like "solo" sequences (Falk and Kello 2017;Kello et al. 2017;Ravignani et al. 2019).
Statistical methods used to infer rhythmic patterns were also quite diverse. Classical, linear statistical methods are of course a first, necessary approach when probing temporal structure in animal interactions (De Gregorio et al. 2019;Ravignani et al. 2019). Phase response curves (Greenfield and Roizen 1993) are quite effective in predicting how the behavior of an individual will be shifted in time depending on the exact time of occurrence of a conspecific behavior (Backwell 2019;Katsu et al. 2019). Circular statistics (Fisher 1995;Berens 2009;Zar 2010) are also increasingly used in animal rhythm research (Backwell 2019; Seki and Tomyta 2019). Ideally, circular statistics can and should be used when 1) two series of temporallystructured behaviors co-occur, 2) one of which is isochronous. If these conditions hold, circular statistics enable testing the relative phase delay of an individual behavior with respect to the constant period length of another individual's behavior (see e.g., Cook et al. 2013; Ravignani and de Reus in press). A possible visual counterpart of circular statistics is the rose plot (e.g., Cook et al. 2013; Ravignani and de Reus in press): a clock-like histogram showing how often an individual phase delay occurs relative to another individual or a metronomic stimulus. Finally, a few techniques from physics, such as the Allan Factor and burstiness, appear quite promising for quantifying rhythmicity and interactivity of behaviors in time (Goh and Barabá si 2008;Kello et al. 2017;Ravignani et al. 2019).
Insights can be gained by comparing the interpretation of results across contributed papers, focusing on 2 in particular. From several perspectives, the indri (De Gregorio et al. 2019) and macaque (Katsu et al. 2019) work are quite similar, both investigating the temporal dynamics of interactive calling. However, while the coordinated vocal behavior in indri is termed "chorusing" (De Gregorio et al. 2019), a similar behavior in macaques is called "turn-taking" (Katsu et al. 2019). Is the difference between chorusing and turn-taking substantial, or does it stem from naming conventions used in different research traditions Ruch et al. 2018)? In particular, similar behaviors consisting of rhythmic vocal interactions are labeled as chorusing by some (e.g., Kotz et al. 2018;Ravignani et al. 2014;Greenfield and Roizen 1993) while as turn-taking by others (e.g., Demartsev et al. 2018;Pika et al. 2018;Takahashi et al. 2013).
Going Forward: What's Next?
All contributions exemplify, in one way or another, some of the current and possibly future trends in this field.
First of all, we see the importance of species' diversity. The study of animal rhythm and synchrony has historically focused on 6 taxonomic groups: primates, birds, anurans, crabs, orthopterans, and fireflies. Primate focus has historically been on Simiiformes (or Anthropoidea), leaving out tarsiers, lorises and lemurs. Likewise, the rhythmicity of songbirds has been mostly investigated, leaving out a large amount of interesting avian species. Beyond the groups discussed here, many other species perform group displays which may have a precise temporal structure and be worth exploring rhythmically. Domestic dogs and wolves howl in groups. These animals are quite common in several parts of the world, so it is surprising that the precise timing of their choruses remains unexplored. Within mammals, rhythm in marsupials and monotremes remains completely unexplored; studying rhythm in these 2 clades would be particularly intriguing, especially considering how different their brains are from those of placental mammals (Suá rez et al. 2018). In particular, work targeting complex, bilateral rhythmic coordination (Sternad et al. 2007) would be particularly interesting due to the acallosal brains of marsupials and monotremes.
Second, we see an increasing emphasis on cross-species comparison and methodological comparability. An example of this is the human-avian comparative experiments in Seki and Tomyta (2019), where instructions to human participants are minimized, making one step towards testing species in comparable ways. Future comparative research should strive to achieve, as much as possible, a good tradeoff among testing (1) top-down, theoretically-driven concepts (2) in an ecologically-relevant, possibly species-specific setup, (3) while achieving cross-species comparability.
Third, in contrast with research in comparative psychology, we see attempts to connect rhythmic behaviors with evolution and ecology (Backwell 2019;De Gregorio et al. 2019;Ravignani et al. 2019). As previously done with other behavioral traits, a long-term The song repertoire of some songbird species goes through different developmental phases of exposure, learning, rehearsal, etc. Although songs have been mostly studied from a spectral and combinatorial perspective, also their rhythmic properties should vary and consolidate as individuals grow (Feher et al. 2009;Norton and Scharff 2016).

Mechanism
Rhythm mechanisms concern the neural and biological predispositions underlying rhythmic, synchronous or coordinated behavior.
The midbrain structures underpinning the metronomic, isochronous tail-wagging of dogs .

Function
Rhythm function concerns the evolutionary pressures that made a particular rhythmic behavior arise in a species.
Rhythmic behavior in some insect species may have evolved as response to pressures for mate attraction, whereas in some primates might have evolved for pair bonding and territorial advertisement (Ravignani et al. 2014). Phylogeny Rhythm phylogeny concerns the evolutionary tree of specific rhythmic behaviors, and whether similar behaviors are present in closely related species.
Most Otariid pinnipeds have a very isochronous vocal rhythm (the typical sea lion barking) and similar across Otariid species. Phocid species have quite diverse vocal rhythms. It appears that phylogeny may play a stronger role in Otarid rather than Phocid pinnipeds (Schusterman 1977;Ravignani 2018a;Terhune 2018). Glossogeny Rhythm glossogeny concerns the cultural transmission and change (often termed "cultural evolution") of rhythmic behaviors, as opposed to the biological predispositions underlying these behaviors.
Although still unclear to which features of animal rhythmic behaviors glossogeny may apply, songs of birds, cetaceans, and pinnipeds are promising candidates (Feher et al. 2009;Rogers 2017;Ravignani 2018a;Schneider and Mercado 2018) Tinbergen's approach (1963) can help answering whether and why a given species "has rhythm" . At least 4 types of questions can be asked about a particular behavior (Tinbergen 1963); this behavior is, in our case, rhythm ). The so-called "proximate causes" are ontogeny and mechanism. The "ultimate causes" are phylogeny and function, which may be more difficult to tackle empirically for the case of rhythm. Tinbergen's (1963) 4 questions can be enriched by Fitch's (2008;2015) "fifth question": glossogeny (last row).
goal of this strand would be to construct "rhythmic phylogenies" , and map their interaction with environmental constraints (Terhune 2018). Fourth, a new strand of animal rhythm experiments seems to focus on spontaneous rhythmic interaction. Seki and Tomyta (2019), in particular, test whether synchrony can be spontaneously achieved in a species whose common behavioral repertoire does not include synchrony. Some parrots in Seki and Tomyta (2019) indeed show a tendency towards synchronizing. Compare this with a recent experiment in a harbor seal pup (Ravignani 2018b, in press), where sound playbacks of conspecific calls elicited antisynchronous (rather than synchronous) vocal responses. In addition, spontaneous rhythmic interaction connects to 2 related themes: learning and ontogeny. Future questions on rhythm learning and ontogeny will include: How much learning do specific rhythmic behaviors require (e.g., Cook et al. 2013), as opposed to mere exposure triggering behavioral predispositions (e.g., Backwell 2019)? How do (possibly learnt) rhythmic behaviors develop over the lifespan (e.g., Ravignani 2018a vs. Ravignani et al. 2019)?
Fifth, rhythmic interactivity appears to have taken center stage (see also Pika et al. 2018;Ravignani and de Reus in press;Ruch et al. 2018). All papers in this issue, together with several others which appeared over the last few years, deal with interactive rhythms. This trend mirrors what has been happening in human cognitive neuroscience, where individual-centered research has gradually left room for group experiments. If we can learn anything from past human work is that maintaining good experimental control in group experiments is hard, but achievable.
Finally, 2 more trends, which are not represented here but due to be more prominent in this field, are neuroscience and genetics. Once a behavior is understood, neuroscientific methods enable a deeper understanding of its nature in light of all of Tinbergen's 4 questions. Neuroscientific approaches are, of course, particularly relevant to understand mechanisms . And they also need not be invasive (e.g., Honing et al. 2018). Likewise, genetics will be helpful to map rhythmic phylogenies. Along these lines, pioneering work has already been performed in insects (e.g., Zhou et al. 2011).
In short, rhythm and synchrony in animal movement and communication is an exciting multidisciplinary field developing at a fast pace. Many questions are open, and numerous low-hanging fruits are ready for grabs to those interested in the topic. In particular, datasets to answer several questions have already been collected, but only analyzed in their spectral dimension. The field of animal rhythms is open to those who want to approach it from their own unique perspective. I am extremely curious and positive about how the field will look like in 20 years from now.