Prognostic impact of retropharyngeal lymphadenopathy in early-stage HPV-associated oropharyngeal cancer: Implications for staging optimization.

OBJECTIVES: We analyzed the prognostic impact of retropharyngeal lymphadenopathy (RPL) in stage I node-positive HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). MATERIALS AND METHODS: We performed a centralized and blinded radiographic review of the pre-treatment images of 234 consecutive patients with AJCC 8th edition stage I cT1-2N1 HPV-associated OPSCC treated with definitive chemoradiation from 2006 to 2016. Five-year disease control and survival outcomes were reported. The prognostic significance of RPL was evaluated through multivariable analysis adjusting for age, smoking history (<10 vs. >10 pack-years), and systemic regimen received. RESULTS: Median follow-up for surviving patients was 49 months (range: 16-121). RPL was associated with increased locoregional recurrence (LRR) (17.0% v. 3.4%, p = 0.01) and distant metastasis (DM) (29.1% v. 5.9%, p = 0.001) and inferior progression-free survival (PFS) (55.6% v. 88.2%, p < 0.001) and overall survival (OS) (60.6% v. 91.2%, p < 0.001). In stage I patients who did not receive high-dose cisplatin (HDC), RPL was associated with worse LRR (p = 0.04), DM (p = 0.03), PFS (p < 0.001), and OS (p < 0.001), whereas in those who did receive HDC, RPL was only associated with increased DM (p = 0.002) and inferior PFS (p = 0.04). CONCLUSION: This study suggests that RPL portends a poor prognosis in stage I node-positive HPV-associated OPSCC. The negative impact on LRR may have been mitigated by receipt of HDC. Outcomes of stage I disease with RPL were comparable to historical reports of patients with more advanced-stage disease. Incorporation of RPL into future disease staging should be considered in order to optimize risk-stratification and exclude unsuitable candidates from treatment de-intensification efforts.

Oropharyngeal Squamous Cell Carcinoma in 390 Patients: Analysis of Clinical and Histological Criteria Which Significantly Impact Outcome.

This study evaluates the prognostic impact of several factors in oropharyngeal squamous cell carcinoma (OPSCC), controlling for human papillomavirus (HPV)-associated tumors and stage (American Joint Committee on Cancer 8th edition). All patients in Southern California Permanente Medical Group diagnosed with OPSCC between 2006 and 2012 tested for p16 immunohistochemistry were included. Review of all pathology materials was combined with central p16 testing. Multivariable analyses were performed. The cohort of 390 patients included 342 p16-positive and 48 p16-negative tumors. For all-comers, on univariate analysis, the following factors, when present, were associated with improved patient survival: p16-positive tumor (n = 324, p < 0.001); crypt versus surface tumor location (n = 312, p = 0.004); nonkeratinizing type (n = 309, p < 0.0001); nonkeratinizing with maturation type (n = 37, p < 0.0001); basaloid pattern (n = 284, p = 0.005); and a broad, pushing border of infiltration (n = 282, p = 0.004). Inferior survival outcomes were observed with: age ≥ 55 years (p < 0.0001); ≥ 10 pack-year smoking history (n = 183, p = 0.003); increasing tumor stage (p < 0.0001); overt radiographic extranodal extension (ORENE) (n = 58, p < 0.0001); low level IV/Vb lymph node involvement (n = 45, p = 0.0002); a jagged pattern of infiltration (n = 76, p = 0.0004); tumor ulceration (n = 76, p = 0.0004); absent lymphocytic infiltrate (p < 0.0001); and concurrent dysplasia (n = 125, p = 0.009). On multivariable analysis, accounting for patient age, smoking history ≥ 10 pack-years, and TNM stage, for patients with p16-positive disease, advanced TNM stage (p = 0.007), the presence of ORENE (p = 0.0002), and low-neck lymphadenopathy (p = 0.0001) were independent negative prognostic factors for disease free survival (DFS). Older age (p < 0.0001), smoking history ≥ 10 pack-years (p = 0.02), advanced TNM stage (p = 0.0002), ORENE (p = 0.004), and low-neck lymphadenopathy (p = 0.002) were independent negative prognostic factors for OS. Among patients with p16-positive OPSCC, older age, smoking history, advanced stage, ORENE, and low-neck lymphadenopathy were significant negative prognostic factors for DFS and/or OS. Further refinement of staging to incorporate additional lymph node findings may be warranted.

Triweekly carboplatin as a potential de-intensification agent in concurrent chemoradiation for early-stage HPV-associated oropharyngeal cancer.

OBJECTIVE: We compared high-dose cisplatin (HDC) vs. triweekly carboplatin (TC)-based chemoradiation in patients with HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). MATERIALS AND METHODS: A retrospective review was conducted from 2006 to 2015 of 421 patients with locally advanced p16-positive OPSCC receiving definitive radiotherapy concurrent with 3 cycles of HDC (100 mg/m2, n = 230) or TC (AUC = 5, n = 191). Three-year locoregional recurrence (LRR), distant metastasis (DM), overall recurrence rate (ORR), overall survival (OS), and cause-specific survival (CSS) are reported. HDC and TC were compared accounting for age, sex, comorbidity index score, smoking history, T stage, and N stage. RESULTS: For all-comers, no difference was observed between HDC and TC for any outcome except for ORR which was lower in patients receiving HDC (12% vs. 17%, p = 0.03). On stage-based analysis, no difference was observed between agents for any outcome for stage I or II disease. However, patients with stage III disease receiving HDC had lower rates of LRR (9% vs. 21%, p = 0.03), DM (7% vs. 28%, p = 0.006), and ORR (14% vs. 40%, p = 0.002), and superior OS (89% vs. 78%, p = 0.04) and CSS (95% vs. 80%, p = 0.02). Patients receiving HDC experienced higher rates of grade 3 leukopenia (25% vs. 11%, p < 0.001), weight loss ≥20% from baseline (21% vs. 8%, p < 0.001), and gastrostomy-tube placements (66% vs. 27%, p < 0.001). CONCLUSION: TC demonstrated comparable outcomes to HDC for stage I or II HPV-associated OPSCC but was inferior to HDC for stage III disease. TC was associated with less toxicity and may be a potential de-intensification agent for early-stage disease.

Impact of chemotherapy regimen on treatment outcomes in patients with HPV-associated oropharyngeal cancer with T4 disease treated with definitive concurrent chemoradiation.

OBJECTIVES: Although human papilloma virus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is typically associated with a good prognosis, patients with T4 disease experience relatively high rates of treatment failure. Our aim was to identify predictors of relapse among patients with clinical T4 disease. MATERIAL & METHODS: A retrospective review was conducted of 93 consecutive patients who underwent definitive concurrent chemoradiation for HPV-associated OPSCC with clinical T4 disease from July 2006 to December 2015. Three-year outcomes, including locoregional recurrence (LRR), distant metastasis (DM), overall survival (OS), and cancer-specific survival (CSS), were examined and reported from the date of treatment completion. Multivariable analysis using a Cox proportional hazards model was performed to test associations between outcome and patient and disease characteristics as well as chemotherapy regimen (high-dose cisplatin (HDC) vs. other). RESULTS: Median follow-up for surviving patients was 50 months (range 18-133). For all-comers, 3-year rates of LRR, DM, OS, and CSS were 15%, 19%, 79%, and 86%, respectively. On multivariable analysis, the only factor prognostic for patient outcomes was the chemotherapy regimen. For patients who received HDC vs. an alternative regimen, 3-year LRR, DM, OS, and CSS, were 9% vs. 20% (p = 0.09), 10% vs. 28% (p = 0.04), 89% vs. 67% (p = 0.04), and 96% vs. 77% (p = 0.02), respectively. CONCLUSION: In patients with HPV-associated OPSCC bearing clinical T4 disease, receipt of a concurrent systemic agent other than HDC resulted in increased treatment failure and inferior survival. This analysis suggests that HDC should remain the preferred concurrent regimen for these patients.

Utilization of a Web-Based Conferencing Platform to Improve Global Radiation Oncology Education and Quality-Proof of Principle Through Implementation in India.

PURPOSE: Chartrounds (www.chartrounds.com) was established in the United States in 2010 as a web-based platform for radiation oncologists to review cases with leading disease-site experts. However, the need for access to experts for peer review and education is not unique to the United States, and the Chartrounds platform was therefore adapted for improved global reach. Chartrounds was first expanded to India, and herein we report our initial experience with this initiative. METHODS AND MATERIALS: The US Chartrounds platform was adapted to create Chartrounds India (ind.chartrounds.com). Through collaboration with the Association of Radiation Oncologists of India, India-based specialists were recruited, and the association's membership list was used to announce sessions to potential participants. RESULTS: Between June 2017 and January 2018, 27 Chartrounds India sessions were completed, led by 21 different specialists (representing 10 centers in India) and covering 11 different disease sites/topics. A total of 240 members from 126 centers (private: 56%; teaching: 36%; public: 8%) across 24 states/territories participated in ≥1 session. Of the 240 members who participated in ≥1 session, 159 (66%) participated in ≥2 sessions and 60 (25%) participated in ≥5 sessions. The average number of participants per session was 34 (range, 13-72). On average, 80% of respondents rated the sessions as high or very high quality; 87% and 95% agreed or strongly agreed that the time was used effectively and that the sessions were relevant to daily practice, respectively. Seventy-six percent agreed or strongly agreed that the sessions will result in a change in their practice. The average feedback survey response rate was 32% (range, 17%-49%). CONCLUSIONS: Chartrounds has proven to be an effective resource for US-based radiation oncologists, and our initial experience with Chartrounds India suggests that an online platform for radiation oncology case review and education can be successfully implemented globally with use of local disease site experts.

Radiographic nodal prognostic factors in stage I HPV-related oropharyngeal squamous cell carcinoma.

BACKGROUND: The updated AJCC Cancer Staging Manual groups all p16-positive oropharyngeal squamous cell carcinoma (OPSCC) with unilateral nodal involvement within 6 cm into the new clinical N1 classification, consolidating a heterogeneous group of disease with varying radiographic findings. METHODS: A central radiological review was conducted identifying 233 patients with stage I node-positive (cT1-2N1) disease who underwent definitive concurrent chemoradiation. Factors evaluated included lymph node size, low-neck lymphadenopathy, retropharyngeal lymphadenopathy, overt radiographic extracapsular extension, and matted lymphadenopathy. RESULTS: On multivariate analysis adjusted for age, smoking history, and chemotherapy regimen, low-neck lymphadenopathy (hazard ratio (HR) = 6.55; P < .001) and retropharyngeal lymphadenopathy (HR = 3.36; P = .009) predicted for inferior progression-free survival (PFS). low-neck lymphadenopathy (HR = 6.38; P = .001) and retropharyngeal lymphadenopathy (HR = 3.32; P = .02) also predicted for inferior overall survival (OS). All other radiographic characteristics showed no prognostic impact for PFS or OS. CONCLUSIONS: This analysis suggests that caution should be advised against de-intensification efforts among patients with stage I node-positive p16-positive OPSCC with low-neck lymphadenopathy or retropharyngeal lymphadenopathy.

Induction chemotherapy followed by concurrent chemoradiation versus concurrent chemoradiation alone in the definitive management of p16-positive oropharyngeal squamous cell carcinoma with low-neck or N3 disease.

OBJECTIVE: The addition of induction chemotherapy (ICT) to concurrent chemoradiation (CCRT) has been investigated as a method of improving outcomes among patients with locally advanced head and neck squamous cell carcinoma. Previous studies have consisted of heterogeneous populations with both p16-positive and p16-negative disease and varying extent of nodal disease burden. We evaluated the role of ICT in p16-positive oropharyngeal squamous cell carcinoma (OPSCC) at high-risk of distant failure. MATERIALS AND METHODS: A retrospective review was conducted of 88 consecutive patients with p16-positive OPSCC with low-neck and/or N3 lymphadenopathy. Among these patients, 44 received ICT followed by CCRT, and 44 received CCRT alone with concurrent agents including Cisplatin, Carboplatin, and Cetuximab. Disease control and survival outcomes were reported after adjusting for age, T stage, N stage, and smoking status. RESULTS: Median follow-up for surviving patients was 47 (range: 13-115) months. Patients who received CCRT alone were older than those who received ICT (61 years vs. 56 years; p = 0.02); the groups were otherwise similarly balanced. 3-year distant metastasis: 38% vs. 18% (adjusted hazard ratio (HR) = 0.32 [0.13-0.82]; p = 0.02). 3-year progression-free survival: 49% vs. 74% (adjusted HR = 0.46 [0.22-0.93]; p = 0.03). 3-year overall survival: 67% vs. 83% (adjusted HR = 0.48 [0.21-1.12]; p = 0.09). CONCLUSION: Among patients with p16-positive OPSCC with low-neck and/or N3 lymphadenopathy, ICT followed by CCRT may reduce the risk for distant failure over CCRT alone and lead to improved progression-free survival. Future trials should concentrate on patients at the highest risk of distant metastasis in order to appropriately assess the benefit of ICT.

Definitive proton radiation therapy and concurrent cisplatin for unresectable head and neck adenoid cystic carcinoma: A series of 9 cases and a critical review of the literature.

BACKGROUND: The primary treatment for head and neck adenoid cystic carcinoma (ACC) is surgery. Infrequently, however, ACC's propensity for perineural and base of skull invasion can preclude definitive surgical management. We present our experience with proton radiation therapy (RT) and concurrent platinum-based chemotherapy. METHODS: Nine patients with unresectable node-negative, nonmetastatic head and neck ACC received definitive proton RT and concurrent cisplatin. Outcomes and toxicities were recorded. A systematic review of the literature was performed. RESULTS: Median follow-up was 27 months (range, 9.2-48.3 months). Four patients achieved complete response at the primary site, and an additional 4 patients achieved stabilization of local disease. Only 1 patient developed local disease progression. Four patients had 5 acute grade 3 (G3) toxicities, and 1 patient developed a chronic G4 optic nerve disorder. CONCLUSION: Our preliminary results suggest proton RT and concurrent chemotherapy is a definitive treatment option for select patients with head and neck ACC. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1472-E1480, 2016.

Postoperative Intensity-Modulated Proton Therapy for Head and Neck Adenoid Cystic Carcinoma.

PURPOSE: Postoperative radiation therapy can improve control for adenoid cystic carcinoma (ACC) of the head and neck; however, delivering adequate dose to the tumor bed must be balanced with limiting dose to nearby critical organs. Intensity-modulated proton therapy (IMPT) may help improve the therapeutic ratio, though concerns exist regarding tissue heterogeneity and other sources of uncertainty in several head and neck subsites. We report control and toxicity outcomes for patients with ACC of the head and neck treated at a single institution with postoperative IMPT and robust planning and analysis. PATIENTS AND METHODS: Sixteen patients with head and neck ACC treated with postoperative IMPT were identified. Intensity-modulated proton therapy was delivered by using multifield optimization. Robust planning and analysis were performed. The median dose was 60 (range, 60 to 70) Gy (RBE) (Gy [relative biological effectiveness]). Adjuvant IMPT was given with (N = 12) or without (N = 4) platinum-based chemotherapy. Tumor control outcomes were recorded from the medical record, and acute and chronic toxicities were graded weekly during treatment and upon follow-up per Common Terminology Criteria for Adverse Events, version 4.0 (CTCAE v4). RESULTS: Median follow-up is 24.9 (range, 9.2 to 40.2) months. One patient developed local and distant recurrence and subsequently died. The remaining 15 patients are alive without evidence of disease. Four patients experienced acute grade 3 toxicities: dermatitis (N = 3) and oral mucositis (N = 1). One patient developed a chronic grade 4 optic nerve disorder. There were no grade 5 toxicities. CONCLUSIONS: Intensity-modulated proton therapy is a feasible option for patients with ACC of the head and neck in the postoperative setting. Robust treatment planning and plan analysis can be performed such that uncertainties and tissue heterogeneities do not appear to limit safe and effective IMPT delivery. Safety and efficacy appear comparable to those of other types of radiation therapy, but further follow-up of clinical outcomes is needed.

Stereotactic Body Radiation Therapy for Prostate Cancer: What is the Appropriate Patient-Reported Outcome for Clinical Trial Design?

PURPOSE: Stereotactic body radiation therapy (SBRT) is increasingly utilized as primary treatment for clinically localized prostate cancer. Consensus regarding the appropriate patient-reported outcome (PRO) endpoints for clinical trials evaluating radiation modalities for early stage prostate cancer is lacking. To aid in clinical trial design, this study presents PROs over a 36-month period following SBRT for clinically localized prostate cancer. METHODS: Between February 2008 and September 2010, 174 hormone-naïve patients with clinically localized prostate cancer were treated with 35-36.25 Gy SBRT (CyberKnife, Accuray) delivered in 5 fractions. Patients completed the validated Expanded Prostate Cancer Index Composite (EPIC)-26 questionnaire at baseline and all follow-ups. The proportion of patients developing a clinically significant decline in each EPIC domain score was determined. The minimally important difference (MID) was defined as a change of one-half the standard deviation from the baseline. Per Radiation Therapy Oncology Group (RTOG) 0938, we also examined the patients who experienced a decline in EPIC urinary domain summary score of >2 points (unacceptable toxicity defined as ≥60% of all patients reporting this degree of decline) and EPIC bowel domain summary score of >5 points (unacceptable toxicity defined as >55% of all patients reporting this degree of decline) from baseline to 1 year. RESULTS: A total of 174 patients at a median age of 69 years received SBRT with a minimum follow-up of 36 months. The proportion of patients reporting a clinically significant decline (MID for urinary/bowel are 5.5/4.4) in EPIC urinary/bowel domain scores was 34%/30% at 6 months, 40%/32.2% at 12 months, and 32.8%/21.5% at 36 months. The patients reporting a decrease in the EPIC urinary domain summary score of >2 points was 43.2% (CI: 33.7%, 54.6%) at 6 months, 51.6% (CI: 43.4%, 59.7%) at 12 months, and 41.8% (CI: 33.3%, 50.6%) at 36 months. The patients reporting a decrease in the EPIC bowel domain summary score of >5 points was 29.6% (CI: 21.9%, 39.3%) at 6 months, 29% (CI: 22%, 36.8%) at 12 months, and 22.4% (CI: 15.7%, 30.4%) at 36 months. CONCLUSION: Following prostate SBRT, clinically significant urinary symptoms are more common than bowel symptoms. Our prostate SBRT treatment protocol meets the RTOG 0938 criteria for moving forward to a Phase III trial comparing it to conventionally fractionated radiation therapy. Notably, between 12 and 36 months, the proportion of patients reporting a significant decrease in both EPIC urinary and bowel domain scores declined, suggesting a late improvement in these symptom domains. Further investigation is needed to elucidate (1) which EPIC domains bear the greatest influence on post-treatment quality of life and (2) at what time point PRO endpoint(s) should be assessed.

Variability in the reported management of pulmonary metastases in osteosarcoma.

Nearly 20% of patients with newly diagnosed osteosarcoma have detectable metastases at diagnosis; the majority of which occur in the lungs. There are no established recommendations for the timing and modality of metastasectomy. Members of the Connective Tissue Oncology Society (CTOS) were emailed an anonymous 10-min survey assessing their management practices for pulmonary findings at the time of an osteosarcoma diagnosis. The questionnaire presented three scenarios and discussed the choice to perform surgery, the timing of resection, and the choice of surgical procedure. Analyses were stratified by medical profession. One hundred and eighty-three physicians responded to our questionnaire. Respondents were comprised of orthopedic surgeons (37%), medical oncologists (31%), pediatric oncologists (22%), and other medical subspecialties (10%). There was variability among the respondents in the management of the pulmonary nodules. The majority of physicians chose to resect the pulmonary nodules following neoadjuvant chemotherapy (46-63%). Thoracotomy was the preferred technique for surgical resection. When only unilateral findings were present, the majority of physicians did not explore the contralateral lung. The majority of respondents did not recommend resection if the pulmonary nodule disappeared following chemotherapy. The survey demonstrated heterogeneity in the management of pulmonary metastases in osteosarcoma. Prospective trials need to evaluate whether these differences in management have implications for outcomes for patients with metastatic osteosarcoma.

Patient-reported outcomes following stereotactic body radiation therapy for clinically localized prostate cancer.

BACKGROUND: Stereotactic body radiation therapy (SBRT) delivers high doses of radiation to the prostate while minimizing radiation to adjacent normal tissues. Large fraction sizes may increase the risk of functional decrements. Treatment-related bother may be more important to a patient than treatment-related dysfunction. This study reports on patient-reported outcomes following SBRT for clinically localized prostate cancer. METHODS: Between August 2007 and July 2011, 228 consecutive hormone-naïve patients with clinically localized prostate cancer were treated with 35-36.25 Gy SBRT delivered using the CyberKnife Radiosurgical System (Accuray) in 5 fractions. Quality of life was assessed using the American Urological Association Symptom Score (AUA) and the Expanded Prostate Cancer Index Composite (EPIC)-26. Urinary symptom flare was defined as an AUA score 15 or more with an increase of 5 or more points above baseline 6 months after treatment. RESULTS: 228 patients (88 low-, 126 intermediate- and 14 high-risk) at a median age of 69 (44-90) years received SBRT with a minimum follow-up of 24 months. EPIC urinary and bowel summary scores declined transiently at 1 month and experienced a second, more protracted decline between 9 months and 18 months before returning to near baseline 2 years post-SBRT. 14.5% of patients experienced late urinary symptom flare following treatment. Patients who experienced urinary symptom flare had poorer bowel quality of life following SBRT. EPIC scores for urinary bother declined transiently, first at 1 month and again at 12 months, before approaching pre-treatment scores by 2 years. Bowel bother showed a similar pattern, but the second decline was smaller and lasted 9 months to 18 months. EPIC sexual summary and bother scores progressively declined over the 2 years following SBRT without recovery. CONCLUSIONS: In the first 2 years, the impact of SBRT on urination and defecation was minimal. Transient late increases in urinary and bowel dysfunction and bother were observed. However, urinary and bowel function and bother recovered to near baseline by 2 years post-SBRT. Sexual dysfunction and bother steadily increased following treatment without recovery. SBRT for clinically localized prostate cancer was well tolerated with treatment-related dysfunction and bother comparable to conventionally fractionated radiation therapy or brachytherapy.

Rationale for stereotactic body radiation therapy in treating patients with oligometastatic hormone-naïve prostate cancer.

Despite advances in treatment for metastatic prostate cancer, patients eventually progress to castrate-resistant disease and ultimately succumb to their cancer. Androgen deprivation therapy (ADT) is the standard treatment for metastatic prostate cancer and has been shown to improve median time to progression and median survival time. Research suggests that castrate-resistant clones may be present early in the disease process prior to the initiation of ADT. These clones are not susceptible to ADT and may even flourish when androgen-responsive clones are depleted. Stereotactic body radiation therapy (SBRT) is a safe and efficacious method of treating clinically localized prostate cancer and metastases. In patients with a limited number of metastatic sites, SBRT may have a role in eliminating castrate-resistant clones and possibly delaying progression to castrate-resistant disease.

Ferritin protein nanocage ion channels: gating by N-terminal extensions.

Ferritin protein nanocages, self-assembled from four-α-helix bundle subunits, use Fe(2+) and oxygen to synthesize encapsulated, ferric oxide minerals. Ferritin minerals are iron concentrates stored for cell growth. Ferritins are also antioxidants, scavenging Fenton chemistry reactants. Channels for iron entry and exit consist of helical hairpin segments surrounding the 3-fold symmetry axes of the ferritin nanocages. We now report structural differences caused by amino acid substitutions in the Fe(2+) ion entry and exit channels and at the cytoplasmic pores, from high resolution (1.3-1.8 Å) protein crystal structures of the eukaryotic model ferritin, frog M. Mutations that eliminate conserved ionic or hydrophobic interactions between Arg-72 and Asp-122 and between Leu-110 and Leu-134 increase flexibility in the ion channels, cytoplasmic pores, and/or the N-terminal extensions of the helix bundles. Decreased ion binding in the channels and changes in ordered water are also observed. Protein structural changes coincide with increased Fe(2+) exit from dissolved, ferric minerals inside ferritin protein cages; Fe(2+) exit from ferritin cages depends on a complex, surface-limited process to reduce and dissolve the ferric mineral. High concentrations of bovine serum albumin or lysozyme (protein crowders) to mimic the cytoplasm restored Fe(2+) exit in the variants to wild type. The data suggest that fluctuations in pore structure control gating. The newly identified role of the ferritin subunit N-terminal extensions in gating Fe(2+) exit from the cytoplasmic pores strengthens the structural and functional analogies between ferritin ion channels in the water-soluble protein assembly and membrane protein ion channels gated by cytoplasmic N-terminal peptides.

Moving metal ions through ferritin-protein nanocages from three-fold pores to catalytic sites.

Ferritin nanocages synthesize ferric oxide minerals, containing hundreds to thousands of Fe(III) diferric oxo/hydroxo complexes, by reactions of Fe(II) ions with O(2) at multiple di-iron catalytic centers. Ferric-oxy multimers, tetramers, and/or larger mineral nuclei form during postcatalytic transit through the protein cage, and mineral accretion occurs in the central cavity. We determined how Fe(II) substrates can access catalytic sites using frog M ferritins, active and inactivated by ligand substitution, crystallized with 2.0 M Mg(II) ± 0.1 M Co(II) for Co(II)-selective sites. Co(II) inhibited Fe(II) oxidation. High-resolution (<1.5 Å) crystal structures show (1) a line of metal ions, 15 Å long, which penetrates the cage and defines ion channels and internal pores to the nanocavity that link external pores to the cage interior, (2) metal ions near negatively charged residues at the channel exits and along the inner cavity surface that model Fe(II) transit to active sites, and (3) alternate side-chain conformations, absent in ferritins with catalysis eliminated by amino acid substitution, which support current models of protein dynamics and explain changes in Fe-Fe distances observed during catalysis. The new structural data identify a ∼27-Å path Fe(II) ions can follow through ferritin entry channels between external pores and the central cavity and along the cavity surface to the active sites where mineral synthesis begins. This "bucket brigade" for Fe(II) ion access to the ferritin catalytic sites not only increases understanding of biological nanomineral synthesis but also reveals unexpected design principles for protein cage-based catalysts and nanomaterials.