Late-Stage Metastatic Melanoma Emerges through a Diversity of Evolutionary Pathways.

Understanding the evolutionary pathways to metastasis and resistance to immune-checkpoint inhibitors (ICI) in melanoma is critical for improving outcomes. Here, we present the most comprehensive intrapatient metastatic melanoma dataset assembled to date as part of the Posthumous Evaluation of Advanced Cancer Environment (PEACE) research autopsy program, including 222 exome sequencing, 493 panel-sequenced, 161 RNA sequencing, and 22 single-cell whole-genome sequencing samples from 14 ICI-treated patients. We observed frequent whole-genome doubling and widespread loss of heterozygosity, often involving antigen-presentation machinery. We found KIT extrachromosomal DNA may have contributed to the lack of response to KIT inhibitors of a KIT-driven melanoma. At the lesion-level, MYC amplifications were enriched in ICI nonresponders. Single-cell sequencing revealed polyclonal seeding of metastases originating from clones with different ploidy in one patient. Finally, we observed that brain metastases that diverged early in molecular evolution emerge late in disease. Overall, our study illustrates the diverse evolutionary landscape of advanced melanoma. SIGNIFICANCE: Despite treatment advances, melanoma remains a deadly disease at stage IV. Through research autopsy and dense sampling of metastases combined with extensive multiomic profiling, our study elucidates the many mechanisms that melanomas use to evade treatment and the immune system, whether through mutations, widespread copy-number alterations, or extrachromosomal DNA. See related commentary by Shain, p. 1294. This article is highlighted in the In This Issue feature, p. 1275.

Who is asked about alcohol consumption? A retrospective cohort study using a national repository of Electronic Medical Records.

Documentation of alcohol use in electronic medical record (EMR) informs interventions to reduce alcohol-related morbidity and mortality. This retrospective cohort study explored EMR data from 960 primary care providers participating in the Canadian Primary Care Sentinel Surveillance Network to describe documentation of alcohol use (e.g. none, current or past use) in the EMR. Included providers represented 700,620 adult patients from across Canada with an encounter between 2015 and 2018. Bivariate comparisons characterized the patients with, and without, documentation of alcohol use. Multivariate generalized estimating equation models with logit function assessed patient and provider characteristics associated with (1) documentation of alcohol and (2) patients with heightened risk for alcohol-related problems. Forty percent of patients had alcohol use documentation in the EMR. Light alcohol consumption was recorded for 43.6% of these patients. Male patients (OR1.09, CI 1.07-1.12), who were older (OR1.26, CI 1.23-1.30), had more frequent visits to their provider (OR1.11, CI 1.09-1.13) and had hypertension (OR1.07, CI 1.06-1.09) or depression (OR1.07, CI 1.09-1.14) had higher odds of alcohol documentation. There were 4.7% of patients with a record indicating heightened risk for alcohol-related problems. Male patients (OR3.27 CI 3.14-3.4), patients with depression (OR2.01 CI1.93-2.1) and rural residency (OR1.35 CI1.29-1.42) was associated with risk for alcohol-related problems. Heavy alcohol consumption is associated with an increased risk of negative health outcomes, particularly for patients with certain chronic conditions. However, these patients do not have alcohol use consistently documented in the EMR. Strategies should be designed and implemented to support more consistent alcohol-screening among high-risk patients.

An immunotherapy survivor population: health-related quality of life and toxicity in patients with metastatic melanoma treated with immune checkpoint inhibitors.

PURPOSE: The immune checkpoint inhibitors (ICIs) have resulted in subgroups of patients with metastatic melanoma achieving high-quality durable responses. Metastatic melanoma survivors are a new population in the era of cancer survivorship. The aim of this study was to evaluate metastatic melanoma survivors in terms of health-related quality of life (HRQoL), immune-related adverse events (irAEs) and exposure to immunosuppressive agents in a large single centre in the UK. METHODS: We defined the survivor population as patients with a diagnosis of metastatic melanoma who achieved a durable response to an ICI and had been followed-up for a minimum of 12 months from initiation of ICI without disease progression. HRQoL was assessed using SF-36. Electronic health records were accessed to collect data on demographics, treatments, irAEs and survival. HRQoL data was compared with two norm-based datasets. RESULTS: Eighty-four metastatic melanoma survivors were eligible and 87% (N = 73) completed the SF-36. ICI-related toxicity of any grade occurred in 92% of patients and 43% had experienced a grade 3 or 4 toxicity. Almost half (49%) of the patients required steroids for the treatment of ICI-related toxicity, whilst 14% required treatment with an immunosuppressive agent beyond steroids. Melanoma survivors had statistically significant lower HRQoL scores with regard to physical, social and physical role functioning and general health compared with the normative population. There was a trend towards inferior scores in patients with previous exposure to ipilimumab compared with those never exposed to ipilimumab. CONCLUSIONS: Our results show that metastatic melanoma survivors have potentially experienced significant ICI-related toxicity and experience significant impairments in specific HRQoL domains. Future service planning is required to meet this population's unique survivorship needs.

Validity and reliability of metrics for translation of regional anaesthesia performance from cadavers to patients.

BACKGROUND: Our goal is to develop metrics that quantify the translation of performance from cadavers to patients. Our primary objective was to develop steps and error checklists from a Delphi questionnaire. Our second objective was to show that our test scores were valid and reliable. METHODS: Sixteen UK experts identified 15 steps conducive to good performance and 15 errors to be avoided during interscalene block on the soft-embalmed cadaver and patients. Thereafter, six experts and six novices were trained, and then tested. Training consisted of psychometric assessment, an anatomy tutorial, volunteer scanning, and ultrasound-guided needle insertion on a pork phantom and on a soft-embalmed cadaver. For testing, participants conducted a single interscalene block on a dedicated soft-embalmed cadaver whilst wearing eye tracking glasses. RESULTS: We developed a 15-step checklist and a 15-error checklist. The internal consistency of our steps measures were 0.83 (95% confidence interval [CI]: 0.78-0.89) and 0.90 (95% CI: 0.87-0.93) for our error measures. The experts completed more steps (mean difference: 3.2 [95% CI: 1.5-4.8]; P<0.001), had less errors (mean difference: 4.9 [95% CI: 3.5-6.3]; P<0.001), had better global rating scores (mean difference: 6.8 [95% CI: 3.6-10.0]; P<0.001), and more eye-gaze fixations (median of differences: 128 [95% CI: 0-288]; P=0.048). Fixation count correlated negatively with steps (r=-0.60; P=0.04) and with errors (r=0.64; P=0.03). CONCLUSIONS: Our tests to quantify ultrasound-guided interscalene nerve block training and performance were valid and reliable.

Donor outcomes in anonymous live liver donation.

BACKGROUND & AIMS: Death rates on liver transplant waiting lists range from 5%-25%. Herein, we report a unique experience with 50 anonymous individuals who volunteered to address this gap by offering to donate part of their liver to a recipient with whom they had no biological connection or prior relationship, so called anonymous live liver donation (A-LLD). METHODS: Candidates were screened to confirm excellent physical, mental, social, and financial health. Demographics and surgical outcomes were analyzed. Qualitative interviews after donation examined motivation and experiences. Validated self-reported questionnaires assessed personality traits and psychological impact. RESULTS: A total of 50 A-LLD liver transplants were performed between 2005 and 2017. Most donors had a university education, a middle-class income, and a history of prior altruism. Half were women. Median age was 38.5 years (range 20-59). Thirty-three (70%) learned about this opportunity through public or social media. Saving a life, helping others, generativity, and reciprocity for past generosity were motivators. Social, financial, healthcare, and legal support in Canada were identified as facilitators. A-LLD identified most with the personality traits of agreeableness and conscientiousness. The median hospital stay was 6 days. One donor experienced a Dindo-Clavien Grade 3 complication that completely resolved. One-year recipient survival was 91% in 22 adults and 97% in 28 children. No A-LLD reported regretting their decision. CONCLUSIONS: This is the first and only report of the characteristics, motivations and facilitators of A-LLD in a large cohort. With rigorous protocols, outcomes are excellent. A-LLD has significant potential to reduce the gap between transplant organ demand and availability. LAY SUMMARY: We report a unique experience with 50 living donors who volunteered to donate to a recipient with whom they had no biological connection or prior relationship (anonymous living donors). This report is the first to discuss motivations, strategies and facilitators that may mitigate physical, social and ethical risk factors in this patient population. With rigorous protocols, anonymous liver donation and recipient outcomes are excellent; with appropriate clinical expertise and system facilitators in place, our experience suggests that other centers may consider the procedure for its significant potential to reduce the gap between transplant organ demand and availability.

Timing the Landmark Events in the Evolution of Clear Cell Renal Cell Cancer: TRACERx Renal.

Clear cell renal cell carcinoma (ccRCC) is characterized by near-universal loss of the short arm of chromosome 3, deleting several tumor suppressor genes. We analyzed whole genomes from 95 biopsies across 33 patients with clear cell renal cell carcinoma. We find hotspots of point mutations in the 5' UTR of TERT, targeting a MYC-MAX-MAD1 repressor associated with telomere lengthening. The most common structural abnormality generates simultaneous 3p loss and 5q gain (36% patients), typically through chromothripsis. This event occurs in childhood or adolescence, generally as the initiating event that precedes emergence of the tumor's most recent common ancestor by years to decades. Similar genomic changes drive inherited ccRCC. Modeling differences in age incidence between inherited and sporadic cancers suggests that the number of cells with 3p loss capable of initiating sporadic tumors is no more than a few hundred. Early development of ccRCC follows well-defined evolutionary trajectories, offering opportunity for early intervention.

Deterministic Evolutionary Trajectories Influence Primary Tumor Growth: TRACERx Renal.

The evolutionary features of clear-cell renal cell carcinoma (ccRCC) have not been systematically studied to date. We analyzed 1,206 primary tumor regions from 101 patients recruited into the multi-center prospective study, TRACERx Renal. We observe up to 30 driver events per tumor and show that subclonal diversification is associated with known prognostic parameters. By resolving the patterns of driver event ordering, co-occurrence, and mutual exclusivity at clone level, we show the deterministic nature of clonal evolution. ccRCC can be grouped into seven evolutionary subtypes, ranging from tumors characterized by early fixation of multiple mutational and copy number drivers and rapid metastases to highly branched tumors with >10 subclonal drivers and extensive parallel evolution associated with attenuated progression. We identify genetic diversity and chromosomal complexity as determinants of patient outcome. Our insights reconcile the variable clinical behavior of ccRCC and suggest evolutionary potential as a biomarker for both intervention and surveillance.

Tracking Cancer Evolution Reveals Constrained Routes to Metastases: TRACERx Renal.

Clear-cell renal cell carcinoma (ccRCC) exhibits a broad range of metastatic phenotypes that have not been systematically studied to date. Here, we analyzed 575 primary and 335 metastatic biopsies across 100 patients with metastatic ccRCC, including two cases sampledat post-mortem. Metastatic competence was afforded by chromosome complexity, and we identify 9p loss as a highly selected event driving metastasis and ccRCC-related mortality (p = 0.0014). Distinct patterns of metastatic dissemination were observed, including rapid progression to multiple tissue sites seeded by primary tumors of monoclonal structure. By contrast, we observed attenuated progression in cases characterized by high primary tumor heterogeneity, with metastatic competence acquired gradually and initial progression to solitary metastasis. Finally, we observed early divergence of primitive ancestral clones and protracted latency of up to two decades as a feature of pancreatic metastases.

Engaging street-involved youth using an evidence-based intervention: A preliminary report of findings.

PROBLEM: Street-involved youth experience many barriers to accessing health and social services. There is a literature gap in the literature regarding evidence-based interventions to facilitate engagement with street-involved youth. METHODS: A qualitative descriptive study of preliminary findings from a large mixed-methods study was undertaken to assess the impact of a resilience-based motivational intervention. This intervention was grounded in frameworks including strengths-based and resilience-based communication using the Seven C's Model of Resilience, positive youth development, and motivational interviewing that are particularly relevant to youth. Individual interviews were conducted with two subsets of youth who participated (n = 3) or did not participate (n = 3) in the intervention. Thematic analysis was conducted to identify themes between the intervention and comparison groups. FINDINGS: Preliminary themes identified across the sample include (1) establishing a trusting relationship, (2) strengthening self-worth and resilience, (3) focusing on goals, and (4) perceiving a sense of hope and possibility. CONCLUSIONS: The themes identified the importance of positive relationships with care providers built upon a foundation of trust to engage youth to remain motivated and focused on their goals.

Optimal utilization of a breast care advanced practice clinician.

BACKGROUND: Incorporation of "lean" business philosophy within health care has the goal of adding value by reducing cost and improving quality. Applying these principles to the role of Advance Practice Clinicians (APCs) is relevant because they have become essential members of the healthcare team. METHODS: An independent surgical breast care clinic directed by an APC was created with measurements of success to include the following: time to obtain an appointment, financial viability, and patient/APC/MD satisfaction. RESULTS: During the study period, there was a trend toward a decreased median time to obtain an appointment. Monthly APC charges increased from $388 to $30,800. The mean provider satisfaction score by Press Ganey was 96% for the APC and 95.8% for the surgeon. Both clinicians expressed significant satisfaction with clinic development. CONCLUSIONS: Overall, initiation of an APC breast clinic met the proposed goals of success. The use of lean philosophy demonstrates that implementation of change can result in added value in patient care.

Multiple cervical lymph node involvement and extra-capsular extension predict for contralateral nodal recurrence after ipsilateral radiotherapy for squamous cell carcinoma of the tonsil.

BACKGROUND: Ipsilateral radiotherapy is an established technique for treating well-lateralised tonsillar tumours. Concerns exist regarding the risk of contralateral nodal failure, particularly in patients with ipsilateral nodal involvement at presentation. In this study, we retrospectively reviewed the clinical outcomes of patients treated with ipsilateral radiotherapy aiming to identify factors that predispose to a higher risk of contralateral nodal recurrence. METHODS: Retrospective case note review of all patients with tonsillar cancer who were treated using ipsilateral radiotherapy between September 1995 and September 2011 was performed. Demographics, T and N stage, involvement of soft palate and/or tongue base, presence of extra-capsular spread (ECS) and treatment details were recorded. Kaplan-Meier curves for treatment outcomes were generated. RESULTS: A total of 136 patients were identified. Median follow-up was 4.2years. Twelve (9%) patients had loco-regional recurrence. Eight patients (6%) had contralateral recurrence. N2b disease, ECS and number of pack-years of smoking were associated with contralateral nodal recurrence. Five-year overall survival was 89%, loco-regional disease-free survival 90%, disease-free survival 86% and distal recurrence-free survival 96%. CONCLUSION: N2b disease, ECS and a greater than 10 pack-year smoking history are risk factors for contralateral nodal recurrence in well-lateralised tonsillar cancers. Prophylactic irradiation of the contralateral neck should be recommended in this group of patients.

Constipation: take it seriously.

A case report of a young, healthy female who presented as an emergency with large bowel obstruction secondary to a rectosigmoid tumour. Acute presentation was preceded by a progressive history of change in bowel habit managed medically in the community with laxative treatment. The family history of note included breast cancer of an identical triplet aged 41 years. An emergency Hartman's procedure was successfully performed the same day as presentation due to the high risk of perforation. The patient was discharged home within a week. Histology confirmed adenocarcinoma, Dukes' Stage C and oncologists have now taken over care of the patient. Discussion points explore whether it is appropriate to refer a young healthy patient presenting with a change in bowel habit if she is resistant to medical management in the community and in the absence of other red flag symptoms. The family history of malignancy and genetic associations are also considered.

Association between statin medication use and improved outcomes during inpatient rehabilitation in older people.

BACKGROUND: statin drugs may induce skeletal myopathy, but might also have the potential to improve rehabilitation outcomes by improving sarcopenia or by preventing intercurrent illness. We examined the association between statin use and functional outcomes in the rehabilitation of older people. METHODS: retrospective cohort study using routinely collected clinical data. Admissions to Royal Victoria Hospital, Dundee for inpatient rehabilitation over a 10-year period were identified. Data were available regarding demographics, statin therapy, antiplatelet therapy, admission and discharge Barthel scores, length of stay and comorbid disease. Multivariate analyses were performed to examine the difference between admission and discharge Barthel score in patients taking statins compared with those not taking statins. RESULTS: a total of 3,422 patients were included. Mean age was 81.4 years; 40% were male. Baseline Barthel scores were similar in the statin/non-statin groups, respectively (10.4/20 versus 10.3/20, P = 0.57). Improvement in the Barthel score between admission and discharge was greater in the statin than non-statin group (3.59 versus 4.30 points, P < 0.001) after adjustment for age, sex, baseline Barthel score and comorbid disease. CONCLUSION: statin use was associated with improved Barthel scores on discharge from rehabilitation. This gain could contribute to improved outcomes as part of the rehabilitation package and requires further prospective investigation.

Fatigue after stroke: the development and evaluation of a case definition.

OBJECTIVE: While fatigue after stroke is a common problem, it has no generally accepted definition. Our aim was to develop a case definition for post-stroke fatigue and to test its psychometric properties. METHODS: A case definition with face validity and an associated structured interview was constructed. After initial piloting, the feasibility, reliability (test-retest and inter-rater) and concurrent validity (in relation to four fatigue severity scales) were determined in 55 patients with stroke. RESULTS: All participating patients provided satisfactory answers to all the case definition probe questions demonstrating its feasibility For test-retest reliability, kappa was 0.78 (95% CI, 0.57-0.94, P<.01) and for inter-rater reliability kappa was 0.80 (95% CI, 0.62-0.99, P<.01). Patients fulfilling the case definition also had substantially higher fatigue scores on four fatigue severity scales (P<.001) indicating concurrent validity. CONCLUSION: The proposed case definition is feasible to administer and reliable in practice, and there is evidence of concurrent validity. It requires further evaluation in different settings.

Evaluation of fatigue scales in stroke patients.

BACKGROUND AND PURPOSE: There is little information on how to best measure poststroke fatigue. Our aim was to identify which currently available fatigue scale is most valid, feasible, and reliable in stroke patients. METHODS: Fatigue scales were identified by systematic search, and the 5 with the best face validity were identified by expert consensus. Feasibility (ie, did patients provide answers?) and internal consistency (an aspect of reliability) of these scales were evaluated by interviewing 55 stroke patients. Test-retest reliability was assessed by reinterviewing 51 patients, interrater reliability was assessed by rerating audio recordings, and convergent validity was assessed by measuring the correlation between scale scores. RESULTS: Of the 52 scales identified, the SF-36v2 (vitality component), the fatigue subscale of the Profile of Mood States, the Fatigue Assessment Scale, the general subscale of the Multidimensional Fatigue Symptom Inventory, and the Brief Fatigue Inventory had the best face validity. The Brief Fatigue Inventory was unfeasible to administer and was omitted. Of the remaining 4 scales, the Fatigue Assessment Scale had the poorest internal consistency. Test-retest reliability for individual scale questions ranged from fair to good; the Fatigue Assessment Scale had the narrowest limits of agreement for the total score, indicating the best test-retest reliability. Interrater reliability for individual questions ranged from good to very good, and there was no significant mean difference in total scores for any scale. Convergent validity was moderate to high for the total scores of the 4 scales. CONCLUSIONS: All four scales were valid and feasible to administer to stroke patients. The Fatigue Assessment Scale had the best test-retest reliability but the poorest internal consistency.

Determination of the lactose content of fluid milk by spectrophotometric enzymatic analysis using weight additions and path length adjustment: collaborative study.

The objective of this collaborative study was to determine the method performance characteristics of a spectrophotometric enzymatic assay for measuring the lactose content of fluid milk. The principle behind the method is similar to that of AOAC Method 984.15 but with significant modifications and added quality control. Additionally, lactose concentration is expressed on a weight/weight (wt/wt) rather than a weight/volume (wt/vol) basis. The principle of the method is the hydrolysis of lactose to D-glucose and D-galactose by beta-galactosidase, followed by the oxidation of beta-D-galactose by nicotinamide adenine dinucleotide (NAD+) in the presence of beta-galactose dehydrogenase. The reaction is catalyzed by the addition of aldose-l-epimerase, which accelerates the mutarotation of alphha-D-galactose to beta-D-galactose. The amount of reduced nicotinamide adenine dinucleotide (NADH) formed is measured at 340 nm and is proportional to the amount of lactose present. Important aspects of the assay include preparing the assay solution by weight (rather than volume), mixing the contents of the spectrophotometric cuvette without losing solution, inclusion of aldose-l-epimerase, specifying spectrophotometer characteristics, and accounting for the optical path length of the spectrophotometric cuvettes. In the collaborative study, 11 laboratories tested one lactose standard and 8 pairs of blind replicate raw, processed, and formulated milks with an anhydrous lactose content between 3.0-7.2%. Statistical performance, in units of g/100 g anhydrous lactose, for the milk materials within the applicability of the method was as follows: mean = 4.4040, Sr = 0.0130, SR = 0.0250, RSDr = 0.29%, RSDR = 0.57%, r = 0.0364, and R = 0.0700. Standard and marginal recoveries were 98.66 and 99.53%, respectively. Method performance represented a significant improvement over what would be achieved if path length was not accounted for or the assay was done volumetrically. The Study Directors recommend that the method for determination of the lactose content of fluid milk by the spectrophotometric enzymatic method using weight additions and path length adjustment be adopted Official First Action.

A multi-centre European study of factors affecting the discharge destination of older people admitted to hospital: analysis of in-hospital data from the ACMEplus project.

OBJECTIVES: to examine the relationship between seven predictor variables (recorded on Day 3 of hospital admission) and discharge destination in non-elective medical patients aged 65+ years. DESIGN: prospective cohort. SETTING: eight centres in six European countries. PREDICTOR VARIABLES: age, gender, living alone, physical function (three categories based on Barthel Index), cognition (Katzman's orientation-memory-concentration test), main body system affected (based on International Classification of Diseases), number of geriatric giants (GGs) involved in the referral (a GG being a problem with falling, mobility, continence or cognition). MAIN OUTCOME MEASURES: discharge destination (by Day 90) in three categories: 'HOMESAME' (return to previous residence), 'INSTIN90' (discharge to alternative residence or still in hospital at 90 days), 'DEADINHO' (death in hospital), RESULTS: in 1,626 patients, discharge destination was HOMESAME in 84.7%, DEADINHO in 8.9% and INSTIN90 in 6.4%. Mean duration of stay was 17.7 days, median 12. Univariate analyses showed a statistically significant relationship between all seven predictor variables and discharge destination. Physical function was the best single predictor with a seven-fold difference in adverse outcome rates between the best and worst categories. On multiple logistic regression, significant predictor variables were as follows. (i) For DEADINHO: physical function, cognition, gender; (ii) for INSTIN90: physical function, living alone, GGs, age, gender. Multiple linear regression identified physical function, GGs and living alone as predictors of loge length of stay. CONCLUSION: case-mix systems to compare risk-adjusted hospital outcome in older medical patients need to incorporate information about physical function, cognition and presenting problems in addition to diagnosis.

Effectiveness of temperature modification in decreasing the bias in milk fat test results between the Babcock and Ether extraction methods.

Both the Babcock (AOAC Method 989.04, revised Final Action 2000) and modified Mojonnier ether extraction (AOAC Method 989.05) methods are used in the dairy industry to determine the fat content of milk. Prior to revision in 1997, the Babcock method gave consistently higher fat test results than did the ether extraction. In 1997, a modification of the Babcock method was introduced to bring the results of the Babcock test into closer agreement with the ether extraction. The Babcock method was modified by lowering the temperatures used at various points in the method from about 57.5 to 48 degrees C to increase the density of the material in the Babcock column. A collaborative study of the modification indicated it was successful in bringing the Babcock and ether extraction results into agreement but suggested that performance of the modified method was not as good as that of the unmodified method. In the present study, substantial evidence is presented to validate the success of the Babcock modification in bringing test results into agreement with ether extraction, and to document that temperature modification does not adversely affect method performance. Data were evaluated from an on-going proficiency testing program where 8-15 laboratories tested 7 milk samples in blind duplicate once every 2 months. Laboratories used the unmodified method from 1995 through 1996 and the modified method from 1998 through 1999. Compared with ether extraction, test results from the unmodified Babcock test were consistently higher by an average of 0.022% fat. For the modified Babcock test, average test results were -0.003% fat lower than with ether extraction and not significantly different from zero. AOAC method performance statistics (within- and between-laboratory precision) were equivalent for both the unmodified (Sr = 0.027, SR = 0.041, RSDr = 0.73%, RSDR= 1.08%) and modified (Sr = 0.023, SR = 0.038, RSDr = 0.60%, RSDR = 1.02%) Babcock methods. Modification of the Babcock method was successful in bringing test results into agreement with those of ether extraction.

Determination of the total nitrogen content of hard, semihard, and processed cheese by the Kjeldahl method: collaborative study.

The objective of this collaborative study was to determine interlaboratory performance statistics for a modified and optimized version of AOAC Method 920.123 for the determination of the total nitrogen content of hard, semihard, and processed cheese by Kjeldahl analysis. Details included addressing the issues of material homogeneity, test portion size (1 g), quantitative transfer (weighing on to filter paper), ensuring system suitability (nitrogen recoveries), and using AOAC Method 991.20 as the basis for nitrogen analysis. Fifteen laboratories tested 18 pairs of blind duplicate cheese materials with a crude protein content between 18 and 36%. Materials represented hard, semihard, and processed commercial cheeses with a wide range of composition. Statistical performance parameters expressed as crude protein (nitrogen x 6.38), g/100 g, with invalid and outlier data removed were mean = 26.461, repeatability standard deviation (Sr) 0.111, reproducibility standard deviation (S(R)) = 0.153, repeatability relative standard deviation (RSDr) = 0.42%, reproducibility relative standard deviation (RSDR) = 0.58%, repeatability (r) = 0.312, and reproducibility (R) = 0.428. The interlaboratory study results were acceptable and comparable to those for the milk Kjeldahl nitrogen method on a relative nitrogen basis. The Study Directors recommend that this modified method for the determination of total nitrogen in hard, semihard, and processed cheese by Kjeldahl analysis be adopted First Action as an improved method to replace Method 920.123.