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To assess the long-term (20-year) endocrine therapy benefit in premenopausal patients with breast cancer.Secondary analysis of the Stockholm trial (STO-5, 1990-1997) randomly assigning 924 premenopausal patients to 2 years of goserelin (3.6 mg subcutaneously once every 28 days), tamoxifen (40 mg orally once daily), combined goserelin and tamoxifen, or no adjuvant endocrine therapy (control) is performed. Random assignment was stratified by lymph node status; lymph node-positive patients (n = 459) were allocated to standard chemotherapy (cyclophosphamide, methotrexate, and fluorouracil). Primary tumor immunohistochemistry (n = 731) and gene expression profiling (n = 586) were conducted in 2020. The 70-gene signature identified genomic low-risk and high-risk patients. Kaplan-Meier analysis, multivariable Cox proportional hazard regression, and multivariable time-varying flexible parametric modeling assessed the long-term distant recurrence-free interval (DRFI). Swedish high-quality registries allowed a complete follow-up of 20 years.In estrogen receptor-positive patients (n = 584, median age 47 years), goserelin, tamoxifen, and the combination significantly improved long-term distant recurrence-free interval compared with control (multivariable hazard ratio [HR], 0.49; 95% CI, 0.32 to 0.75, HR, 0.57; 95% CI, 0.38 to 0.87, and HR, 0.63; 95% CI, 0.42 to 0.94, respectively). Significant goserelin-tamoxifen interaction was observed (This study shows 20-year benefit from 2 years of adjuvant endocrine therapy in estrogen receptor-positive premenopausal patients and suggests differential treatment benefit on the basis of tumor genomic characteristics. Combined goserelin and tamoxifen therapy showed no benefit over single treatment. Long-term follow-up to assess treatment benefit is critical.

Background: Women with estrogen receptor (ER)-positive disease have a long-term risk of distant recurrence. The poor survival of premenopausal women diagnosed with breast cancer combined with the otherwise long life expectancy makes it especially important to identify tumor characteristics associated with long-term survival. Intra-tumor heterogeneity, having cancer cells of varying characteristics across the tumor, may promote therapeutic resistance and metastatic capacity. We have previously shown that high intra-tumor heterogeneity of ER increases the risk of fatal breast cancer. To our knowledge, the relation between intra-tumor heterogeneity of progesterone receptor (PR) expression and long-term survival is not known. We aimed to study the association between intra-tumor heterogeneity of PR and long-term survival of premenopausal women in the Stockholm tamoxifen trial (STO-5), with 20-years complete follow-up. Methods: This study was a secondary analysis of 504 ER-positive/PR-positive premenopausal women from the STO-5 trial (1990-1997). 451 women had human epidermal growth factor receptor 2 (HER2)-negative tumors. Patients were randomized to receive either 2 years of endocrine treatment (tamoxifen and/or goserelin) or no endocrine treatment. Lymph node-positive patients (n=251) also received standard chemotherapy (CMF). Immunohistochemical analysis, including estimating the proportion of tumor cells for each PR intensity level (0, 1+, 2+, or 3+), was completed in 2020. Intra-tumor heterogeneity of PR was calculated using Rao’s quadratic entropy and then categorized into low and high intra-tumor heterogeneity groups, using a predefined cutoff at the second tertile. Complete long-term (20 year) follow-up was obtained from high-quality Swedish registries. Long-term distant recurrence-free interval (DRFI) was assessed using univariate Kaplan-Meier analysis and multivariable Cox proportional hazard modeling, adjusting for patient and tumor characteristics. Results: A statistically significant difference in 20-year DRFI was seen between patients with high and low intra-tumor heterogeneity of PR in the univariate Kaplan-Meier analysis (log-rank P< 0.01). Survival proportions for DRFI at 20 years were 60.2% (95% CI, 53.2%-68.1%) and 73.3% (95% CI, 68.6%-78.3%) for patients with high and low PR intra-tumor heterogeneity, respectively. Analysis in patients with HER2-negative tumors yielded similar results. In the multivariable analysis, women with high intra-tumor heterogeneity of PR had a significantly increased long-term risk of distant recurrence, compared to women with low intra-tumor heterogeneity, hazard ratio (HR)=1.49 (95% CI, 1.08-2.06). The same pattern was seen in HER2-negative women, HR=1.50 (95% CI, 1.06-2.12), see Table. Conclusions: This study suggests an increased long-term risk of distant recurrences in ER-positive/PR-positive premenopausal women with high intra-tumor heterogeneity of PR as compared to women with low intra-tumor heterogeneity of PR, independent of HER2 status. Ongoing analyses include using deep learning for image analysis of breast cancer tumors to examine intra-tumor heterogeneity at higher resolution and for various tumor characteristics. Better understanding of tumor characteristics associated with long-term risk in premenopausal women is needed, given the poor prognosis and early onset of the disease. Long-term risk of distant recurrence by PR intra-tumor heterogeneity and HER2 status Multivariable Cox proportional hazard regression modeling of 20-year distant recurrence-free interval (DRFI) by high and low PR intra-tumor heterogeneity. ER-positive/PR-positive and ER-positive/PR-positive/HER2-negative premenopausal women were analyzed separately. The crude model was adjusted for age, randomization year, lymph node status, and endocrine treatment. The full model was adjusted for age, randomization year, lymph node status, endocrine treatment, tumor size, Ki-67 status, and HER2 status. Citation Format: Oscar Danielsson, Huma Dar, Gizeh Perez-Tenorio, Anna Nordenskjöld, Christina Yau, Christopher C. Benz, Laura J. Esserman, Bo Nordenskjöld, Olle Stål, Tommy Fornander, Annelie Johansson, Linda S. Lindström. Long-term survival and intra-tumor heterogeneity of progesterone receptor expression in estrogen receptor-positive/progesterone receptor-positive premenopausal women with breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-03.

Background: Tamoxifen is a standard endocrine therapy for both pre- and postmenopausal ER-positive breast cancer patients. Patients with ER-positive disease have a long-term risk of distant recurrence, thus, long-term follow-up studies are essential to understand true treatment benefit. Clinically used tumor characteristics are prognostic 5-10 years after primary diagnosis, however, whether these characteristics are predictive of long-term tamoxifen benefit is largely unexplored. Therefore, we aimed to determine the long-term tamoxifen therapy benefit by the clinically used tumor characteristics in pre- vs postmenopausal patients in the Stockholm tamoxifen (STO)-trials with 20-years complete follow-up. Methods: Secondary analysis of 1242 ER-positive/HER2-negative patients from the STO-trials, randomized to at least 2 years of 40 mg tamoxifen vs no endocrine therapy (control). Premenopausal lymph node-positive patients were allocated to chemotherapy as standard of care and postmenopausal high-risk patients were further randomized to chemotherapy vs radiotherapy. Tumor immunohistochemical analysis was recently conducted. Complete 20-year follow-up was obtained from Swedish high-quality registries. Long-term distant recurrence-free interval (DRFI) was assessed by multivariable Cox proportional hazard regression and time-varying analysis using flexible parametric modelling. Results: Premenopausal patients showed significantly improved long-term DRFI from tamoxifen vs control if they were lymph node-negative (Hazard Ratio [HR]=0.46; 95% CI, 0.24-0.87), PR-positive (HR=0.61; 95% CI, 0.41-0.91), or of genomic low risk (HR=0.47; 95% CI, 0.26-0.85), see Table. In postmenopausal patients, significantly improved long-term DRFI from tamoxifen vs control was seen for all good prognosis tumor characteristics, i.e. small tumor size (pT≤20mm: HR=0.55; 95% CI, 0.39-0.77), tumor grade 1-2 (HR=0.55; 95% CI, 0.41-0.73), lymph node-negative (HR=0.44; 95% CI, 0.30-0.64), PR-positive (HR=0.60; 95% CI, 0.44-0.80), Ki-67-low (< 15%: HR=0.51; 95% CI, 0.38-0.68), and genomic low risk (HR=0.53; 95% CI, 0.37-0.74), see Table. Also, postmenopausal patients with large tumor size (pT>20mm: HR=0.64; 95% CI, 0.44-0.94) and PR-negative tumors (HR=0.51; 95% CI, 0.32-0.81) showed significant long-term tamoxifen benefit. Time-varying analysis in premenopausal patients indicated that tamoxifen therapy benefit diminished over time. Significant tamoxifen benefit until year 5, 10, and 15 after primary diagnosis was observed for PR-positive, lymph node-negative, and genomic low-risk patients, respectively. Postmenopausal patients had a significant long-term tamoxifen benefit if they had tumors of small or large tumor size, tumor grade 1-2, lymph node-negative status, PR-positive status, low Ki-67 levels, or genomic low risk. Conclusions: This study suggests a differential long-term tamoxifen therapy benefit in pre- vs postmenopausal patients. Clinically defined low-risk postmenopausal patients have long-term tamoxifen benefit, whereas the benefit is absent or diminish over time for premenopausal patients. Improved long-term prognostic and endocrine therapy predictive markers in premenopausal breast cancer patients with poor prognosis and long life-expectancy is needed, which could involve molecular tools. Long-term tamoxifen benefit in premenopausal and postmenopausal breast cancer patients by the clinically used tumor characteristics. Table Multivariable Cox proportional hazard regression analysis of 20-year distant recurrence-free interval (DRFI) for patients with ER-positive/HER2-negative tumors, comparing patients randomized to tamoxifen vs patients randomized to no endocrine therapy (control). Adjusted for age, randomization year, tumor size, tumor grade, lymph node status, PR status, Ki-67 status, chemotherapy, radiotherapy, and type of surgery. Citation Format: Annelie Johansson, Huma Dar, Anna Nordenskjöld, Gizeh Perez-Tenorio, Christina Yau, Christopher C. Benz, Laura J. Esserman, Laura Van’t Veer, Bo Nordenskjöld, Olle Stål, Tommy Fornander, Linda S. Lindström. Differential Long-Term Benefit from Adjuvant Tamoxifen Therapy in Estrogen Receptor (ER)-Positive/Human Epidermal Growth Factor Receptor 2 (HER2)-Negative Premenopausal and Postmenopausal Breast Cancer Patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-11.

Background: The risk for patients with estrogen receptor (ER)-positive breast cancer remains stable decades after primary diagnosis, with a large proportion of distant metastatic events occurring late. Thus, long-term follow-up studies are essential to understand true treatment benefit. Tamoxifen (TAM) therapy is a fundamental endocrine treatment for ER-positive breast cancer. We have previously shown a long-term tamoxifen therapy benefit for patients with less aggressive tumor characteristics in ER-positive/HER2-negative patients. However, ER-positive breast cancer is highly heterogenous and can be separated into different molecular subpopulations that behave differently during extended follow-up. The long-term tamoxifen benefit by molecular subtype is largely unexplored. We therefore aimed to investigate the long-term benefit from tamoxifen by clinically used tumor characteristics in Luminal A vs Luminal B patients in the Stockholm tamoxifen (STO)-trials with complete 20-years follow-up. Methods: Secondary analysis of ER-positive/HER2-negative patients with Luminal A or B molecular subtype (n=952) from the STO-trials (1976-1997) randomized to TAM (40 mg) vs no endocrine therapy (control). Gene expression data was generated using custom designed Agilent arrays from FFPE breast cancer tumor tissue and was used to define PAM50 molecular subtypes. The clinically used markers were reannotated in 2014 and 2020. Complete 20-year follow-up was obtained from Swedish high-qualitative registries. The long-term distant recurrence-free interval (DRFI) was assessed by Kaplan-Meier, multivariable Cox proportional hazard regression and time-varying analysis, using flexible parametric modelling. Results: Multivariable analysis showed significantly improved long-term DRFI from TAM vs control for both Luminal A (HR=0.59; 95% CI, 0.44-0.81) and Luminal B (HR=0.67; 95% CI, 0.46-0.99) patients. Time-varying analysis showed that patients with Luminal A subtype significantly benefitted from TAM for 15 years (HR=0.60, 95% CI, 0.39-0.94 at year 15), whereas patients with Luminal B subtype had a significant TAM benefit for 5 years (HR=0.64, 95% CI, 0.42-0.98 at year 5), see Table. Furthermore, a significant long-term TAM benefit for patients with large tumor size (pT>20mm: HR=0.46; 95% CI, 0.25-0.84), tumor grade 1-2 (HR=0.55; 95% CI, 0.40-0.77), lymph node-negative (HR=0.51, 95% CI, 0.34-0.78), PR-positive (HR=0.57, 95% CI, 0.40-0.81) and Ki-67-low tumors (HR=0.55, 95% CI, 0.39-0.77) was seen for Luminal A patients. In Luminal B patients, significantly improved long-term DRFI from TAM vs control was seen for small tumor size (pT≤20mm: HR=0.44; 95% CI, 0.24-0.80), tumor grade 1-2 (HR=0.51; 95% CI, 0.29-0.90), lymph node-negative (HR=0.40, 95% CI, 0.19-0.83), PR-positive (HR=0.59, 95% CI, 0.38-0.93) and Ki-67-low tumors (HR=0.45, 95% CI, 0.25-0.84). Conclusions: This study suggests a 15 years tamoxifen benefit for patients with Luminal A subtype tumors, whereas the benefit for patients with Luminal B tumors is up to five years after diagnosis. Furthermore, our study suggests a long-term tamoxifen benefit for patients with less aggressive tumor characteristics regardless of tumor subtype, except for tumor size in Luminal A patients. ER-positive breast cancer is a heterogeneous disease and long-term follow-up studies in molecular subtypes are essential to understand differences in treatment benefit. Table. Time-varying analysis of long-term tamoxifen therapy benefit by PAM50 molecular subtype. Time-varying analysis of distant recurrence-free interval (DRFI) to assess how tamoxifen benefit varied over the 20 year of follow-up using flexible parametric survival modelling. Patients included in analyses with no missing values. Estimated hazard ratios (HR) at year 5, 10, 15 and 20 for patients with Luminal A or Luminal B molecular subtype tumors randomized to tamoxifen therapy, as compared with patients randomized to no endocrine therapy (control). Adjusted for age, period of primary breast cancer diagnosis, tumor size, tumor grade, lymph node status, PR status, Ki-67 status, chemotherapy, radiotherapy, type of surgery, and menopausal status. *indicates significant findings P Citation Format: Huma Dar, Annelie Johansson, Anna Nordenskjöld, Gizeh Perez-Tenorio, Christina Yau, Christopher C. Benz, Laura J. Esserman, Bo Nordenskjöld, Olle Stål, Tommy Fornander, Linda S. Lindström. Long-Term Benefit from Adjuvant Tamoxifen in Luminal A and Luminal B Breast Cancer Patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-04.

Clinically used breast cancer markers, such as tumor size, tumor grade, progesterone receptor (PR) status, and Ki-67 status, are known to be associated with short-term survival, but the association of these markers with long-term (25-year) survival is unclear.To assess the association of clinically used breast cancer markers with long-term survival and treatment benefit among postmenopausal women with lymph node-negative, estrogen receptor [ER]-positive and ERBB2-negative breast cancer who received tamoxifen therapy.This study was a secondary analysis of data from a subset of 565 women with ER-positive/ERBB2-negative breast cancer who participated in the Stockholm tamoxifen (STO-3) randomized clinical trial. The STO-3 clinical trial was conducted from 1976 to 1990 and comprised 1780 postmenopausal women with lymph node-negative breast cancer who were randomized to receive adjuvant tamoxifen therapy or no endocrine therapy. Complete 25-year follow-up data through December 31, 2016, were obtained from Swedish national registers. Immunohistochemical markers were reannotated in 2014. Data were analyzed from April to December 2020.Patients in the original STO-3 clinical trial were randomized to receive 2 years of tamoxifen therapy vs no endocrine therapy. In 1983, patients who received tamoxifen therapy without cancer recurrence during the 2-year treatment and who consented to continued participation in the STO-3 study were further randomized to receive 3 additional years of tamoxifen therapy or no endocrine therapy.Distant recurrence-free interval (DRFI) by clinically used breast cancer markers was assessed using Kaplan-Meier and multivariable Cox proportional hazards analyses adjusted for age, period of primary diagnosis, tumor size (T1a and T1b [T1a/b], T1c, and T2), tumor grade (1-3), PR status (positive vs negative), Ki-67 status (low vs medium to high), and STO-3 clinical trial arm (tamoxifen treatment vs no adjuvant treatment). A recursive partitioning analysis was performed to evaluate which markers were able to best estimate long-term DRFI.The study population comprised 565 postmenopausal women (mean [SD] age, 62.0 [5.3] years) with lymph node-negative, ER-positive/ERBB2-negative breast cancer. A statistically significant difference in long-term DRFI was observed by tumor size (88% for T1a/b vs 76% for T1c vs 63% for T2 tumors; log-rank P .001) and tumor grade (81% for grade 1 vs 77% for grade 2 vs 65% for grade 3 tumors; log-rank P = .02) but not by PR status or Ki-67 status. Patients with smaller tumors (hazard ratio [HR], 0.31 [95% CI, 0.17-0.55] for T1a/b tumors and 0.58 [95% CI, 0.38-0.88] for T1c tumors) and grade 1 tumors (HR, 0.48; 95% CI, 0.24-0.95) experienced a significant reduction in the long-term risk of distant recurrence compared with patients with larger (T2) tumors and grade 3 tumors, respectively. A significant tamoxifen treatment benefit was observed among patients with larger tumors (HR, 0.53 [95% CI, 0.32-0.89] for T1c tumors and 0.34 [95% CI, 0.16-0.73] for T2 tumors), lower tumor grades (HR, 0.24 [95% CI, 0.07-0.82] for grade 1 tumors and 0.50 [95% CI, 0.31-0.80] for grade 2 tumors), and PR-positive status (HR, 0.38; 95% CI, 0.24-0.62). The recursive partitioning analysis revealed that tumor size was the most important characteristic associated with long-term survival, followed by clinical trial arm among patients with larger tumors.This secondary analysis of data from the STO-3 clinical trial indicated that, among the selected subgroup of patients, tumor size followed by tumor grade were the markers most significantly associated with long-term survival. Furthermore, a significant long-term tamoxifen treatment benefit was observed among patients with larger tumors, lower tumor grades, and PR-positive tumors.

Background Invasive lobular carcinoma (ILC) comprises 8–15 % of all invasive breast cancers and large population-based studies with >10 years of follow-up are rare. Whether ILC has a long-time prognosis different from that of invasive ductal carcinoma, (IDC) remains controversial. Purpose To investigate the excess mortality rate ratio (EMRR) of patients with ILC and IDC and to correlate survival with clinical parameters in a large population-based cohort. Material and methods From 1989 through 2006, we identified 17,481 patients diagnosed with IDC (n = 14,583) or ILC (n = 2898), younger than 76 years from two Swedish Regional Cancer Registries. Relative survival (RS) during 20 years of follow up was analysed. Results ILC was significantly associated with older age, larger tumours, ER positivity and well differentiated tumours. We noticed an improved survival for patients with ILC during the first five years, excess mortality rate ratio (EMRR) 0.64 (CI 95 % 0.53–0.77). This was shifted to a significant decreased survival 10–15 years after diagnosis (EMRR 1.49, CI 95 % 1.16–1.93). After 20 years the relative survival rates were similar, 0.72 for ILC and 0.73 for IDC. Conclusions During the first five years after surgery, the EMRR was lower for patients with ILC as compared to patients with IDC, but during the years 10–15 after surgery, we observed an increased EMRR for patients with ILC as compared to IDC. These EMRR between ILC and IDC were statistically significant but the absolute difference in excess mortality between the two groups was small., Highlights • This is a large population-based study with more than 17,000 patients with a follow up exciding 20 years. • There is clinically important differences between invasive lobular and ductal carcinoma of the breast. • Lobular carcinoma shows better survival during the first period but significantly worse at the late period of observation.

During the recent decades, breast cancer survival has gradually improved but there is limited knowledge on the improvement in population-based studies of patients diagnosed with different stages of the disease and in different age groups.In two Swedish health care regions a total of 42,220 female breast cancer patients below 90 years of age were diagnosed between 1989 and 2013. They were treated and followed according to national and regional guidelines and formed a population-based cohort.Using patients diagnosed in 1989-1993 as a reference to the relative risk, 5-year mortality decreased with 49% for patients diagnosed at the end of the observation period (CI 95% 45-58). The mortality tended to decrease for patients with all stages of breast cancer and test for trend resulted in a statistically significant improvement over time in 5-year relative survival in stage III and IV and in 10-year survival in stage I and III. For each operable stage of disease, patients aged below 40 years or more than 70 years when diagnosed tended to have less favorable survival than patients diagnosed between 40-69 years of age. Test for trend resulted in statistically significant improvements over time for patients diagnosed at ages below 40, 40-54 and 54-69, but less marked improvements for patients older than 70 when diagnosed.During the period 1989-2013 the relative risk of 5-year mortality decreased with 49%. Improvements were seen in all age groups but were unevenly distributed between stages and age groups pointing to the need for further improvements for younger and elderly patients.

Background In published radiotherapy trials, the failure rate in the control arm among patients with one to three positive nodes is high compared with that seen with modern adjuvant treatments. Therefore, the generalizability of the results has been questioned. The aim of the present study was to compare relative survival in breast cancer patients between two Swedish regions with screening mammography programs and adjuvant treatment guidelines similar with the exception of the indication of radiotherapy for patients with one to three positive nodes. Patients and methods Between 1989 and 2006, breast cancer patients were managed very similarly in the west and southeast regions, except for indication for postoperative radiotherapy. In patients with one to three positive nodes, postmastectomy radiotherapy was generally given in the southeast region (89% of all cases) and generally not given in the west region (15% of all cases). For patients with one to three positive nodes who underwent breast-conserving surgery, patients in the west region had breast radiotherapy only, while patients in the southeast region had both breast and lymph nodes irradiated. Results The 10-year relative survival for patients with one to three positive lymph nodes was 78% in the west region and 77% in the southeast region (P = 0.12). Separate analyses depending on type of surgery, as well as number of examined nodes, also revealed similar relative survival. Conclusion Locoregional postoperative radiotherapy has well-known side-effects, but in this population-based study, there was little or no influence of this type of radiotherapy on survival when one to three lymph nodes were involved.

The independent predictive information from progesterone receptor (PgR) positivity for breast cancer treated with tamoxifen has been questioned after an overview by the Early Breast Cancer Trialists Collaborative Group (EBCTCG). However, the studies in the overview were to a large content performed before modern PgR immunohistochemistry (IHC) was developed. We therefore investigated the predictive value of PgR determined with IHC in estrogen receptor (ER)-positive tumors from patients participating in the Stockholm trial of adjuvant tamoxifen therapy. The Stockholm Breast Cancer Study Group conducted a randomized trial during 1976 through 1990 comparing adjuvant tamoxifen versus control. The patients were stratified according to tumor size and lymph node status in high-risk and low-risk groups. In this study, we evaluated 618 patients with ER-positive "low-risk" breast cancer (size aecurrency sign 30 mm, lymph node-negative) for whom PgR was determined by IHC at one pathology laboratory. The median time of follow-up was 21 years. Patients with ER-positive tumors that were also PgR-positive by IHC did benefit from tamoxifen, while we could not show any long-term benefit for those with tumors positive for ER only (recurrence rate ratio 0.43, 95 % CI 0.29-0.62 and 0.87, 95 % CI 0.52-1.46, respectively). We further investigated the influence of different levels of PgR positivity on recurrence risk. The results show that at all receptor levels with aeyen10 % stained PgR-positive cells, the patients did benefit from tamoxifen. There was no clear linear trend in benefit with increasing proportion of stained cells. PgR positivity determined by IHC is a marker indicating long-term benefit from adjuvant tamoxifen in patients with ER-positive tumors. Funding Agencies|Swedish Cancer Society; Swedish Breast Cancer Association; Cancer Research Foundations of Radiumhemmet; Cancer Society in Stockholm; King Gustav V Jubilee Clinical Research Foundation; County Council of Ostergotland; Onkologiska Klinikernas i Linkoping Forskningsfond

Three key enzymes in the anabolic phosphorylation of deoxyribonucleosides and deoxyribonucleoside analogs were purified i.e. cytoplasmic thymidine kinase (TK1), mitochondrial thymidine kinase (TK2) and cytoplasmic deoxycytidine kinase (dCK) from human, mouse and monkey liver and spleen. Their subunit structure and substrate specificities were compared. Extensive purification of TK1 and dCK from mouse spleen and TK2 from mouse and monkey livers revealed major polypeptide bands of 25,30 and 28 kD, respectively, on sodium dodecyl sulphate-polyacrylamide gel electrophoresis which are very similar to the subunit molecular weights of the corresponding human enzymes. Affinity purified polyclonal antibodies against human dCK also cross-reacted with 30 kD bands in extracts from both mouse and monkey spleen. Thus, the molecular weights of the subunits of these three enzymes appeared to be very similar in all three species. TK1 and TK2 from these different sources appeared to have similar substrate specificities against several deoxyribonucleoside analogs. However, mouse dCK differed significantly from monkey and human dCK in its capacity to phosphorylate dAdo and 2',3'-dide-oxycytidine (ddCyd) with a V max , approximately 10-fold lower than that of the two latter enzymes. The K m and V max values for dCyd and arabinocytosine appeared to be very similar with the enzymes from all three species. The fact that mouse dCK shows low activity with dAdo and ddCyd explains differences reported previously in the metabolism of dAdo and ddCyd in mouse compared to that in human lymphocytes. These results argue against the use of mice as model systems for human deoxynucleoside metabolism.