Cytoplasmic Cyclin E and Phospho-Cyclin-Dependent Kinase 2 Are Biomarkers of Aggressive Breast Cancer.

Cyclin E and its co-activator, phospho-cyclin-dependent kinase 2 (p-CDK2), regulate G1 to S phase transition and their deregulation induces oncogenesis. Immunohistochemical assessments of these proteins in cancer have been reported but were based only on their nuclear expression. However, the oncogenic forms of cyclin E (low molecular weight cyclin E or LMW-E) in complex with CDK2 are preferentially mislocalized to the cytoplasm. Here, we used separate nuclear and cytoplasmic scoring systems for both cyclin E and p-CDK2 expression to demonstrate altered cellular accumulation of these proteins using immunohistochemical analysis. We examined the specificity of different cyclin E antibodies and evaluated their concordance between immunohistochemical and Western blot analyses in a panel of 14 breast cell lines. Nuclear versus cytoplasmic staining of cyclin E readily differentiated full-length from LMW-E, respectively. We also evaluated the expression of cyclin E and p-CDK2 in 1676 breast carcinoma patients by immunohistochemistry. Cytoplasmic cyclin E correlated strongly with cytoplasmic p-CDK2 (P < 0.0001), high tumor grade, negative estrogen/progesterone receptor status, and human epidermal growth factor receptor 2 positivity (all P < 0.0001). In multivariable analysis, cytoplasmic cyclin E plus phosphorylated CDK2 (as one variable) predicted breast cancer recurrence-free and overall survival. These results suggest that cytoplasmic cyclin E and p-CDK2 can be readily detected with immunohistochemistry and used as clinical biomarkers for aggressive breast cancer.

Inflammatory Breast Cancer: Understanding the Patient Experience.

BACKGROUND: Inflammatory breast cancer (IBC) is an aggressive, locally advanced cancer with a 5-year survival rate of approximately 40%. Although patients with IBC likely experience significant and variable symptom burden from diagnosis through survivorship, the description of the symptom burden in this population is limited. OBJECTIVES: The purpose of this study was to describe the experience of patients with IBC and define the content domain for a patient-reported outcome measure of IBC symptom burden. METHODS: Twenty patients with IBC described their experience in single qualitative interviews. Content analysis was used to define the symptom burden content domain. Relevance ratings by a panel of experts reduced the number of items for a preliminary patient-reported outcome symptom burden measure. RESULTS: The mean (SD) participant age was 52.8 (12.0) years; 50.0% had distant metastatic disease, and 85.0% were currently receiving treatment. Content analysis revealed 45 symptoms, with 20 symptoms reported by greater than or equal to 20% of participants. All participants described localized disease-related symptoms. Treatment-related symptoms varied among participants based on the modalities received. CONCLUSION: Patients with IBC experience symptom burden that is distinct from the symptom burden experienced by patients with non-IBC. IMPLICATIONS FOR PRACTICE: Differentiating the disease-related symptoms of IBC may assist clinicians in making timely and accurate diagnoses for IBC. A disease- and treatment-specific measure of the symptom burden of IBC should be incorporated in clinical practice to allow for regular assessment and evaluation of symptom burden and implementation of evidence-based interventions for symptom management.

Maintenance Pembrolizumab Therapy in Patients with Metastatic HER2-negative Breast Cancer with Prior Response to Chemotherapy.

PURPOSE: Accumulating toxicities hinder indefinite chemotherapy for many patients with metastatic/recurrent HER2-negative breast cancer. We conducted a phase II trial of pembrolizumab monotherapy following induction chemotherapy to determine the efficacy of maintenance immunotherapy in patients with metastatic HER2-negative inflammatory breast cancer (IBC) and non-IBC triple-negative breast cancer (TNBC) and a biomarker study. PATIENTS AND METHODS: Patients with a complete response, partial response, or stable disease (SD) after at least three cycles of chemotherapy for HER2-negative breast cancer received pembrolizumab, regardless of programmed death-ligand 1 expression. Pembrolizumab (200 mg) was administered every 3 weeks until disease progression, intolerable toxicity, or 2 years of pembrolizumab exposure. The endpoints included the 4-month disease control rate (DCR), progression-free survival (PFS), overall survival, and response biomarkers in the blood. RESULTS: Of 43 treated patients, 11 had metastatic IBC and 32 non-IBC TNBC. The 4-month DCR was 58.1% [95% confidence interval (CI), 43.4-72.9]. For all patients, the median PFS was 4.8 months (95% CI, 3.0-7.1 months). The toxicity profile was similar to the previous pembrolizumab monotherapy study. Patients with high T-cell clonality at baseline had a longer PFS with pembrolizumab treatment than did those with low T-cell clonality (10.4 vs. 3.6 months, P = 0.04). Patients who achieved SD also demonstrated a significant increase in T-cell clonality during therapy compared with those who did not achieve SD (20% vs. 5.9% mean increase, respectively; P = 0.04). CONCLUSIONS: Pembrolizumab monotherapy achieved durable treatment responses. Patients with a high baseline T-cell clonality had prolonged disease control with pembrolizumab.

ATM signals to TSC2 in the cytoplasm to regulate mTORC1 in response to ROS.

Ataxia-telangiectasia mutated (ATM) is a cellular damage sensor that coordinates the cell cycle with damage-response checkpoints and DNA repair to preserve genomic integrity. However, ATM also has been implicated in metabolic regulation, and ATM deficiency is associated with elevated reactive oxygen species (ROS). ROS has a central role in many physiological and pathophysiological processes including inflammation and chronic diseases such as atherosclerosis and cancer, underscoring the importance of cellular pathways involved in redox homeostasis. We have identified a cytoplasmic function for ATM that participates in the cellular damage response to ROS. We show that in response to elevated ROS, ATM activates the TSC2 tumor suppressor via the LKB1/AMPK metabolic pathway in the cytoplasm to repress mTORC1 and induce autophagy. Importantly, elevated ROS and dysregulation of mTORC1 in ATM-deficient cells is inhibited by rapamycin, which also rescues lymphomagenesis in Atm-deficient mice. Our results identify a cytoplasmic pathway for ROS-induced ATM activation of TSC2 to regulate mTORC1 signaling and autophagy, identifying an integration node for the cellular damage response with key pathways involved in metabolism, protein synthesis, and cell survival.

Osteoprogenitor-GMP crosstalk underpins solid tumor-induced systemic immunosuppression and persists after tumor removal.

Remote tumors disrupt the bone marrow (BM) ecosystem (BME), eliciting the overproduction of BM-derived immunosuppressive cells. However, the underlying mechanisms remain poorly understood. Herein, we characterized breast and lung cancer-induced BME shifts pre- and post-tumor removal. Remote tumors progressively lead to osteoprogenitor (OP) expansion, hematopoietic stem cell dislocation, and CD41- granulocyte-monocyte progenitor (GMP) aggregation. The tumor-entrained BME is characterized by co-localization between CD41- GMPs and OPs. OP ablation abolishes this effect and diminishes abnormal myeloid overproduction. Mechanistically, HTRA1 carried by tumor-derived small extracellular vesicles upregulates MMP-13 in OPs, which in turn induces the alterations in the hematopoietic program. Importantly, these effects persist post-surgery and continue to impair anti-tumor immunity. Conditional knockout or inhibition of MMP-13 accelerates immune reinstatement and restores the efficacies of immunotherapies. Therefore, tumor-induced systemic effects are initiated by OP-GMP crosstalk that outlasts tumor burden, and additional treatment is required to reverse these effects for optimal therapeutic efficacy.

PTEN deficiency is fully penetrant for prostate adenocarcinoma in C57BL/6 mice via mTOR-dependent growth.

The tumor suppressor phosphatase and tensin homolog (PTEN) is frequently involved in human prostate carcinoma. PTEN is therefore an attractive target for the development of preclinical animal models. Prostate intraepithelial neoplasia lesions develop in mice with Pten heterozygosity, but disease progression has been reported only in combination with either other tumor suppressor gene alterations or the conditional inactivation of both Pten alleles in prostate epithelial cells. We report that on a C57BL/6 background, in contrast to previous studies on mixed 129 genetic backgrounds, Pten locus heterozygosity is fully penetrant for the development of prostate adenocarcinoma. Grossly observable tumors were detected at 6 months of age, and, by 10 to 12 months, 100% of examined mice developed adenocarcinoma of the anterior prostate. Furthermore, double heterozygotes carrying both Pten and Tsc2-null alleles showed no increase relative to Pten(+/-) heterozygotes in either lesion development or progression. Lesions in both Pten(+/-); Tsc2(+/-), and Pten(+/-) mice exhibited loss of PTEN expression and activation of PI3K signaling. PI3K activation occurred early in prostate intraepithelial neoplasia lesion formation in these animals, consistent with loss of PTEN function, and contributed to the etiology of tumors that developed in Pten(+/-) mice. Furthermore, prostate lesion growth in Pten(+/-) mice was dependent on mTOR, as evidenced by a reduction in both phospho-S6 levels and proliferative index after rapamycin treatment.

Cytoplasmic sequestration of p27 via AKT phosphorylation in renal cell carcinoma.

PURPOSE: p27 localization and expression has prognostic and predictive value in cancer. Little is known regarding expression patterns of p27 in renal cell carcinoma (RCC) or how p27 participates in disease progression or response to therapy. EXPERIMENTAL DESIGN: RCC-derived cell lines, primary tumors, and normal renal epithelial cells were analyzed for p27 expression, phosphorylation (T157 of the NLS), and subcellular localization. RCC-derived cell lines were treated with phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors and effects on p27 localization were assessed. The potential contribution of cytoplasmic p27 to resistance to apoptosis was also evaluated. RESULTS: p27 was elevated in tumors compared with matched controls, and cytoplasmic mislocalization of p27 was associated with increasing tumor grade. Cytoplasmic localization of p27 correlated with phosphorylation at T157, an AKT phosphorylation site in the p27 NLS. In RCC cell lines, activated PI3K/AKT signaling was accompanied by mislocalization of p27. AKT activation and phosphorylation of p27 was associated with resistance to apoptosis, and small interfering RNA knockdown of p27 or relocalization to the nucleus increased apoptosis in RCC cells. Treatment with the PI3K inhibitors LY294002 or wortmannin resulted in nuclear relocalization of p27, whereas mTOR inhibition by rapamycin did not. CONCLUSIONS: In RCC, p27 is phosphorylated at T157 of the NLS, with increasing tumor grade associated with cytoplasmic p27. PI3K inhibition (which reduces AKT activity) reduces T157 phosphorylation and induces nuclear relocalization of p27, whereas mTOR inhibition does not. Clinical testing of these findings may provide a rational approach for use of mTOR and PI3K/AKT pathway inhibitors in patients with RCC.

Longitudinal Evaluation of the Healthy Living Assessment as an Experiential Learning Activity Provided On-Campus.

Objective. To implement and evaluate the effectiveness of healthy living assessments (HLA) conducted on campus by undergraduate pharmacy students. Methods. Because of a shortage of workplace-based placements for undergraduate pharmacy students, a program was developed for students to conduct HLAs on campus for volunteer patients. Pharmacy students underwent training and completed a competency assessment before being approved to conduct HLAs. Staff members and students were recruited to serve as participants. Following the HLA, pharmacy students completed a quantitative and qualitative questionnaires to assess their perceived educational gains and opinions about the experience. Participants who underwent an HLA were asked to complete a questionnaire about the quality of the service they received. Results. From 2011-2019, 896 HLAs were conducted by 764 undergraduate pharmacy students. The students reported that completing an HLA improved their clinical knowledge, counselling skills, professionalism, and confidence when talking to participants. They believed the HLA delivered an authentic learning experience, similar to that achieved during workplace-based placements. The HLA service was rated as good or outstanding by 99% of the participants, and the majority stated that they intended to make lifestyle changes as a result of attending the HLA. Conclusion. Conducting healthy living assessments provided undergraduate pharmacy students a valuable, quality-assured opportunity for experiential learning. The HLAs were well received by participants as they informed them about their current health status and gave them useful advice about making health improvements.

Use of Wearable Activity Tracker in Patients With Cancer Undergoing Chemotherapy: Toward Evaluating Risk of Unplanned Health Care Encounters.

PURPOSE: Unplanned health care encounters (UHEs) such as emergency room visits can occur commonly during cancer chemotherapy treatments. Patients at an increased risk of UHEs are typically identified by clinicians using performance status (PS) assessments based on a descriptive scale, such as the Eastern Cooperative Oncology Group (ECOG) scale. Such assessments can be bias prone, resulting in PS score disagreements between assessors. We therefore propose to evaluate PS using physical activity measurements (eg, energy expenditure) from wearable activity trackers. Specifically, we examined the feasibility of using a wristband (band) and a smartphone app for PS assessments. METHODS: We conducted an observational study on a cohort of patients with solid tumor receiving highly emetogenic chemotherapy. Patients were instructed to wear the band for a 60-day activity-tracking period. During clinic visits, we obtained ECOG scores assessed by physicians, coordinators, and patients themselves. UHEs occurring during the activity-tracking period plus a 90-day follow-up period were later compiled. We defined our primary outcome as the percentage of patients adherent to band-wear ≥ 80% of 10 am to 8 pm for ≥ 80% of the activity-tracking period. In an exploratory analysis, we computed hourly metabolic equivalent of task (MET) and counted 10 am to 8 pm hours with > 1.5 METs as nonsedentary physical activity hours. RESULTS: Forty-one patients completed the study (56.1% female; 61.0% age 40-60 years); 68% were adherent to band-wear. ECOG score disagreement between assessors ranged from 35.3% to 50.0%. In our exploratory analysis, lower average METs and nonsedentary hours, but not higher ECOG scores, were associated with higher 150-day UHEs. CONCLUSION: The use of a wearable activity tracker is generally feasible in a similar population of patients with cancer. A larger randomized controlled trial should be conducted to confirm the association between lower nonsedentary hours and higher UHEs.

Survivorship and Advocacy in Inflammatory Breast Cancer.

In February 2017, the Morgan Welch Inflammatory Breast Cancer (IBC) Research Program and Clinic hosted a scientific conference in Houston to commemorate the tenth anniversary of the opening of the first IBC-dedicated clinic in the world. Attendees included basic science researchers, clinicians who treat IBC, as well as patients and their caregivers. Several US-based and international IBC-focused nonprofit organizations were also represented. In this third paper from the conference, we report on the breakout session regarding survivorship and advocacy issues related to IBC, sharing an overview of the educational content presented and discussions regarding the future of IBC advocacy. Panelists focused on lymphedema research and clinical solutions, integrative medicine, and social work, with time provided for questions in small groups. IBC nonprofits that are leading advocacy efforts were introduced, and ways to become involved in these initiatives were discussed. Priorities for future advocacy and clinical care needs were also highlighted. In addition to summarizing these topics, we provide a suggested integrated IBC-specific plan of care that could be provided to the patient at the beginning of care and referred to throughout treatment and follow-up.

Cyclin E Overexpression Sensitizes Triple-Negative Breast Cancer to Wee1 Kinase Inhibition.

PURPOSE: Poor prognosis in triple-negative breast cancer (TNBC) is due to an aggressive phenotype and lack of biomarker-driven targeted therapies. Overexpression of cyclin E and phosphorylated-CDK2 are correlated with poor survival in patients with TNBC, and the absence of CDK2 desensitizes cells to inhibition of Wee1 kinase, a key cell-cycle regulator. We hypothesize that cyclin E expression can predict response to therapies, which include the Wee1 kinase inhibitor, AZD1775. EXPERIMENTAL DESIGN: Mono- and combination therapies with AZD1775 were evaluated in TNBC cell lines and multiple patient-derived xenograft (PDX) models with different cyclin E expression profiles. The mechanism(s) of cyclin E-mediated replicative stress were investigated following cyclin E induction or CRISPR/Cas9 knockout by a number of assays in multiple cell lines. RESULTS: Cyclin E overexpression (i) is enriched in TNBCs with high recurrence rates, (ii) sensitizes TNBC cell lines and PDX models to AZD1775, (iii) leads to CDK2-dependent activation of DNA replication stress pathways, and (iv) increases Wee1 kinase activity. Moreover, treatment of cells with either CDK2 inhibitors or carboplatin leads to transient transcriptional induction of cyclin E (in cyclin E-low tumors) and result in DNA replicative stress. Such drug-mediated cyclin E induction in TNBC cells and PDX models sensitizes them to AZD1775 in a sequential treatment combination strategy.Conclusions: Cyclin E is a potential biomarker of response (i) for AZD1775 as monotherapy in cyclin E-high TNBC tumors and (ii) for sequential combination therapy with CDK2 inhibitor or carboplatin followed by AZD1775 in cyclin E-low TNBC tumors.

International Consensus on the Clinical Management of Inflammatory Breast Cancer from the Morgan Welch Inflammatory Breast Cancer Research Program 10th Anniversary Conference.

National and international experts in inflammatory breast cancer (IBC) from high-volume centers treating IBC recently convened at the 10th Anniversary Conference of the Morgan Welch Inflammatory Breast Cancer Research Program at The University of Texas MD Anderson Cancer Center in Houston Texas. A consensus on the clinical management of patients with IBC was discussed, summarized, and subsequently reviewed. All participants at the conference (patients, advocates, researchers, trainees, and clinicians) were queried using the MDRing electronic survey on key management issues. A summary of the expert consensus and participant voting is presented. Bilateral breast and nodal evaluation, breast magnetic resonance imaging, positron emission tomography/computed tomography, and medical photographs were endorsed as optimal. Neoadjuvant systemic therapy, modified radical mastectomy and level I and II ipsilateral axillary node dissection, post-mastectomy radiotherapy, adjuvant targeted therapy and hormonal therapy as indicated, and delayed reconstruction were agreed-upon fundamental premises of standard non-protocol-based treatment for IBC. Consideration for local-regional therapy in de novo stage IV IBC was endorsed to provide local control whenever feasible. Variation across centers and special circumstances were discussed.

CDK4/6 Inhibitors Sensitize Rb-positive Sarcoma Cells to Wee1 Kinase Inhibition through Reversible Cell-Cycle Arrest.

Research into the biology of soft tissue sarcomas has uncovered very few effective treatment strategies that improve upon the current standard of care which usually involves surgery, radiation, and chemotherapy. Many patients with large (>5 cm), high-grade sarcomas develop recurrence, and at that point have limited treatment options available. One challenge is the heterogeneity of genetic drivers of sarcomas, and many of these are not validated targets. Even when such genes are tractable targets, the rarity of each subtype of sarcoma makes advances in research slow. Here we describe the development of a synergistic combination treatment strategy that may be applicable in both soft tissue sarcomas as well as sarcomas of bone that takes advantage of targeting the cell cycle. We show that Rb-positive cell lines treated with the CDK4/6 inhibitor palbociclib reversibly arrest in the G1 phase of the cell cycle, and upon drug removal cells progress through the cell cycle as expected within 6-24 hours. Using a long-term high-throughput assay that allows us to examine drugs in different sequences or concurrently, we found that palbociclib-induced cell-cycle arrest poises Rb-positive sarcoma cells (SK-LMS1 and HT-1080) to be more sensitive to agents that work preferentially in S-G2 phase such as doxorubicin and Wee1 kinase inhibitors (AZD1775). The synergy between palbociclib and AZD1775 was also validated in vivo using SK-LMS1 xenografts as well as Rb-positive patient-derived xenografts (PDX) developed from leiomyosarcoma patients. This work provides the necessary preclinical data in support of a clinical trial utilizing this treatment strategy. Mol Cancer Ther; 16(9); 1751-64. ©2017 AACR.

Cyclin E overexpression as a biomarker for combination treatment strategies in inflammatory breast cancer.

Inflammatory breast cancer (IBC) is a virulent form of breast cancer, and novel treatment strategies are urgently needed. Immunohistochemical analysis of tumors from women with a clinical diagnosis of IBC (n = 147) and those with non-IBC breast cancer (n = 2510) revealed that, whereas in non-IBC cases cytoplasmic cyclin E was highly correlated with poor prognosis (P < 0.001), in IBC cases both nuclear and cytoplasmic cyclin E were indicative of poor prognosis. These results underscored the utility of the cyclin E/CDK2 complex as a novel target for treatment. Because IBC cell lines were highly sensitive to the CDK2 inhibitors dinaciclib and meriolin 5, we developed a high-throughput survival assay (HTSA) to design novel sequential combination strategies based on the presence of cyclin E and CDK2. Using a 14-cell-line panel, we found that dinaciclib potentiated the activity of DNA-damaging chemotherapies treated in a sequence of dinaciclib followed by chemotherapy, whereas this was not true for paclitaxel. We also identified a signature of DNA repair-related genes that are downregulated by dinaciclib, suggesting that global DNA repair is inhibited and that prolonged DNA damage leads to apoptosis. Taken together, our findings argue that CDK2-targeted combinations may be viable strategies in IBC worthy of future clinical investigation.

Scientific Summary from the Morgan Welch MD Anderson Cancer Center Inflammatory Breast Cancer (IBC) Program 10th Anniversary Conference.

In 2006, a remarkable collaboration between University of Texas MD Anderson Cancer Center clinicians and Texas and New Mexico State legislators led to the formation of a dedicated IBC Research Program and Clinic at MD Anderson. This initiative provided funding and infrastructure to foster coordination of an IBC World Consortium of national and international experts, and launch the first ever IBC international conference in 2008, which brought together experts from around the world to facilitate collaborations and accelerate progress. Indeed great progress has been made since then. National and international experts in IBC convened at the 10th Anniversary Conference of the MD Anderson IBC Clinic and Research Program and presented the most extensive sequencing analysis to date comparing IBC to non-IBC, gene- and protein-based immunoprofiling of IBC versus non-IBC patients, and converging lines of evidence on the specific role of the microenvironment in IBC. Novel models, unique metabolic mechanisms, and prominent survival pathways have been identified and were presented. Multiple clinical trials based on the work of the last decade are in progress or in development. The important challenges ahead were discussed. This progress and a coordinated summary of these works are presented herein.

Indoleamine 2,3-dioxygenase expression in transplanted NOD Islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the breakdown of the amino acid tryptophan into kyneurenine. It has been shown that IDO production by placental trophoblasts prevents the attack of maternal T-cells activated in response to the paternal HLA alleles expressed by the tissues of the fetus. In this article, we show that adenoviral gene transfer of IDO to pancreatic islets can sufficiently deplete culture media of tryptophan and consequently inhibit the proliferation of T-cells in vitro. Experiments in vivo have also demonstrated that transplantation of IDO-expressing islets from prediabetic NOD mouse donors into NODscid recipient mice is associated with a prolongation in islet graft survival after adoptive transfer of NOD diabetogenic T-cells. This protection is attributed to the depletion of tryptophan at the transplantation site beneath the kidney capsule. These results suggest that local modulation of tryptophan catabolism may be a means of facilitating islet transplantation as a therapy for type 1 diabetes.

Gene transfer of manganese superoxide dismutase extends islet graft function in a mouse model of autoimmune diabetes.

Islet transplantation is a promising cure for diabetes. However, inflammation, allorejection, and recurrent autoimmune damage all may contribute to early graft loss. Pancreatic islets express lower levels of antioxidant genes than most other tissues of the body, and beta-cells in particular are sensitive to oxidative damage. Therefore, damage from oxidative stress may pose a major obstacle to islet replacement therapy in that both the islet isolation and transplantation processes generate oxygen radicals. To determine whether antioxidant gene overexpression in isolated pancreatic islets can prevent oxidative damage and prolong islet function after transplantation, we used the NOD mouse model to study oxidative stress encountered during both transplantation and autoimmune attack. We transferred an antioxidant gene, manganese superoxide dismutase (MnSOD), by adenoviral infection into isolated islets that were transplanted into streptozotocin-treated NODscid recipient mice. Functioning islet grafts were subsequently exposed to diabetogenic spleen cells and monitored until graft failure. The results show that islet grafts overexpressing MnSOD functioned approximately 50% longer than control grafts. This significant prolongation of graft function suggests that the antioxidant activity of MnSOD is beneficial to transplanted islet survival and may be used in combination with other strategies aimed at islet graft protection.

SOP3: a web-based tool for selection of oligonucleotide primers for single nucleotide polymorphism analysis by Pyrosequencing.

SOP3 is a web-based software tool for designing oligonucleotide primers for use in the analysis of single nucleotide polymorphisms (SNPs). Accessible via the Internet, the application is optimized for developing the PCR and sequencing primers that are necessary for Pyrosequencing. The application accepts as input gene name, SNP reference sequence number, or chromosomal nucleotide location. Output can be parsed by gene name, SNP reference number, heterozygosity value, location, chromosome, or function. The location of an individual polymorphism, such as an intron, exon, or 5' or 3' untranslated region is indicated, as are whether nucleotide changes in an exon are associated with a change in an amino acid sequence. SOP3 presents for each entry a set of forward and biotinylated reverse PCR primers as well as a sequencing primer for use during the analysis of SNPs by Pyrosequencing. Theoretical pyrograms for each allele are calculated and presented graphically. The method has been tested in the development of Pyrosequencing assays for determining SNPs and for deletion/insertion polymorphisms in the human genome. Of the SOP3-designed primer sets that were tested, a large majority of the primer sets have successfully produced PCR products and Pyrosequencing data.