Pesticide mobility: determination by soil thin-layer chromatography.

Pesticide movement was evaluated by the comparison of R(F) values on thin layers of soils. Results from the new technique correlated well with existing information on pesticide movement, facilitating the grouping of pesticides into classes on the basis of mobility. Thin-layer chromatography may have broad applicability in soils research.

Expression of AP-2 transcription factors in human breast cancer correlates with the regulation of multiple growth factor signalling pathways.

The AP-2 transcription factors are required for normal growth and morphogenesis during mammalian development. Previous in vitro studies have also indicated that the AP-2 family of proteins may be involved in the etiology of human breast cancer. The AP-2 genes are expressed in many human breast cancer cell lines, and critical AP-2-binding sites are present in both the ERBB-2 (HER2/neu) and estrogen receptor promoters. We have now characterized immunological reagents that enable specific AP-2 family members, including AP-2alpha and AP-2gamma, to be detected in human breast cancer epithelium. Data obtained with these reagents demonstrate that whereas AP-2alpha and AP-2gamma are both present in benign breast epithelia, there is a significant up-regulation of AP-2gamma expression in breast cancer specimens (P = 0.01). There was also a significant correlation between the presence of the AP-2alpha protein and estrogen receptor expression (P = 0.018) and between specimens containing both AP-2alpha/AP-2gamma proteins and ERBB-2 expression (P = 0.003). Furthermore, we detected an association (P = 0.04) between the expression of AP-2gamma and the presence of an additional signal transduction molecule implicated in breast cancer, the insulin-like growth factor I receptor. Analysis of the proximal promoter of the insulin-like growth factor I receptor revealed a novel AP-2-binding site. Thus, AP-2 proteins may directly regulate the transcription of this growth factor receptor. Taken together, these data strongly support a role for the AP-2 gene family in the control of cell growth and differentiation in breast cancer.

Elevated frequency and functional activity of a specific germ-line p53 intron mutation in familial breast cancer.

Previous studies have determined that the frequency of germ-line p53 mutations in familial breast cancer patients is 1% or less, but these reports have not investigated the importance of polymorphic intron base changes in the p53 gene. Therefore, we investigated the frequency of both exon and intron germ-line p53 base changes in 42 breast cancer patients with a strong family history of breast cancer. The mean age of presentation of these patients was 44.0 years (range, 29-69), and 12 of 42 (29%) were of known Ashkenazi ancestry. Purified DNA obtained from the 42 index cases was screened for germ-line p53 mutations in exons 2-11 and surrounding introns using a combination of intron based primers for PCR-single strand conformation polymorphism analysis, direct sequencing, and microarray sequencing using the Affymetrix p53 gene chip methodology. Morphological analysis of apoptosis and cell survival determination were performed on EBV-immortalized lymphoblastoid cell lines from two patients with the p53 intron 6 mutation. A germ-line mutation in the p53 gene at nucleotide 13964 with a G to C base change (13964GC) was identified in 3 of 42 (7.1%) hereditary breast cancer patients. Two patients were heterozygous for this mutation, and one patient had a homozygous mutation. In comparison, 0 of 171 (0%) of sporadic breast cancer patients had the p53 13964GC mutation (P = 0.0003). We found that 0 of 42 (0%) of these hereditary breast cancer patients had other germ-line p53 mutation in exons 2-11. However, pedigree analysis demonstrated that all three patients had strong family histories of multiple types of cancers consistent with Li-Fraumeni syndrome but with late age of onset. Comprehensive BRCA1 and BRCA2 nucleotide analysis from patients with the p53 13964GC mutation revealed no concomitant deleterious BRCA1 or BRCA2 mutations, although they were found in the other hereditary breast cancer patients. Functional analysis of two immortalized lymphoblastoid cell lines derived from patients with the p53 13964GC mutation demonstrated prolonged in vitro survival in response to cisplatinum treatment and showed decreased chemotherapy-induced apoptosis. Immunohistochemical analysis of breast tumors from these patients revealed high levels of mutant p53 protein, suggesting a functional mutation in the p53 gene. In summary, we have identified a single p53 intron mutation in familial breast cancer patients that is present at elevated frequency and has functional activity.

Insulin-like growth factor-I receptor overexpression mediates cellular radioresistance and local breast cancer recurrence after lumpectomy and radiation.

The insulin-like growth factor-I receptor (IGF-IR) plays a critical role in cell growth regulation and transformation. The radiosensitivity of NIH 3T3 fibroblasts overexpressing either wild-type or mutant IGF-IR was examined. High levels of wild-type IGF-IR conferred radioresistance, and mutational analysis revealed that this effect correlated with the transforming capacity but not the mitogenic activity of the receptor. The radioresistant phenotype was reversed when the cells were incubated with antisense oligonucleotides targeted to IGF-IR mRNA, demonstrating that IGF-IR directly influences radioresistance. The clinical significance of these findings was examined in an immunohistochemical analysis of primary breast tumors, revealing that high levels of IGF-IR in tumor samples were highly correlated with ipsilateral breast tumor recurrence (IBTR) following lumpectomy and radiation therapy (P = 0.001). Subgroup analysis revealed that, for early breast tumor relapses (within 4 years of initial breast tumor diagnosis), elevated levels of IGF-IR were strongly associated with IBTR (P = 0.004) but IGF-IR expression was not prognostic for IBTR from breast cancer patients with late relapses (P was not significant). These studies provide evidence for the influence of IGF-IR on cellular radioresistance and response to therapy and raise the possibility that the radiocurability of selected tumors may be improved by pharmaceutical strategies directed toward the IGF-IR.

BRCA1/BRCA2 germline mutations in locally recurrent breast cancer patients after lumpectomy and radiation therapy: implications for breast-conserving management in patients with BRCA1/BRCA2 mutations.

PURPOSE: Breast cancer patients treated conservatively with lumpectomy and radiation therapy (LRT) have an estimated lifetime risk of local relapse (ipsilateral breast tumor recurrence [IBTR]) of 10% to 15%. For breast cancer patients carrying BRCA1 or BRCA2 (BRCA1/2) mutations, the outcome of treatment with LRT with respect to IBTR has not been determined. In this study, we estimate the frequency of BRCA1/2 mutations in a study of breast cancer patients with IBTR treated with LRT. PATIENTS AND METHODS: Between 1973 and 1994, there were 52 breast cancer patients treated with LRT who developed an IBTR within the prior irradiated breast and who were willing to participate in the current study. From our database, we also identified 52 control breast cancer patients treated with LRT without IBTR. The control patients were individually matched to the index cases with respect to multiple clinical and pathologic parameters. Lymphocyte DNA specimens from all 52 locally recurrent patients and 15 of the matched control patients under age 40 were used as templates for polymerase chain reaction amplification and dye-primer sequencing of exons 2 to 24 of BRCA1, exons 2 to 27 of BRCA2, and flanking intron sequences. RESULTS: After LRT, eight (15%) of 52 breast cancer patients had IBTR with deleterious BRCA1/2 mutations. By age, there were six (40%) of 15 patients with IBTR under age 40 with BRCA1/2 mutations, one (9.0%) of 11 between ages 40 and 49, and one (3.8%) of 26 older than age 49. In comparison to the six (40%) of 15 of patients under age 40 with IBTR found to have BRCA1/2 mutations, only one (6.6%) of 15 matched control patients without IBTR and had a BRCA1/2 mutation (P =.03). The median time to IBTR for patients with BRCA1/2 mutations was 7.8 years compared with 4.7 years for patients without BRCA1/2 mutations (P =.03). By clinical and histologic criteria, these relapses represented second primary tumors developing in the conservatively treated breast. All patients with BRCA1/2 mutations and IBTR underwent successful surgical salvage mastectomy at the time of IBTR and remain alive without evidence of local or systemic progression of disease. CONCLUSION: In this study, we found an elevated frequency of deleterious BRCA1/2 mutations in breast cancer patients treated with LRT who developed late IBTR. The relatively long time to IBTR, as well as the histologic and clinical criteria, suggests that these recurrent cancers actually represent new primary breast cancers. Early onset breast cancer patients experiencing IBTR have a disproportionately high frequency of deleterious BRCA1/2 mutations. This information may be helpful in guiding management in BRCA1 or BRCA2 patients considering breast-conserving therapy.

BAG-1: a novel biomarker predicting long-term survival in early-stage breast cancer.

PURPOSE: Among women with early-stage breast cancer treated with lumpectomy and radiation therapy, 30% to 40% will develop metastatic disease, which is often fatal. A need exists therefore for biomarkers that distinguish patients at high risk of relapse. We performed a retrospective correlative analysis of BAG-1 protein expression in breast tumors derived from a cohort of early-stage breast cancer patients. PATIENTS AND METHODS: Archival paraffin blocks from 122 women with stages I to II breast cancer treated with lumpectomy and radiation therapy (median follow-up, 12.1 years) were analyzed by immunohistochemical methods using monoclonal antibodies recognizing BAG-1 and other biomarkers, including Bcl-2, estrogen receptor, progesterone receptor, p53, and HER2/Neu. Immunostaining data were correlated with distant metastasis-free survival (DMFS) and overall survival (OS). RESULTS: Cytosolic immunostaining for BAG-1 was upregulated in 79 (65%) of 122 invasive breast cancers (P <.001) compared with normal breast. Elevated BAG-1 was significantly associated with longer DMFS and OS, overall (stages 1 and II) and in node-negative (stage I only) patients, on the basis of univariate and multivariate analyses (DMFS, P =.005; OS, P =.01, in multivariate analysis of all patients; DMFS, P =.005; OS, P =.001, in multivariate analysis of node-negative patients). All other biomarkers failed to reach statistical significance in multivariate analysis. Clinical stage was an independent predictor of OS (P =.04) and DMFS (P =.02). CONCLUSION: These findings provide preliminary evidence that BAG-1 represents a potential marker of improved survival in early-stage breast cancer patients, independent of the status of axillary lymph nodes.

Euploid derivatives of duplications from a translocation in neurospora.

Nontandem terminal chromosome duplications derived from N. crassa translocation T(I-->VI)NM103 give rise mitotically to some daughter nuclei which have become euploid by loss of one or the other of the two duplicated segments. Loss of the segment in normal sequence occurs as often as loss of the translocated segment. This is in contrast to all of several other Neurospora duplications that have been studied, where loss of the segment in normal sequence is absent or rare.--T(NM103) has the distal two thirds of linkage group IR exchanged with the right tip of VI. Crosses to normal sequence produce a class of morphologically distinct progeny with IR chromosome duplications. For a few days after germination, test crosses of these progeny are barren (make perithecia but few or no spores, as observed commonly with Neurospora duplications). Growing duplication cultures become fertile by accumulating nuclei which have been reduced to either normal sequence (by loss of the segment in translocation sequence) or translocation sequence (by loss of the segment in normal sequence). Both types usually appear within the first week of growth. Naturally formed mixtures or heterokaryons of NM103 duplication nuclei and their reduced euploid products have been studied by plating and by progeny testing. Determination of nuclear type is based on culture morphology, expression of genetic markers, and crossing behavior. Within the limits of testing, loss is found to begin precisely at the interchange points. The unique finding of frequent breakdown of normal-sequence linkage group I chromosomes is not dependent on the strain from which the chromosome was derived. Many different strains were tested, and for each one evidence was found that nuclei reduced to translocation sequence had been produced from duplication nuclei by loss of the segment in normal sequence.

Spore killer, a chromosomal factor in neurospora that kills meiotic products not containing it.

Three chromosomal factors called Spore killer (Sk) have been found in wild populations of Neurospora sitophila and N. intermedia. Sk resembles other examples of meiotic drive such as Segregation Distorter in Drosophila, Pollen killer in wheat, and Gamete eliminator in tomato. In crosses heterozygous for Sk, each ascus contains four viable black ascospores and four inviable, undersize, clear ascospores, with second-division segregations infrequent. The survivors contain the killer allele Sk(K), while unlinked markers segregate normally. Reciprocal crosses are identical. When crosses are homozygous for an allele of Sk, all eight ascospores are viable and black in most asci. (Many homozygous crosses have a background level of randomly occurring inviable spores; however, the pattern of 4 viable: 4 small clear ascospores is not found in any of the asci of Sk-homozygous crosses.)--Killer (Sk-1(K)) and sensitive (Sk-1(S)) alleles occur in about equal numbers among a worldwide sample of N. sitophila strains, following no geographic pattern. No killer allele has been found in N. crassa. Sk-2(K) and Sk-3(K), found in N. intermedia, are rare. Most N. intermedia strains are Sk-2(S) and Sk-3(S), but some are wholly or partially resistant to one or both of the killer alleles, while not themselves acting as killers. Sk-2(K) and Sk-2(R) are both specific in conferring resistance to Sk-2(K), but not to Sk-3(K). Likewise Sk-3(K) and Sk-3(R) are resistant specifically to Sk-3(K), but not to Sk-2(K). Resistance segregates as an allele of Sk(K).--Sk-2 and Sk-3 have been mapped near the centromere of linkage group III after introgression into N. crassa, where crossing over is normally 11% between the proximal III markers acr-2 and leu-1. But crossing over is absent in this region when either of the killer alleles is heterozygous (Sk-2(K)xSk-2(S), Sk-3(K)xSk-3(S) and Sk-2(K)xSk-2(R) have been examined).

The effect of evening alcohol consumption on next-morning glucose control in type 1 diabetes.

OBJECTIVE: Alcohol is associated with acute hypoglycemia in patients with type 1 diabetes. After drinking alcohol in the evening, delayed hypoglycemia has also been described, although its cause is unknown. We performed a controlled study to investigate this phenomenon. RESEARCH DESIGN AND METHODS: We admitted six men with type 1 diabetes (aged 19-51 years, HbA(1c) 7.0-10.3%) on two occasions, from 5:00 P.M. to 12:00 noon the following day. They received regular insulin injections before standardized meals, at 6:00 P.M. and 8:00 A.M., and a basal insulin infusion (0.15 mU x kg(-1) x min(-1)) from 11:00 P.M. They drank either dry white wine (0.75 g/kg alcohol) or mineral water at 9:00 P.M. over 90 min. Blood glucose, alcohol, insulin, cortisol, growth hormone, and glucagon levels were measured. RESULTS: Blood ethanol reached a mean (SEM) peak of 19.1 (1.2) mmol/l and was undetectable by 8:00 A.M. There were no significant differences in evening or overnight blood glucose levels between the studies. In the morning, fasting and postprandial blood glucose levels were significantly lower after consumption of wine (postprandial peak 8.9 [1.7] vs. 15 [1.5] mmol/l, P < 0.01), and from 10:00 A.M., five subjects required treatment for hypoglycemia (nadir 1.9-2.9 mmol/l). None of the subjects had hypoglycemia after consumption of water. After consumption of wine, growth hormone secretion was significantly reduced between midnight and 4:00 A.M. (area under the curve 2.1 [1.1] vs. 6.5 [2.1] microg. l(-1) x h(-1), P = 0.04). There were no differences in insulin or other hormone levels. CONCLUSIONS: In type 1 diabetes, moderate consumption of alcohol in the evening may predispose patients to hypoglycemia after breakfast the next morning. This is associated with reduced nocturnal growth hormone secretion. Patients should be informed of this risk and advised regarding appropriate preventative measures.

Prognostic significance of apoptosis regulators in breast cancer.

Dysregulation of normal programmed cell death mechanisms plays an important role in the pathogenesis and progression of breast cancer, as well as in responses of tumors to therapeutic intervention. Overexpression of anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-X(L) has been implicated in cancer chemoresistance, whereas high levels of pro-apoptotic proteins such as Bax promote apoptosis and sensitize tumor cells to various anticancer therapies. Though the mechanisms by which Bcl-2 family proteins regulate apoptosis are diverse, ultimately they govern decision steps that determine whether certain caspase family cell death proteases remain quiescent or become active. To date, approximately 17 cellular homologs of Bcl-2 and at least 15 caspases have been identified in mammals. Other types of proteins may also modulate apoptotic responses through effects on apoptosis-regulatory proteins, such as BAG-1-a heat shock protein 70 kDa (Hsp70/Hsc70)-binding protein that can modulate stress responses and alter the functions of a variety of proteins involved in cell death and division. In this report, we summarize our attempts thus far to explore the expression of several Bcl-2 family proteins, caspase-3, and BAG-1 in primary breast cancer specimens and breast cancer cell lines. Moreover, we describe some of our preliminary observations concerning the prognostic significance of these apoptosis regulatory proteins in breast cancer patients, contrasting results derived from women with localized disease (with or without node involvement) and metastatic cancer.

Cyclin D1 expression and early breast cancer recurrence following lumpectomy and radiation.

PURPOSE: The purpose of this study was to determine the prognostic significance of cyclin D1 (cycD1) levels in ipsilateral breast tumor recurrence (IBTR) following lumpectomy and radiation therapy. METHODS AND MATERIALS: A total of 98 patients (49 patients with IBTR and 49 matched cases without IBTR) selected from our conservatively treated breast cancer population served as the patient population for the current study. All patients were treated with lumpectomy followed by radiation therapy to the intact breast to a total median dose of 64 Gy. The patients were followed in our clinic with a median follow-up of 13 years. Immunohistochemical analysis of cycD1 in these 98 early-stage breast cancer patients was performed using a polyclonal antibody generated against the human cycD1 protein. All clinical, pathologic, and molecular variables were entered into a computerized data base for statistical analysis. RESULTS: Low levels of immunohistochemically detected cycD1 protein correlated with IBTR (p = 0.001), but there was no association between cycD1 protein levels and metastatic disease, axillary lymph node involvement, distant disease-free survival (DDFS), and overall survival (OS). Subgroup analysis revealed that for early breast tumor relapses (within 4 years of initial breast tumor diagnosis), low levels of cycD1 were associated with IBTR (p = 0.004), but cycD1 expression was not prognostic for IBTR from breast cancer patients with late relapses (p = NS). CONCLUSION: These studies provide in vivo evidence for the prognostic and biologic significance of cycD1 expression in determining response to radiation therapy in breast cancer patients.

True recurrence vs. new primary ipsilateral breast tumor relapse: an analysis of clinical and pathologic differences and their implications in natural history, prognoses, and therapeutic management.

PURPOSE: The purpose of this study was to classify all ipsilateral breast tumor relapses (IBTR) in patients treated with conservative surgery and radiation therapy (CS+RT) as either new primary tumors (NP) or true local recurrences (TR) and to assess the prognostic and therapeutic implications of this classification. METHODS AND MATERIALS: Of the 1152 patients who have been treated at Yale-New Haven Hospital before 1990, 136 patients have experienced IBTR as their primary site of failure. These relapses were classified as either NP or TR. Specifically, patients were classified as NP if the recurrence was distinctly different from the primary tumor with respect to the histologic subtype, the recurrence location was in a different location, or if the flow cytometry changed from aneuploid to diploid. This information was determined by a detailed review of each patient's hospital and/or radiotherapy record, mammograms, and pathologic reports. RESULTS: As of 2/99, with a mean follow-up of 14. 2 years, the overall ipsilateral breast relapse-free rate for all 1152 patients was 86% at 10 years. Using the classification scheme outlined above, 60 patient relapses were classified as TR, 70 were classified as NP and 6 were unable to be classified. NP patients had a longer mean time to breast relapse than TR patients (7.3 years vs. 3.7 years, p < 0.0001) and were significantly younger than TR patients (48.9 years vs. 54.5 years, p < 0.01). Patients developed both TR and NP at similar rates until approximately 8 years, when TR rates stabilized but NP rates continued to rise. By 15 years following original diagnosis, the TR rate was 6.8% compared to 13.1% for NP. Of the patients who had been previously tested for BRCA1/2 mutations, 17% (8/52) had deleterious mutations. It is noteworthy that all patients with deleterious mutations had new primary IBTR, while patients without deleterious mutations had both TR and NP (p = 0.06). Ploidy was evenly distributed between TR and NP but NP had a significantly lower S phase fraction (NP 13.1 vs. TR 22.0, p < 0.05). The overall survival following breast relapse was 64% at 10 years and 49% at 15 years. With a mean follow-up of 10.4 years following breast relapse, patients with NP had better 10-year overall survival (TR 55% vs. NP 75%, p < 0.0001), distant disease-free survival (TR 41% vs. NP 85%, p < 0.0001), and cause-specific survival (TR 55% vs. NP 90%, p < 0.0001). CONCLUSION: It appears that a significant portion of patients who experience ipsilateral breast tumor relapse following conservative surgery and radiation therapy have new primary tumors as opposed to true local recurrences. True recurrence and new primary tumor ipsilateral breast tumor relapses have different natural histories, different prognoses, and, in turn, different implications for therapeutic management.

Effective treatment of stage I uterine papillary serous carcinoma with high dose-rate vaginal apex radiation (192Ir) and chemotherapy.

PURPOSE: Uterine papillary serous carcinoma (UPSC) is a morphologically distinct variant of endometrial carcinoma that is associated with a poor prognosis, high recurrence rate, frequent clinical understaging, and poor response to salvage treatment. We retrospectively analyzed local control, actuarial overall survival (OS), actuarial disease-free survival (DFS), salvage rate, and complications for patients with Federation International of Gynecology and Obstetrics (FIGO) (1988) Stage I UPSC. METHODS AND MATERIALS: This retrospective analysis describes 38 patients with FIGO Stage I UPSC who were treated with the combinations of radiation therapy, chemotherapy, total abdominal hysterectomy, and bilateral salpingo-oophorectomy (TAH/BSO), with or without a surgical staging procedure. Twenty of 38 patients were treated with a combination of low dose-rate (LDR) uterine/vaginal brachytherapy using 226Ra or 137Cs and conventional whole-abdomen radiation therapy (WART) or whole-pelvic radiation therapy (WPRT). Of 20 patients (10%) in this treatment group, 2 received cisplatin chemotherapy. Eighteen patients were treated with high dose-rate (HDR) vaginal apex brachytherapy using 192Ir with an afterloading device and cisplatin, doxorubicin, and cyclophosphamide (CAP) chemotherapy (5 of 18 patients). Only 6 of 20 UPSC patients treated with combination LDR uterine/vaginal brachytherapy and conventional external beam radiotherapy underwent complete surgical staging, consisting of TAH/BSO, pelvic/para-aortic lymph node sampling, omentectomy, and peritoneal fluid analysis, compared to 15 of 18 patients treated with HDR vaginal apex brachytherapy. RESULTS: The 5-year actuarial OS for patients with complete surgical staging and adjuvant radiation/chemotherapy treatment was 100% vs. 61% for patients without complete staging (p = 0.002). The 5-year actuarial OS for all Stage I UPSC patients treated with postoperative HDR vaginal apex brachytherapy and systemic chemotherapy was 94% (18 patients). The 5-year actuarial OS for Stage I UPSC patients treated with HDR vaginal apex brachytherapy and chemotherapy who underwent complete surgical staging was 100% (15 patients). The 5-year actuarial OS for the 20 Stage I UPSC patients treated with combinations of pre- and postoperative LDR brachytherapy and postop WART was 65%. None of the 6 surgically staged UPSC patients treated with LDR radiation and WART/WPRT developed recurrent disease. For patients with FIGO Stage IA, IB, and IC UPSC who underwent complete surgical staging, the 5-year actuarial DFS by depth of myometrial invasion was 100, 71, and 40%, respectively (p = 0.006). The overall salvage rate for local and distant recurrence was 0%. Complications following HDR vaginal apex brachytherapy included only Radiation Therapy Oncology Group (RTOG) grade 1 and 2 toxicity in 16% of patients. However, complications from patients treated with WART/WPRT, and/or LDR brachytherapy, included RTOG grade 3 and 4 toxicity in 15% of patients. CONCLUSION: Patients with UPSC should undergo complete surgical staging, and completely surgically staged FIGO Stage I UPSC patients can be effectively and safely treated with HDR vaginal apex brachytherapy and chemotherapy. Both OS and DFS of patients with UPSC are dependent on depth of myometrial invasion. The salvage rate for both local and distant UPSC recurrences is extremely poor. Complications from HDR vaginal apex brachytherapy were minimal.

Impaired absorption and omission of insulin: a novel method of detection using the diabetes advisory system computer model.

The Diabetes Advisory System (DIAS) is a decision-support program developed to assist insulin dose adjustment in type 1 diabetes. In this paper, we show how it might be used to identify impaired absorption or omission of insulin in patients with poorly controlled blood glucose. An evaluation of glucose results from four outpatients with persistent hyperglycemia is presented (age 19-48 years with type 1 diabetes for 13-18 years of duration, HbA1c 9.4-13.6%). Each had completed a 4-day record of blood glucose (BG, pre-meal and bedtime), dietary (carbohydrate) intake, and insulin doses (with injection sites). From these data, DIAS modeled a glucose profile (simulated glucose, SG) for the same period. Qualitative assessments were made of differences between BG and SG, and selective reduction or complete removal of insulin doses where BG >> SG. Large improvements in modeling were attributed to either impaired absorption or omission of insulin. Confirmation of these problems was sought from the patients by detailed consultation and physical examination. Impaired insulin absorption was suspected in two patients, both having significant injection site abnormalities. Insulin omission was suspected in the other two subjects. Both had normal injection sites, and one admitted to missing doses. Following retraining, data from three patients showed noticeable improvements in overall modeling as well as glucose control. Using DIAS in the evaluation of patients with type 1 diabetes may highlight previously unrecognized injection site abnormalities or insulin dose omission. This could assist rational optimization of insulin therapy in cases of persistently poor glucose control.