Thymidine Labeling Index Research Articles

GATA2 Mutation in Twins with Transient Aplastic Anemia after Chloramphenicol Exposure; A Follow up to Nagao and Mauer 1969

A case of identical twins who developed transient aplastic anemia after chloramphenicol exposure was reported by Nagao and Mauer in the New England Journal of Medicine in 1969. Bone marrow (BM) morphology and tritiated thymidine labeling indices were suggestive of a failure of delivery cells from the undifferentiated stem cell compartment. They concluded that an underlying genetic factor which predisposed to the development of drug induced aplastic anemia was highly likely. We now report long term follow up on the twins from that article and have identified that they both harbour a mutation in GATA2. Twin 1 presented with severe pancytopenia at age 8 months, 3 months after chloramphenicol exposure. His leukocytes and erythrocytes had normalized with supportive care, but platelet count (PC) remained low at 92,000/mm3. Later in life, he was diagnosed with transfusion acquired hepatitis C, treated with Sofosbuvir/Velpatasvir. He did not develop recurrent infections, warts, lymphedema, or pulmonary symptoms. At age 51, he was referred to Hematology for asymptomatic thrombocytopenia with a PC of 17,000/mm3. BM aspirate and biopsy showed a variably cellular marrow with relative erythroid hyperplasia and megaloblastoid features. Cytogenetics, FISH for myelodysplastic syndrome (MDS) related abnormalities and a 33 gene panel for MDS/chronic myelomonocytic leukemia related mutations by next generation sequencing were all normal. A next generation sequencing panel of 59 genes associated with hereditary BM failure syndromes was remarkable only for a previously unreported heterozygous mutation in GATA2 (c1040C>A, pThr347Asn). Twin 2 was also exposed to chloramphenicol at 5 months of age and was found to be pancytopenic when evaluated for BM donation for his brother. His pancytopenia spontaneously resolved at the end of observation. Subsequent medical problems included partial complex seizures, dysautonomia, vertigo, chronic pain syndrome and recurrent Clostridium difficile colitis. He had no history of other recurrent infections, warts, lymphedema, or pulmonary symptoms. He had mild asymptomatic cytopenias noted with a white blood cell count of 3600/mm3, a hemoglobin of 12.9 g/dl and a PC of 97,000/mm3 at the age of 50 years. At the age of 52, he was referred to hematology and was asymptomatic with a normal hemogram. DNA sequencing confirmed an identical GATA2 mutation. GATA2, a DNA binding transcription factor with two zinc finger domains, plays a significant role in gene regulation, cell fate decisions and maintenance of the hematopoietic stem cell pool. Excessive GATA2 activity is reported to prevent stem cells from leaving the pool, while insufficient GATA2 activity causes stem cells to differentiate at increased rates thereby depleting the pool. Homozygous GATA deletions/mutations are lethal. Reported mutations include deletions, frame shift mutations, nonsense mutations and point mutations. Many point mutations appear to act by causing haplo-insufficiency. The point mutation in these patients maps in proximity to the C-zinc finger domain and could potentially disrupt an exonic splicing enhancer causing an in-frame deletion of exon six. Clinical syndromes associated with GATA2 mutations include familial BM failure and cytopenia; familial myelodysplastic syndromes and acute myeloid leukemia; immunodeficiency with monocytopenia and recurrent viral, fungal and mycobacterial infections; immunodeficiency with decreased natural killer, dendritic, B cells, and monocytes; Emberger syndrome presenting with myelodysplastic syndrome, warts and lymphedema; and pulmonary alveolar proteinosis. Cytopenia has been the only clinically obvious manifestation in these twins. Chloramphenicol has been associated with two forms of hematological toxicity. The first is a dose related reversible bone marrow suppression presenting as anemia, and the second is idiosyncratic, often fatal, aplastic anemia. It was proposed that these may be opposite ends of a spectrum with an underlying genetic predisposition leading to the more severe forms. The finding of a GATA2 mutation in these patients supports Nagao and Maurer's speculation that an underlying genetic factor affecting stem cell regulation predisposed to the development of chloramphenicol induced aplastic anemia in these twins. Figure Disclosures No relevant conflicts of interest to declare.

The impact of progesterone receptor expression on prognosis of patients with rapidly proliferating, hormone receptor-positive early breast cancer: a post hoc analysis of the IBIS 3 trial.

Background:In the Italian Breast Cancer Intergroup Studies (IBIS) 3 phase III trial, we compared cyclophosphamide, methotrexate, 5-fluorouracil (CMF) alone to sequential epirubicin/CMF regimens in patients with rapidly proliferating early breast cancer (RPEBC). We performed a post hoc analysis in the subgroup of patients with hormone-receptor-positive RPEBC on the prognostic role of progesterone receptor (PgR) status.Methods:RPEBC was defined by thymidine labeling index (TLI) >3% or grade 3 or S-phase >10% or Ki67 >20%. We analyzed 466 patients with hormone-receptor-positive RPEBC receiving sequential epirubicin/CMF regimens followed by tamoxifen, and for whom the status of ER and PgR was available.Results:Considering both cut-off values of 10% and 20%, PgR expression was significantly associated with age, menopausal status, and ER expression; HER2 status was associated with PgR status only at a cutoff value of 20% PgR. Upon univariate analysis, tumor size, nodal status, and PgR were significantly associated with disease-free survival (DFS) and overall survival (OS), while age class and local treatment type were associated only with DFS. Patients with PgR <20% showed lower 5- and 10-year DFS [hazard ratio (HR) = 1.48; 95%CI: 1.01–2.18; p = 0.044] and OS (HR = 1.85; 95%CI: 1.08–3.19, p = 0.025) rates compared with patients with PgR ⩾20%. Upon multivariate analysis, only tumor size, nodal status, and PgR were independent prognostic factors.Conclusions:Our results highlight the independent prognostic relevance of PgR expression in patients with hormone-receptor-positive RPEBC treated with adjuvant chemotherapy and endocrine therapy, where the definition of prognostic subgroups is still a major need.

The prognostic role of progesterone receptor in patients with rapidly proliferating, hormone receptor-positive early breast cancer.

545 Background: The prognostic role of progesterone receptor (PgR) in highly proliferating early breast cancer (BC) is not well established. We retrospectively explored this biomarker in a cohort of patients with highly proliferating tumors enrolled in a phase III trial of adjuvant therapy. Methods: 1066 patients with N- or 1-3 N+ BC were randomized to receive: epirubicin followed by CMF, CMF followed by epirubicin, or CMF alone. Rapidly proliferating tumors were defined by thymidine labeling index (TLI) &gt; 3% or histological grade 3 or S-phase &gt; 10% or Ki67 &gt; 20%. We analyze the subgroup of 466 patients with hormone receptor (HR)-positive tumors treated with sequential epirubicin/CMF regimens followed by tamoxifen and for whom immunohistochemical assessments of estrogen receptor (ER), PgR, HER2 and Ki67 were available. Disease-free (DFS) and overall survival (OS) curves were built with the Kaplan–Meier estimator and compared by logrank test and Cox regression models. Results: PgR expression was significantly associated with ER expression, HER2 status, age and menopausal status, but not with Ki67, tumor size and nodal status. PgR cutoff values of 10% and 20% were chosen based on a Receiver Operating Characteristics analysis and the literature data. DFS and OS figures at 5 and 10 years, as well as the relative hazard ratios, according to the different PgR cutoff values, are reported in the table. Conclusions: Our results confirm the prognostic relevance of PgR expression in a cohort of patients with highly proliferating, HR-positive early BC treated with adjuvant chemotherapy and endocrine therapy. [Table: see text]

Time to initiation of adjuvant chemotherapy in patients with rapidly proliferating early breast cancer

AimTo evaluate the optimal time interval from definitive surgery to commencing chemotherapy in early breast cancer (EBC). Patients and methodsThe relationship between time to initiation of adjuvant chemotherapy (TTC), calculated in weeks, and disease-free (DFS) or overall survival (OS), was assessed in 921 EBC patients with rapidly proliferating tumours (thymidine labelling index >3% or G3 or Ki67 >20%), randomised in a phase III clinical trial (NCT01031030) to receive chemotherapy with or without anthracyclines (epirubicin→cyclophosphamide, methotrexate and fluorouracil (CMF) versus CMF→epirubicin versus CMF). DFS, OS and 95% confidence intervals (95% confidence interval (CI)) were calculated by the Kaplan–Meier method. Multivariate Cox analysis was performed in relation with nodal involvement, oestrogen receptor and human epidermal growth factor receptor 2 (HER2) status, Ki67 value, type of adjuvant chemotherapy, menopausal status and tumour size. ResultsAt a median follow-up of 105months (range 2–188), a prolonged TTC resulted in a significant increase in the risk of relapse: hazard ratio (HR) 1.15 (95% CI 1.02–1.30, p=0.019). Using a backward elimination procedure, TTC, tumour size and nodal involvement remained significantly associated with DFS. A time-dependent receiver-operating characteristic (ROC) curve analysis was subsequently utilised to evaluate the best cut-off for TTC, identifying 7weeks as the best threshold for longer OS (p=0.043): 8-year OS 88% (95% CI 85–90) for patients with a TTC <7weeks and 78% (95% CI 68–87) for the other group. ConclusionsOur results confirm that a shorter TTC may reduce relapses and possibly also improve clinical outcome in patients with highly proliferating EBC.

263PD - Time to Initiation of Adjuvant Chemotherapy in Patients with Rapidly Proliferating Early Breast Cancer

ABSTRACT Introduction: The optimal time from surgery to commencing chemotherapy in early breast cancer (EBC) remains unclear. We assessed the influence of time to initiation of adjuvant chemotherapy (TTC) on the outcome of EBC patients enrolled onto a phase III clinical trial (NCT01031030). Methods: The relationship between TTC, calculated as the time (in weeks) from definitive surgery to initiation of adjuvant chemotherapy, and disease-free (DFS) or overall survival (OS) was assessed in 1066 EBC patients with rapidly proliferating tumors (thymidine labeling index > 3% or G3 or Ki67 > 20%), randomized to receive adjuvant chemotherapy with or without anthracyclines (epirubicin → CMF vs CMF → epirubicin vs CMF). DFS, OS and their 95% confidence intervals (95% CI) were calculated by the Kaplan-Meier method. Multivariate Cox analysis was performed in relation to nodal involvement, estrogen receptor and HER2 status, Ki67 value, type of adjuvant chemotherapy, menopausal status and tumor size. Results: Information on TTC was available for 713 women. At a median follow-up of 105 months (range 2-188), a prolonged TTC resulted in a significant increase of 16% in the risk of relapse (95% CI 1.03–1.30, p = 0.016) in a multivariable Cox regression model (Table 1). The impact on OS was not significant. Using a backward elimination procedure, TTC, tumor size and nodal involvement remained significantly associated with DFS (Hazard ratio [HR] = 1.15, 95% CI 1.02-1.29, p = 0.018; HR = 1.44, 95% CI 1.08-1.92, p = 0.012; HR = 1.44, 95% CI 1.08-1.92, p = 0.012, respectively). Again, nodal involvement and Ki67 were associated with OS (HR = 1.66, 95% CI 1.11-2.49, p = 0.014; HR = 1.63, 95% CI 1.03-2.59, p = 0.039, respectively). DFS OS HR (95% CI) p HR (95% CI) p TTC 1.16 (1.03–1.30) 0.016 1.14 (0.97–1.35) 0.121 Age 0.99 (0.97–1.02) 0.829 0.99 (0.96–1.03) 0.615 ER Positive 1.00 1.00 Negative 1.20 (0.89–1.61) 0.242 1.20 (0.78–1.84) 0.397 HER2 Positive 1.00 1.00 Negative 0.99 (0.73–1.33) 0.929 0.92 (0.60–1.42) 0.713 Lymph node status Negative 1.00 1.00 Positive 1.53 (1.14–2.04) 0.004 1.71 (1.13–2.59) 0.011 Menopausal status Premenopausal 1.00 1.00 Postmenopausal 1.32 (0.85–2.04) 0.219 1.56 (0.82–2.97) 0.177 Tumor size 1.00 1.00 ≥2 cm 1.41 (1.05–1.88) 0.020 1.44 (0.95–2.18) 0.085 Ki67 ≤20% 1.00 1.00 >20% 1.06 (0.77–1.44) 0.724 1.54 (0.96–2.48) 0.073 Treatment arm E»CMF 0.72 (0.49–1.04) 0.079 0.69 (0.41–1.16) 0.161 CMF»E 0.79 (0.55–1.14) 0.202 0.75 (0.45–1.25) 0.273 CMF 1.00 1.00 Conclusions: Our results suggest that patients with rapidly proliferating EBC should be treated as soon as possible once their recovery from surgery is complete. Disclosure: All authors have declared no conflicts of interest.

Early colon cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Early colon cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Impact of body mass index (BMI) on the prognosis of women with high-risk early breast cancer (BC) receiving adjuvant chemotherapy (CT).

552 Background: Obesity has been shown to impact the prognosis of early BC; this effect was not consistently observed across the different biological subtypes, and is less clear when aggressive tumor phenotypes are considered. The aim of this study was to evaluate the influence of BMI (kg/m2) on the prognosis of women with high risk early BC enrolled into a phase III clinical trial of adjuvant CT. Methods: The relationship between BMI and Disease Free (DFS) or Overall Survival (OS) was assessed in 1066 early BC patients with rapidly proliferating tumors (Thymidine Labeling Index &gt; 3% or G3 or Ki67 &gt; 20%), randomized to receive adjuvant CT with or without anthracyclines (Epirubicin → CMF vs CMF → Epirubicin vs CMF). BMI was defined as follows: normal &lt; 25 kg/m2, overweight 25-30 kg/m2, obese &gt;30 kg/m2. DFS and OS were calculated by Kaplan-Meier estimation; multivariate Cox analysis was performed according to menopausal status, type of CT, hormonal, HER-2 and nodal status. Results: Information on BMI at baseline, was available on 959 women. Of these, 430/959 (44.8%) were normal, 331 (34.5%) were overweight and 198 (20.7%) were obese. Median age was 52 years (range 26 to 70); 48% was node positive, 62% was ER positive and 33% was HER-2 positive. At a median follow-up of 69 mos (range 1-119), 5-year DFS was 80% (95% CI 78-83) and 5-year OS was 94% (95% CI 90–94). 5-year DFS was 81% (95% CI 77-85), 82% (95% CI 77-86) and 76% (95% CI 70-83), in normal, overweight and obese women, respectively (p 0.6). 5-year OS was 92% (95% CI 89-95), 94% (95% CI 91-96) and 89% (95% CI 84-93), respectively (p 0.4). By multivariate analysis only ER, HER-2 and nodal status were significantly associated with differences in DFS and/or OS. Conclusions: BMI at baseline was not associated with the prognosis of early BC patients with rapidly proliferating tumors, receiving adjuvant CT. These results confirm those achieved in triple negative BC and suggest that neither dietary restriction or medical interventions aimed at reducing BMI and/or underlying insulin resistance nor specific anticancer strategies seem to be appropriate in this subgroup.

Effect of neutropenia with adjuvant epirubicin-CMF on survival in patients with node-negative or 1 to 3 node-positive rapidly proliferating breast cancer.

e11566 Background: Patients who experience at least some degree of neutropenia whilst receiving adjuvant chemotherapy for breast cancer show improved survival. Tumor proliferation is a prognostic marker in breast cancer. We therefore hypothesized that patients with rapidly proliferating breast cancer and evidence of neutropenia whilst on adjuvant chemotherapy would have a better survival than that of similar patients in whom there was no evidence of myelotoxicity. Methods: We reviewed the case notes of 173 women treated in Wide Area of Romagna cancer centers with epirubicin followed by the intravenous CMF (cyclophosphamide, methotrexate and 5-fluorouracil) regimen or the inverse sequence for rapidly proliferating breast cancer, defined by thymidine labeling index (TLI) >3%, to identify patient- and treatment-related factors influencing outcome. Results: The actuarial 5-year overall survival for these women was 94%, with the anticipated poorer outcome for those with higher TLI (≥10% vs <10%, P=0.032). There was no evidence that ER-negative tumors, HER-2 positive disease or older age at presentation resulted in poorer survival. Of note, the absolute survival advantage was confined to the 47% of patients who had grade 3/4 neutropenia (P<0.001) with respect to those with grade 0-2 neutropenia (P=0.268). Conclusions: Our results suggest that neutropenia has a greater influence on outcome than age and tumor biological factors in patients with rapidly proliferating breast cancer.

Randomized phase III trial of adjuvant epirubicin followed by cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) versus CMF followed by epirubicin in patients with node-negative or 1–3 node-positive rapidly proliferating breast cancer

Adjuvant cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) have proven highly effective in rapidly proliferating breast cancer (RPBC). It has also been seen that sequential administration of doxorubicin and CMF is superior to their alternation, especially in indolent tumors. In a phase III study, we evaluated whether adjuvant epirubicin (E) followed by CMF is superior to the inverse sequence in RPBC. Patients with node-negative or 1-3 node-positive RPBC (Thymidine Labeling Index>3% or histological grade 3 or S-phase>10% or Ki67>20%) were randomized to receive E (100 mg/m(2) i.v. d1, q21 days for 4 cycles) followed by CMF (600, 40, 600mg/m(2) i.v. d1 and 8, q28 days for 4 cycles) (E→CMF) or CMF followed by E (CMF→E) or CMF for 6 cycles. From November 1997 to December 2004, 1066 patients were enrolled: E→CMF 440, CMF→E 438, and CMF 188. At a median follow-up of 69months, 5-year OS was 91% (95% CI 88-94) for E→CMF and 93% (95% CI 90-95) for CMF→E, with adjusted hazard ratio of 0.88 (95% CI 0.58-1.35), and DFS was 80% in both arms, with adjusted hazard ratio of 0.99 (95% CI 0.73-1.33, Cox model). Adverse events were similar, apart from a higher rate of neutropenia in the CMF→E arm. No important differences in clinical outcome were observed between the two different sequences, making both a valid option in early breast cancer. Further molecular characterization of the tumors might help to identify subgroups achieving higher benefit from either sequence.

Randomized phase III trial of adjuvant epicirubicin (E) followed by cyclophosphamide, methotrexate, and fluorouracil (CMF) or CMF followed by E in patients with N- or ≤ 3 N+ rapidly proliferating breast cancer (RPBC)

560 Background: Antimetabolites are active in proliferating cells, and the adjuvant schedule CMF is highly effective in RPBC, whereas the sequential administration of doxorubicin (D) and CMF is superior to CMF–&gt;D, especially in indolent tumors. In a phase III study, we evaluated whether adjuvant E followed by CMF is superior to the inverse sequence in RPBC. Methods: Patients with N-, T &gt; 1 cm or ≤ 3 N+ and any T RPBC (defined by thymidine labeling index or grade or S-phase or Ki67/MIB1) were randomized to receive E (100 mg/m2 i.v. d 1, q 21 days for 4 cycles) followed by CMF (600, 40, 600 mg/m2 i.v. d 1 and 8, q 28 days for 4 cycles) (arm A) or CMF followed by E (arm B) or CMF (600, 40, 600 mg/m2 i.v. d 1 and 8, q 28 days for 6 cycles) (arm C). Arm C was closed after the EBCTCG 2000 meta-analysis (data not shown). The main endpoint was overall survival (OS), and the study had 80% power to detect a 7% absolute increase in 5-year OS with 400 patients per arm. Results: From November 1997 to December 2004, 1066 patients were enrolled (arms A/B/C: 440/438/188): N- 53%, estrogen receptor positive 63%, grade 3, 77%. At a median follow up of 69 months, 5-year disease-free survival was 80% in both arms (A and B) (p = 0.93, logrank test), with adjusted hazard ratio (AHR) 0.99 (95% CI 0.73–1.33, Cox model), and OS was 91% in arm A and 93% in arm B (p = 0.66, logrank), with AHR 0.88 (95% CI 0.58–1.35, Cox model). Adverse events were similar, apart from a small increase in grade 4 neutropenia in arm B. Conclusions: No relevant differences in clinical outcome were observed with the 2 different sequences. Further subgroup analyses are ongoing to verify the efficacy of each sequence as a function of biomolecular and hormonal profiles. No significant financial relationships to disclose.

Temperature and germ cell regulation of Leydig cell proliferation stimulated by Sertoli cell-secreted mitogenic factor: a possible role in cryptorchidism.

Local control of Leydig cell morphology and function by seminiferous tubules was suggested in previous in vivo studies, especially those that used experimental cryptorchid rat testis as a model. These studies reported changes in morphology, increases in cell number and mitotic index and decreases in testosterone formation and luteinizing hormone/human chorionic gonadotropin receptor levels of Leydig cells. However, little is known about how these changes are mediated. We recently observed that a novel Sertoli cell-secreted mitogenic factor stimulated proliferation, decreased testosterone formation and luteinizing hormone/human chorionic gonadotropin receptor levels, and dramatically altered the morphology of Leydig cells in culture. In the present studies, we demonstrate that an increase in coculture temperature from 33 to 37 degrees C increased [3H]-thymidine incorporation (5.6- vs. 19.2-fold) and labelling index (4.3% vs. 15.8%), and accelerated proliferation (2.1- vs. 3.9-fold) of cultured immature Leydig cells. In addition, testosterone formation and luteinizing hormone/human chorionic gonadotropin receptor levels of Leydig cells cocultured with Sertoli cells were further decreased following a 4 degrees C increase in coculture temperature. This elevation in culture temperature increased both the secretion of this factor by Sertoli cells and responsiveness of Leydig cells to this factor. In addition, the presence of germ cells, especially pachytene spermatocytes, inhibited the secretion of the mitogenic factor by Sertoli cells. These temperature- and germ cell-associated effects mimicked the morphological and functional changes of Leydig cells reported following experimental cryptorchidism. These observations suggest a possible role of this Sertoli cell-secreted mitogenic factor in explaining Leydig cell changes following experimental cryptorchidism.

Is the thymidine labeling index a good prognostic marker in breast cancer?

BackgroundThe aim of the present study was to determine the prognostic relevance of thymidine labeling index (TLI) in patients with breast cancer.MethodsTLI of the primary tumor was measured in 268 patients at the time of the surgical biopsy by an in vitro method.ResultsFifty-four patients had stage I disease, and 138 patients had stage II disease, and 76 patients had stage III disease. One hundred-four patients were found to have low TLI-index (<3%), and 164 patients had high TLI-index (≥3%). The median follow-up was 71.5 months (range, 6–138 months). The 5-year overall survival (OS) and disease free survival (DFS) rates was 84% and 74%, respectively. Lymph node involvement, tumor size more than 2 cm, high nuclear grade and estrogen receptor negativity were found to be associated with poorer DFS and OS rates. On subgroup analysis, however, the 5-year OS rate was significantly higher in the low TLI-group than in the high TLI-group in patients with stage I disease (100% vs 76%, p = 0.05).ConclusionOur findings suggest that the prognostic significance of TLI appears to be limited to early breast cancer that might help to distinguish patients who need more aggressive adjuvant treatment.

Phase III randomized multicenter study on the effects of adjuvant CMF in patients with node-negative, rapidly proliferating breast cancer: twelve-year results and retrospective subgroup analysis

The randomized multicenter study on rapidly proliferating breast cancer, assessed according to thymidine labelling index (TLI), was activated at the end of the 1980s. The present work investigated whether and to what degree the short-term advantages observed from adjuvant CMF (cyclophosphamide, methotrexate, 5-fluorouracil) were maintained at a longer follow-up. Two hundred and eighty-one patients with node-negative and high TLI tumors were randomized to receive six cycles of CMF or no further treatment. At a median follow-up of 12 years, CMF produced a 25% and 20% relative reduction in relapse and death cumulative incidence, respectively. A breakdown analysis identified a subgroup of patients with intermediate proliferating tumors for whom a 70% and 73% reduction in relapse and death was observed in the intention-to-treat population. An even higher reduction of 80% and 84% in relapse and death was seen for the patients who had received the full CMF dose. We identified a subgroup of patients with intermediate proliferating tumors in whom the high benefit obtained from adjuvant CMF was maintained at a long-term follow up.

Cell Proliferation Measurements by Bromodeoxyuridine or Thymidine Incorporation: Clinical Correlates.

T he thymidine or bromodeoxyuridine labeling index (LI) is measured by determining the incorporation of precursor by viable cells in DNA synthesis (S-phase) and is expressed as percent. LI relates closely to cell proliferative rate. More precise measurement requires determination of S-phase duration (Ts), currently not practical for clinical application. LI correlates with mitotic index (MI), 1,2 but LI is much easier to measure accurately, particularly when the proliferative rate is low, because the ratio of labeled to mitotic cells may approach 100 to i. 3 Measurements of LI with tritiated thymidine (3H-dThd) or 5-bromo-2'-deoxyuridine (BrdUrd) administered intravenously or used in vitro with fresh tissue slices are similar. 4 Labeled cells in S-phase are visualized by autoradiography (3H-dThd) or immunohistochemistry (BrdUrd) in tissue sections (Fig 1). Histological identification of cells circumvents problems that can be troublesome with flow cytometry. Our purpose is to describe LI distributions of human tumors that we have studied by a single, standardized technique of in vitro labeling of fresh tissue slices with 3H-dThd or BrdUrd. 5,6 Results show that certain cancers have characteristic high or low proliferative rates, while others have a wide spectrum. The latter probably represent several discrete tumor types that have not been adequately classified by histological criteria. For the former, cell kinetic characteristics can be construed from the histological classification. For the latter, kinetic measurements can provide useful data for subclassification. LIs of some normal tissues are listed in Table 1. Certain tissues are in continuous renewal and have high LIs, whereas others have very low LIs. Some of the latter can respond to cell loss with proliferation

Correlations between clonogenicity and prognostic factors in human breast cancer.

The pursuit of more effective systemic treatments in breast cancer has been limited by a failure to maintain or grow tumour cells in vitro. This study reports our experience in obtaining clonogenic growth in 54 primary breast cancers obtained at mastectomy. Of 47 assays free of infection, clonogenic growth was achieved in 22 (41 per cent). Only 5 of 23 oestrogen receptor positive tumours grew compared to 14 of 21 oestrogen receptor negative tumours chi 2 = 9.03; P less than 0.01). None of the 5 cytosolic receptor positive tumours contained a nuclear receptor for oestrogen. Growth was not related to tumour stage, menopausal status, age or histological grade. Receptor negative tumours had higher thymidine labelling indices, but these were no different to the tumours grown in the assay.

Prognostic factors and in vitro cytotoxic sensitivity in colorectal cancer

The poor response rates to chemotherapy for colorectal cancer justify attempts to rationalize selection of patients for treatment, and the development of systems to evaluate new cytotoxic agents. Refinement of prognostic indices may identify colorectal cancer patients at a higher risk of recurrence who merit more aggressive treatment. We report our experience with the stem cell assay and pulse thymidine labelling in 43 primary colorectal cancers. Thirty-six tumours were evaluable, and clonogenic growth was obtained in 30 (83 per cent). In 24 tumours (67 per cent) growth was adequate for meaningful interpretation of a cytotoxic drug assay. Frequency of growth and colony forming efficiency did not correlate with histopathological grade, Dukes' stage or tumour cell kinetic indices. Thymidine labelling indices correlated with Dukes' stage (A and B versus C and D, P less than 0.01, Mann-Whitney U test). Cytotoxic assays with 5-fluorouracil and 5'-deoxy-5-fluorouridine were undertaken in 18 cases (14 primary carcinomas, 4 malignant ascites), of which 14 were evaluable and 3/14 (21.5 per cent) were chemosensitive in vitro. Both drugs were equally effective in vitro at clinically attainable plasma concentrations. This is in accordance with the response rates observed clinically with 5-FU chemotherapy in colorectal cancer.

Possible test for selection of adjuvant systemic therapy in early cancer of the breast.

The estrogen receptor content and the thymidine labelling index have been correlated in a series of primary cancers of the breast. Receptor was assayed in tumours of 409 patients with operable breast cancer. Recurrence rates were significantly higher in patients whose tumours did not contain receptors than in those whose tumours did. Women without axillary node involvement whose tumours lacked oestrogen receptors showed the same high rate of recurrence as all women with axillary involvement. The oestrogen receptor status of metatases was similar to that of the primary tumours. The thymidine labelling of 83 tumours was inversely related to their receptor content. Tumours without receptors had the highest indices and therefore the highest potential tumour growth. These results have been discussed in relation to selection of systemic adjuvant therapy.

Fibroblast growth factor (FGF) 2 and FGF9 mediate mesenchymal–epithelial interactions of peritubular and Sertoli cells in the rat testis

The role of fibroblast growth factor (FGF) 2 and FGF9 as mediators of cell-cell interactions between Sertoli cells (SCs) and peritubular cells (PCs) was investigated. Using RT-PCR, we demonstrated that SCs and PCs recovered from 20-day-old rats expressed several of the seven FGF receptors (FGFRs), and more specifically the FGFR1 IIIc. FGF2 and FGF9 did not elicit any morphological changes in primary cultures of SCs, nor did they alter the number of SCs in culture. By contrast, changes in shape were observed in FGF2- and FGF9-treated PCs. In addition, FGF2 but not FGF9 enhanced significantly and dose-dependently the number of PCs in culture, indicating that FGF2 was a survival factor for these cells. It was also mitogenic because it enhanced the [3H]thymidine labeling index in PCs. We next examined the effects of FGF2 and FGF9 in a coculture system using 20-day-old rat SCs and PCs, and in an organotypic culture system using XY rat embryonic gonads. In both models, FGF2 and FGF9 were found to promote cellular interactions as evidenced by the extent of cellular reorganization in the coculture system, and cord morphogenesis and growth in the organotypic culture system. A key feature in SC-PC interactions is the synthesis and remodeling of the basement membrane which is co-elaborated by the two cell types. Since basement membrane homeostasis depends upon the coordinated activity of proteinases and inhibitors, the proteinases and inhibitors produced by PCs and SCs degrading or opposing degradation of the major components of the basement membrane were further studied. Specifically, we monitored the metalloproteinases (MMP)-2 and -9 and the tissue inhibitors -1, -2 and -3, the plasminogen activators (PAs) and the PA inhibitor-1, using zymography for the proteinases and Western blots for the cognate inhibitors. Cocultures received FGF or an analog of cAMP in order to prevent cellular reorganization. We found that FGF2 was unique in inducing MMP-9 in coculture. Also, the enhanced levels of the PA inhibitor-1 and the 30 kDa band glycosylated form of tissue inhibitor-3 correlated with the enhanced SC-PC reorganization. It was concluded that FGF2 and FGF9 are morphogens for the formation of testicular cords.

Breast carcinoma malignancy grading by Bloom–Richardson system vs proliferation index: reproducibility of grade and advantages of proliferation index

Questions of reproducibility and efficacy of histologic malignancy grading relative to alternative proliferation index measurements for outcome prediction remain unanswered. Microsections of specimens from the Cooperative Breast Cancer Tissue Resource (CBCTR) were evaluated by seven pathologists for reproducibility of grade and classification. Nuclear figure classification was assessed using photographs. Grade was assigned by the Bloom–Richardson method, Nottingham modification. Proliferation index was evaluated prospectively by deoxyribose nucleic acid precursor uptake with thymidine (autoradiographic) or bromodeoxyuridine (immunohistochemical) labeling index using fresh tissue from 631 node-negative breast cancer patients accessioned at St Luke’s Hospital. A modified Nottingham–Bloom–Richardson grade was derived from histopathologic data. Median post-treatment observation was 6.4 years. Agreement on classification of nuclear figures (N=43) was less than good by kappa statistic (κ=0.38). Grade was moderately reproducible in four trials (N=10,10,19, 10) with CBCTR specimens (κ=0.50–0.59). Of components of Bloom–Richardson grade, agreement was least for nuclear pleomorphism (κ=0.37–0.50), best for tubularity (κ=0.57–0.83), and intermediate for mitotic count (κ=0.45–0.64). Bloom–Richardson grade was a univariate predictor of prognosis in node-negative St Luke’s patients, and was improved when mitotic count was replaced by labeling index (low, mid, or high). When labeling index was added to a multivariate model containing tumor size and vessel invasion, grade was no longer a significant predictor of tumor-specific relapse-free or overall survival. Mitotic index predicted best when intervals were lowered to 0–2, 3–10, and >10 mitotic figures per ten 0.18 mm2 high-power fields. We conclude that Nottingham–Bloom–Richardson grades remain only modestly reproducible. Prognostically useful components of grade are mitotic index and tubularity. The Nottingham–Bloom–Richardson system can be improved by lowering cutoffs for mitotic index and by counting 20–30 rather than 10 high-power fields. Measurement of proliferation index by immunohistochemically detectable markers will probably give superior prognostic results in comparison to grade.

Tumour resistance to cisplatin: a modelling approach

Although chemotherapy has revolutionized the treatment of haematological tumours, in many common solid tumours the success has been limited. Some of the reasons for the limitations are: the timing of drug delivery, resistance to the drug, repopulation between cycles of chemotherapy and the lack of complete understanding of the pharmacokinetics and pharmacodynamics of a specific agent. Cisplatin is among the most effective cytotoxic agents used in head and neck cancer treatments. When modelling cisplatin as a single agent, the properties of cisplatin only have to be taken into account, reducing the number of assumptions that are considered in the generalized chemotherapy models. The aim of the present paper is to model the biological effect of cisplatin and to simulate the consequence of cisplatin resistance on tumour control. The ‘treated’ tumour is a squamous cell carcinoma of the head and neck, previously grown by computer-based Monte Carlo techniques. The model maintained the biological constitution of a tumour through the generation of stem cells, proliferating cells and non-proliferating cells. Cell kinetic parameters (mean cell cycle time, cell loss factor, thymidine labelling index) were also consistent with the literature. A sensitivity study on the contribution of various mechanisms leading to drug resistance is undertaken. To quantify the extent of drug resistance, the cisplatin resistance factor (CRF) is defined as the ratio between the number of surviving cells of the resistant population and the number of surviving cells of the sensitive population, determined after the same treatment time. It is shown that there is a supra-linear dependence of CRF on the percentage of cisplatin-DNA adducts formed, and a sigmoid-like dependence between CRF and the percentage of cells killed in resistant tumours. Drug resistance is shown to be a cumulative process which eventually can overcome tumour regression leading to treatment failure.