DNA ploidy of spindle cell soft-tissue tumors and its relationship to histology and clinical outcome

With the use of fresh cell suspensions, the DNA ploidy of 11 benign and 27 malignant spindle cell soft-tissue lesions was determined by flow cytometry (37 cases) and/or image cytometry (35 cases). Of the 27 malignant lesions, 10 were low- or intermediate-grade sarcomas, and 17 were high-grade sarcomas. In the malignant lesions, the DNA ploidy was correlated with the histologic grade and the clinical outcome. Of the 11 benign lesions, six had a diploid DNA ploidy pattern, and five were nondiploid by either flow cytometry or image cytometry. All benign cases had a favorable outocome regardless of ploidy. Eight of the 10 low- or intermediate-grade malignant lesions were diploid, whereas two were nondiploid. Of the 17 high-grade sarcomas, 15 were nondiploid by either method of measurement and nine by both. Of the 10 diploid malignant tumors, only one had an unfavorable outcome (malignant mesothelioma with biopsy only), whereas of the 17 malignant lesions that were nondiploid, five had no evidence of recurrent disease, three cases recurred, eight patients died of disease, and one patient died of an unrelated cause. We concluded that (1) ploidy does not differentiate benign from malignant spindle cell soft-tissue tumors, (2) the nondiploid DNA pattern is more common in high-grade than in low- or intermediate-grade sarcomas, and (3) there is a statistically significant relationship between nondiploidy and a worse clinical outcome in sarcomas.     

High endothelial venules in B-cell non-Hodgkin malignant lymphomas

The number of high endothelial venules (HEVs) per unit area was determined in 55 B-cell non-Hodgkin malignant lymphomas (nHMLs) classified according to the Kiel classification. The number of HEVs per unit area was significantly different between low- and high-grade B-cell nHMLs. In the low-grade group, a large number of HEVs with preserved structure were present, whereas in high-grade tumours the HEVs appeared damaged. The present findings support the previous observation attributing prognostic relevance to lymphocyte recirculation in nHMLs.     

Proliferation, ploidy and prognosis in uterine smooth muscle tumours

DNA ploidy, mitotic rate (per 10 high power fields), mitotic index (per 1000 tumour nuclei), Ki-67 labelling index and S phase fraction were measured in 23 uterine leiomyosarcomas and 10 tumours of uncertain malignant potential. Correlations were calculated by Spearmann rank correlation. Univariate survival analysis was performed by log rank analysis and multivariate analysis performed by the Cox linear regression method. Ki-67 index and S phase fraction were significantly higher in leiomyosarcomas than in tumours of uncertain malignant potential. There was significant correlation between mitotic rate, mitotic index, Ki-67 index and S phase fraction in cases of leiomyosarcoma. Fifteen of 22 leiomyosarcomas and one of 10 tumours of uncertain malignant potential were DNA aneuploid. On univariate analysis of all the smooth muscle tumours, DNA ploidy, presence of significant nuclear atypia and presence of coagulative tumour cell necrosis were associated with outcome. Only DNA ploidy was associated with outcome in the group of leiomyosarcomas. On multivariate analysis of all of the smooth muscle tumours. DNA ploidy, age and grade of atypia were independently associated with outcome. No single factor was independently predicitive of outcome in the group of leiomyosarcomas. Alternative indices of cell proliferation correlate with mitotic rate in uterine leiomyosarcoma and do not provide additional useful prognostic information. DNA ploidy, age and grade of atypia are independently associated with outcome in uterine smooth muscle tumours and measurement of DNA ploidy may be useful in identification of cases with an adverse prognosis.     

p53, cellular proliferation, and apoptosis-related factors in thymic neuroendocrine tumors.

Thymic neuroendocrine tumors are biologically aggressive neoplasms with extensive local invasion and high mortality. Although various markers of cellular proliferation and apoptosis have correlated with degrees of tumor differentiation in pulmonary neuroendocrine neoplasms, they have not been systematically studied in thymic neuroendocrine tumors. We immunostained 21 cases of thymic neuroendocrine tumors for p53, MIB-1, and the apoptosis-related markers Bcl-2, Bcl-x, and Bax. By histological classification the cases were low-grade (nine cases), intermediate-grade (eight cases), and high-grade (four cases) thymic neuroendocrine tumors. p53 was expressed in five cases: 1/9 low grade, 3/8 intermediate grade, and 2/4 high grade. The mean cellular proliferation (MIB-1) was 7.1% (range 2-12%) in low-grade thymic neuroendocrine tumors, 6.1% (range 2-15%) in intermediate-grade thymic neuroendocrine tumors, and 34.2% (range 2-80%) in high-grade thymic neuroendocrine tumors. Bcl-2 was expressed in 16 cases: 7/9 low grade, 5/8 intermediate grade, and 4/4 high grade. Bcl-x was expressed in 16 cases: 7/9 low grade, 6/8 intermediate grade, and 3/4 high grade. Bax was expressed in 13 cases: 5/9 low grade, 4/8 intermediate grade, and 4/4 high grade. The presence of mutant p53 in the tumor was associated with a statistically significant decreased mean survival (P<0.05). In contrast, either by positive or negative staining or by the score technique (staining intensity x percentage of cells staining), the presence of Bcl-x was associated with an increased mean survival (P<0.05). Finally, a Bcl-x : Bax ratio >or=1 was also associated with an increased mean survival, as compared to a Bcl-x : Bax ratio >or=1 (P<0.05). Our study shows that p53 expression and certain apoptosis markers correlate with survival. The expression of these markers may account for differences in biological behavior.     

Ovarian carcinoma cells in effusions show increased S-phase fraction compared to corresponding primary tumors

The objective of this study was to analyze large-scale genomic patterns during disease progression from primary tumor to effusion in ovarian carcinoma, and to study the association between DNA ploidy parameters in effusions, proliferation/survival markers, and clinicopathologic characteristics. DNA ploidy status, DNA index (DI), and S-phase fraction (SPF) were compared in 22 matched primary carcinomas (all prechemotherapy specimens) and effusions (14 prechemotherapy and 8 postchemotherapy specimens) using image analysis. The association between these parameters and previously studied cell survival/proliferation biomarkers, previous administration of chemotherapy, chemotherapy response and survival was analyzed in a larger series of 54 effusions. The majority of specimens were aneuploid irrespective of anatomic site, with no significant differences in DI. SPF was significantly higher in effusions compared to matched primary tumors (P = 0.007 for all 22 pairs, P = 0.011 for 14 matched prechemotherapy specimens). Higher SPF was related to higher Ki-67 score (P = 0.045), and both SPF and DI were directly associated with higher level of Survivin (P < 0.001 for both). DI and SPF in effusions showed no association with histological grade, FIGO stage, residual disease volume, previous chemotherapy, response to chemotherapy at primary disease, recurrence or survival. Ovarian carcinoma cells in effusions have increased proliferation compared to corresponding primary tumors, as evidence of disease progression. DNA ploidy parameters in cancer cells in effusions are unaltered by chemotherapy and appear to be unrelated to chemotherapy response and to survival, suggesting that large-scale genomic patterns at this anatomic site are not useful in segregating patients into prognostic groups.     

Decreased annexin I expression in prostatic adenocarcinoma and in high-grade prostatic intraepithelial neoplasia

AIMS: To analyse annexin I expression in prostatic carcinoma. Annexin I belongs to a family of structurally related calcium and phospholipid-binding proteins implicated in signal transduction, DNA replication, cell proliferation and apoptosis. The decreased expression of annexin I, II and VII proteins has been reported in different types of cancer. METHODS AND RESULTS: Using immunohistochemistry, we analysed annexin I expression in 77 cases of prostatic adenocarcinoma (Gleason score 6, N = 40; Gleason scores 7-8, N = 27; and Gleason scores 9-10, N = 10) and high-grade prostatic intraepithelial neoplasia (PIN, N = 50). Immunoreactivity of annexin I in tumour cells was evaluated as negative (< 5% of cells), focally positive (5-25% of cells) or positive (> 25% of cells). In contrast to positive staining in adjacent benign prostatic epithelium, annexin I expression was decreased (focally positive) in 76% of cases of high-grade PIN (P < 0.0001) and was decreased or absent in 81% of prostatic adenocarcinomas (P < 0.0001). Annexin I expression in all higher grade tumours (Gleason scores 7-10) was only focally positive or absent. CONCLUSIONS: Expression of annexin I inversely correlates with the increasing histological grade of prostatic adenocarcinoma. By showing a progressive loss of annexin I expression in high-grade PIN, intermediate-grade and high-grade cancer, our findings suggest that the loss of annexin I expression occurs early in prostatic tumorigenesis and becomes more prominent throughout tumour progression. The loss of expression of annexin I may serve as a useful marker of prostate cancer development and progression.     

Malignant lymphomas and the acquired immunodeficiency syndrome. Evaluation of 30 cases using a working formulation

Malignant lymphomas occurring in 29 homosexual men and one thalassemic woman with the acquired immunodeficiency syndrome or the acquired immunodeficiency syndrome-related complex are reported using a working formulation for non-Hodgkin's lymphomas (NHLs). The patients' ages ranged from 25 to 59 years, with an average age of 42 years. Ninety percent of the cases were extranodal; 67% were exclusively extranodal. One case of Hodgkin's disease was encountered. All NHLs were of the diffuse types in both the intermediate- and high-grade categories, with the largest single group (49%) being of the diffuse, large, follicular-center-cell types. The NHLs in this series were classifiable as B-cell neoplasms and were aggressive as evidenced by markedly reduced median survivals. The morphological diagnosis as defined in the working formulation, especially for the intermediate-grade lesions, offered little significant prognostic information.     

Histologic grading of noninvasive papillary urothelial tumors: validation of the 1998 WHO/ISUP system by immunophenotyping and follow-up

Cytokeratin (CK) 20, Ki-67, and p53 were applied to 84 noninvasive papillary urothelial tumors graded by the 1973 World Health Organization (WHO) and 1998 WHO/International Society of Urological Pathology (ISUP) systems. In the WHO/ISUP classification, all benign lesions showed normal CK20 staining and all carcinomas showed abnormal staining. The Ki-67 index was significantly different between benign and malignant lesions (P < .05) and between low- and high-grade carcinomas (P < .001). p53 was negative in all benign lesions, with a significant difference between low- and high-grade carcinomas (P < .001). Tumor recurrence was significantly different between low- and high-grade carcinomas (no recurrences among the papillary urothelial neoplasms of low malignant potential). By the 1973 WHO classification, normal CK20 staining was present both in benign lesions and in carcinomas. Ki-67 staining did not distinguish between grade 2 and grade 3 carcinomas (P > .05), and there was no difference in p53 staining in grades 1 and 2 carcinomas (P > .05). Recurrences were not different between grades 1, 2, and 3 carcinomas. All biologic markers studied and tumor recurrences were significantly different among papillary lesions classified by the WHO/ISUP system but not by the 1973 WHO system, validating the predictive value of the WHO/ISUP system and providing objective markers for the grading of papillary urothelial tumors.     

DNA study of bladder papillary tumours chemically induced by N-butyl-N-(4-hydroxybutyl) nitrosamine in Fisher rats

To examine DNA abnormalities in bladder papillary tumours induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in female rats, using image cytometric DNA analysis and cytogenetics. Thirty female rats were exposed to BBN in their drinking water for 20 weeks. One group of 10 animals served as controls. The animals exposed to BBN were killed at a rate of two per week, with the bladder being collected under aseptic conditions and those tumours with exophytic growth removed. The nuclear DNA content of the tumours was evaluated using image cytometric analysis. In two rats part of the tumour pieces was stipulated for culturing. Cytogenetic analysis was performed on at least 30 cells from each cell population and on both tumours. Papillary carcinomas were classified as low grade and high grade. DNA ploidy studies were carried out on 28 low-grade and 21 high-grade papillary carcinomas. Histograms obtained by image analysis showed that a normal urothelium was diploid; 28.6% and 100% of low-and high-grade papillary carcinomas were aneuploid respectively. Both tumours used for cell culture showed multiple numerical and structural chromosome alterations and several marker chromosomes. Image cytometric DNA analysis proved to be a good and reliable method for examining DNA alterations in papillary bladder carcinomas. The present findings establish that the DNA content is statistically different between low-grade and high-grade papillary carcinomas and that deviation from the diploid number is markedly higher in the high-grade ones. In addition, the occurrence of marker chromosomes seems to be related to the aggressiveness of the tumour.     

DNA flow cytometry of follicular non-Hodgkin''s lymphoma.

S-phase fraction, an index of cellular proliferation, and DNA ploidy were measured by DNA flow cytometry in a retrospective study of lymph node biopsy specimens from 83 cases (before treatment) of follicular non-Hodgkin's lymphoma, Working Formulation categories B and C. The correlations between these measures and survival, clinical stage, symptoms and histopathological factors were investigated. Aneuploidy was rare (n = 16) and had no effect on length of survival or transformation to high grade lymphoma. The overall mean S-phase fraction was 3.6%; for the whole series increasing S-phase fraction was associated with decreased survival. A high S-phase fraction (more than 5%) in initial biopsy specimens was also associated with an increased risk of subsequent high grade transformation at relapse. There was no difference between the survival or proliferative activity of tumours composed of mainly small cleaved cells compared with those composed of mixed small and large cells. There was no difference in survival or proliferative activity between tumours showing a pure follicular growth pattern and those with a mixed follicular and diffuse growth pattern. Multifactorial analysis showed that an S-phase fraction of more than 5% and B symptoms were the most important factors determining survival in these follicular non-Hodgkin's lymphomas.     

Activated T-cell subsets in benign lymphoid hyperplasias and B-cell non-Hodgkin''s lymphomas.

Activated tumor-infiltrating T lymphocytes (TIL-T) were quantitated prospectively in excisional biopsy specimens of 49 B-cell non-Hodgkin's lymphomas (NHL) of various grades and compared with eight benign lymphoid hyperplasias (BLH) to identify any potential difference in host T-cell response. Immunotyping of tissue-cell suspensions was done by three-color flow cytometry, which was complemented by immunocytology by using cytocentrifuged preparations. Two activated T-cell subsets were studied: acutely activated TIL-T (CD3+ CD25+ HLADr-) and chronically activated TIL-T (CD3+ CD25- HLADr+). Results showed an association of the more aggressive intermediate/high-grade B-cell NHL with a higher percentage of late-phase activated TIL-T and a progressive increase with the grade of malignancy: 10.29%, 23.25%, 33.87%, and 47.78% (means) for BLH and for low-, intermediate- and high-grade B-cell NHL, respectively. Differences for this subset were significant (P less than 0.050) for the following comparisons: hyperplasia versus intermediate-grade NHL (P less than 0.0012), hyperplasia versus high-grade NHL (P less than 0.0002), and low versus high-grade NHL (P less than 0.0080). The percentage of acutely activated TIL-T cells did not show a statistically significant difference between the groups. The results suggest a host T-cell response to proliferating neoplastic cells in B-cell NHL. Paradoxically, the response does not appear to be protective for the host since the intensity is directly proportional to the grade of malignancy. However, the recognition of this response may have clinical applications since its amplification with biological response modifiers may result in effective adoptive immunotherapy of B-cell NHL. Further clarification of the specificity and biologic significance of host T-cell activation in B-cell NHL will require functional studies of isolated lymphocytic subpopulations from neoplastic tissue.     

Correlation between DNA flow cytometric and nucleolar organizer region data in non-Hodgkin's lymphomas

The argyrophilic staining (AgNOR) technique, novel in histopathology, was applied to a series of 20 non-Hodgkin's lymphomas (NHL) of established Kiel subtype. The method demonstrates nucleolar organizer regions (NORs) by virtue of sulphydryl groups on their associated proteins and the enumeration of AgNOR foci has been previously shown to discriminate between NHL of low- and high-grade histological types. This finding was confirmed and the results were compared with those obtained by means of DNA flow cytometry performed on paraffin wax-embedded tissue from the same lymphomas. There was a very good linear correlation between the mean numbers of AgNOR sites per nucleus and the percentage of S-phase cells for each case, both values being high in high-grade NHL and low in low-grade lesions. Conversely there was no significant correlation between the DNA index, representing DNA aneuploidy, and AgNOR counts. It is suggested that the numbers of AgNORs in a lymphoma may be related to the dividing fraction of cells rather than, as might be expected, to ploidy alone. It is also proposed that the AgNOR technique, which is rapid, simple, and inexpensive, may provide, at least, an adjunct to DNA flow cytometry in the assessment of neoplasm in histopathology.     

Working formulation of the non-Hodgkin's lymphomas: a study of cell size and mitotic indices in cytologic subtypes

Cell size and mitotic indices were studied in 243 non-Hodgkin's lymphomas (NHL) diagnosed cytologically and classified according to the Working Formulation. Low-, intermediate-, and high-grade lymphomas constituted 14.8%, 18.9%, and 61.7% of the cases, respectively. Measurement of the diameter of 100 lymphoid cells in each case showed that in most of the low-grade malignant lymphomas, 50-80% of the cells were small (less than 10 microns). A majority of the cells (an average of 45-60%) in intermediate- and high-grade lymphomas were in the range of 11-15 microns. Only the intermediate-grade large noncleaved and high-grade immunoblastic lymphomas had a majority of their cells (greater than 50%) larger than 15 microns. The mitotic indices (number of mitotic figures per 500 lymphoid cells) showed a highly significant difference between low- (0.7 +/- 1.14), intermediate- (3.0 +/- 2.67), and high-grade (5.4 +/- 3.40) lymphomas using the Wilcoxon's signed rank test (P less than 0.0001). Nearly two-thirds of the cases in each grade were within specific ranges of mitotic indices, i.e., less than 1 for low-, 1-3 for intermediate-, and greater than 3 for high-grade lymphomas.     

POSITIVE CORRELATION BETWEEN APOPTOTIC AND PROLIFERATIVE INDICES IN GASTROINTESTINAL LYMPHOMAS OF MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT)

To understand the role of deregulation of apoptosis in the pathogenesis of gastrointestinal MALT lymphoma, apoptosis has been quantitatively studied in paraffin sections from 40 cases (19 low grade, 21 high grade). The extent of apoptosis was correlated with histological grade, proliferative activity as measured by immunostaining of Ki67 proliferation antigen, and the expression of bcl-2 and p53 oncoproteins, which are known to participate in the regulation of apoptosis. Both apoptotic and proliferative indices were significantly ( P <0·00001) higher in high-grade than in low-grade tumours. Overall, apoptotic indices were negatively correlated with bcl-2 expression, particularly in low-grade tumours in which both strong bcl-2 expression and low levels of apoptosis were observed. Thus, the slow expansion of low-grade MALT lymphoma may partly result from a prolonged life-span of tumour cells, due to bcl-2-mediated blockage of apoptosis. No difference in apoptotic indices was found between p53-positive and p53-negative cases. Furthermore, correlation analysis revealed a significantly positive association between apoptotic and proliferative indices. This supports the current belief that the mechanisms controlling apoptosis and proliferation are both activated during the cell cycle and whether a cell enters the proliferation cycle or the apoptotic process depends on survival factors.     

Low versus high cell turnover in diffusely growing non-Hodgkin's lymphomas

Cell loss, perhaps as important as cell production in determining the size of an expanding cell population, has not usually been registered in quantitative cellular kinetic analyses of neoplastic disorders. The present retrospective study on various types and subtypes of non-Hodgkin's lymphomas (NHLs; n=170) was designed to test the usefulness of a novel additional parameter, the ‘turnover index’ (TI), which is the sum per case of the mitotic index and the apoptotic index. Results document that TIs clearly distinguished between categories and subtypes of NHLs according to the Kiel classification. Cluster analysis of TIs plotted against the percentage of Ki-67-positive cells per case revealed that about one-third of the high-grade malignancy lymphomas actually belonged to the low-turnover lymphomas. Overall survival was longer in the low- than in the high-turnover group of lymphomas. Assessment of TIs can, for practical diagnostic purposes, be replaced by counting mitotic figures and apoptotic cells in several high-power fields. The TI concept may help to interpret the kinetics of NHLs in terms of accumulation vs. proliferation of cells.     

Immunohistochemical staining of cytologic smears with MIB-1 helps distinguish low-grade from high-grade neuroendocrine neoplasms

Neuroendocrine neoplasms (NENs) of the lung and gastrointestinal tract constitute a pathologic and biologic spectrum of tumors. Accurate cytologic diagnosis of a neuroendocrine neoplasm is important since definitive treatment frequently is based on low- and high-grade categories without histologic sampling. In many instances, however, low- and high-grade NENs share cytologic features, hindering a precise classification. Since the histologic diagnostic criteria for separation of low- from high-grade categories can be based on the proliferation rate, we proposed to evaluate the usefulness of the immunocytochemical stain for the proliferation marker MIB-1 in the grading of NENs. Cytologic preparations of 63 NENs were retrieved from the files of Memorial Sloan-Kettering Cancer Center, New York, NY. One representative alcohol-fixed slide from each case was destained and restained immunocytochemically for MIB-1. When MIB-1 immunoreactivity was considered, all low-grade NENs showed immunoreactivity in fewer than 25% of the neoplastic cells, and all high-grade NENs demonstrated immunoreactivity in more than 50% of neoplastic cells. Our study demonstrates that MIB-1 dramatically stratifies NENs as low-grade or high-grade. Therefore, the proliferation index also correlates with grade of NEN in cytology specimens.     

Short-term significance of DNA ploidy and cell proliferation in breast carcinoma: a multivariate analysis of prognostic markers in a series of 308 patients

AIM: To determine the importance of tumour DNA ploidy and cell proliferation, as measured by the S phase fraction (SPF), in relation to other established clinicopathological indicators of prognosis in breast cancer. METHODS: A prospective study of 308 patients. Tumours were staged following the TNM system criteria and were classified according to the histological type and grade. DNA flow cytometry was performed on fresh/frozen samples stained with propidium iodide. Hormone receptors were analyzed by immunocytochemistry. A Cox proportional hazards regression model was used for statistical evaluation of the prognostic factors. RESULTS: Median follow up time was 39.6 months (range 3 to 84). A DNA diploid pattern was found in 134 tumours (43.5%) and aneuploid in 174 (56.5%). Median SPF value was 6.1% (range 1% to 27.8%). DNA ploidy and SPF were strongly correlated (p < 0.001), and both were related to histological type (p < 0.001), grade of differentiation (p < 0.001), tumour size (p = 0.006 and p = 0.002), and hormone receptor activity (p < 0.001). DNA ploidy was also related to node status (p = 0.022), but SPF was not. In univariate analysis, there were significant correlations between disease-free survival and age, histological grade, tumour size, node status, DNA ploidy, SPF, and hormone receptor activity; age, tumour size, node status, DNA ploidy, and hormone receptors were predictors of overall survival. In multivariate analysis, only node status (p = 0.001) and DNA ploidy (p = 0.006) retained independent prognostic significance in relation with overall survival, while node status (p < 0.001) and SPF (p < 0.001) were predictors of disease-free survival. DNA ploidy and SPF continued to predict disease-free and overall survival in lymph node positive (pN1) patients but not in the lymph node negative (pN0) group. CONCLUSIONS: DNA ploidy and SPF are strongly intercorrelated and have independent prognostic value for predicting the short term clinical outcome of breast carcinoma patients.     

Allelic imbalance analysis of chromosome 16q shows that grade I and grade III invasive ductal breast cancers follow different genetic pathways.

It remains controversial as to whether low- and high-grade invasive ductal breast cancers are related by progression through grade, or generally follow distinct genetic pathways. It has previously been shown by comparative genomic hybridization (CGH) that the latter is more likely to be correct, based on the high frequency of chromosome 16q deletion in grade I cancers, but not in grade III. This study used microsatellite markers on 16q to confirm the differences between grade I and grade III tumours and to exclude the possibility that grade III tumours generally arise from grade I tumours through a process involving regain of 16q. The concordance between the CGH and microsatellite data is good but imperfect, probably reflecting the different sensitivities and specificities of the two techniques and the varying mechanisms of allele loss in each tumour type.     

Flow cytometry in the evaluation of the prognosis and course of malignant soft-tissue tumors

Investigation of DNA content by flow cytometry method (determination of ploidy and cell proliferation activity) carried out in 45 patients with malignant and 19 patients with benign tumours revealed diploid distribution of DNA in 18 cases and aneuploid in one case of benign tumours. DNA distribution in malignant tumours was diploid in 23 cases (51.1%) and aneuploid in 22 regardless of histological variant of the tumour. Diploid cells were always present in the aneuploid tumours and their number was higher in primary tumours than in recurrent tumours (only tumour cells were found in the cytological control smears). No full correlation is observed between indices of cell proliferation activity obtained by morphological and cytometric methods. Ploidy and proliferation activity (number of cells in S-phase of cell cycle) are prognostic criteria of the survival and duration of disease without recurrence.