Abstract Background: SPARC (Secreted Protein Acidic and Rich in Cysteine) is known to regulate cell growth and to inhibit cell-cycle progression. It has high affinity in binding albumin and hence has been suggested to predict benefit of albumin-bound cytotoxic agents. We conducted a pooled analysis to elucidate SPARC expression according to BC molecular subtypes and its association with clinical outcome in early BC. Methods: We used publically available datasets and normalized microarray data as published by the original studies. Eligible patients were those who received no adjuvant systemic therapy (untreated series), were treated with tamoxifen (tam-treated series) or were treated with neoadjuvant anthracyclines ± paclitaxel or docetaxel (neoadjuvant series). We computed 2 SPARC modules, SPARC7 and SPARC8 that were composed of genes with an absolute correlation above 0.7 and 0.8 with SPARC, respectively. In the untreated series, we examined the expression of SPARC according to BC subtype defined by PAM50. We investigated the correlation with other gene modules representing diverse biological processes; proliferation (AURKA, GGI), immune (STAT1, IRM), and stroma (DCN, PLAU). We investigated the association with relapse-free survival (RFS) in univariate and multivariate models, both in the untreated and tam-treated series. This was performed in all patients and according to BC subtype. In the neoadjuvant series, we investigated the association with pathological complete response (pCR) in all patients and according to BC subtype. All multivariate models were adjusted for tumor size, nodal status, age and histological grade. Results: 1008, 393 and 996 patients were included in the untreated, tam-treated and neoadjuvant series, respectively. SPARC expression was highest in luminal-A, small (< 2 cm) and low histological grade tumors (all p < 0.0001). We found high positive correlation between SPARC, SPARC modules and stroma-related modules (r = 0.90) but a negative correlation with proliferation (r = −0.66). No correlation was observed with immune-related modules. In the untreated series, SPARC was not associated with prognosis in the univariate model. However, restricting the analysis to the HER2 molecular subtype, high SPARC expression was associated with short RFS in both univariate and multivariate models (HR: 3; 95% CI [1.5–6.3], p = 0.002). In the tam-treated series, SPARC expression was not associated with clinical outcome neither in luminal-A nor luminal-B. In the neoadjuvant series, SPARC expression was not associated with pCR (p = 0.37) except in the HER2 subtype in which high expression was independently associated with low pCR rates in the multivariate model (OR: 0.32; 95% CI [0.1–0.9], p = 0.043). Similar associations were observed with SPARC7 and SPARC8 modules. Conclusion: Two main conclusions could be drawn from our analysis 1) SPARC is highly expressed in low proliferative ER+ tumors; 2) High SPARC expression is associated with short RFS and poor response to neoadjuvant therapy in patients with HER2+ BC. These results could be relevant in the clinical development of albumin-bound compounds, in which SPARC expression is believed to identify patients who could benefit from these agents. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-07-22.

Abstract Background: Translational research studies in the context of international clinical trials may incur prolonged transportation time of tumor specimens. The effect of prolonged transportation time and sample preservation method on RNA quality and on reliability of derived gene expression profiling (GEP) is unknown. Methods: Tumor material from surgical specimens of patients with primary breast cancer (BC) was obtained with cold ischemia time (CIT) recorded for each patient. Each sample was divided into 8 aliquots 4 that were placed in RNAlater and 4 that were frozen. To simulate prolonged transportation duration, samples were kept 1 week in a bucket of dry ice replenished daily, with temperature monitoring. The bucket was stored at room temperature. RNA extraction and GEP using Affymetrix HG-U133 Plus2 were performed at baseline and at day 2, 4 and 7. Linear mixed effect models were used to explore effects of transportation time and preservation method on RNA quality (RNA Integrity Number-RIN). Pre-defined single genes (n = 12), gene modules (n = 25) and molecular subtypes (PAM50) were evaluated. P < 0.01 was considered for significance. Results: Samples were collected from 13 patients with an average CIT of 37 minutes (range: 20–85). During the simulation period, the mean temperature at which samples were stored was −77.8°C (SD+/−1.6°C). RIN values decreased significantly over time (1-day change −0.08, CI = −0.12 to −0.04, p < 0.001) and by RNAlater (−0.29, CI = −0.07 to −0.51, p < .01), but remained always above 6.5. RNA degradation evaluated with actin 3/5 and gapdh 3/5 ratios was significantly higher over time for samples preserved in RNAlater compared with frozen (difference in changes 0.68, CI=0.49 to 0.87, p < 0.001 and 0.33,0.25 to 0.4, p < 0.001, respectively), and for 5 RNAlater (38%) samples these ratios exceeded the Affymetrix thresholds (3 and 1.25, respectively) at least at 1 time-point. Comparing tissue preservation methods, only the expression of PTEN gene and CASP3 module changed significantly more over time in RNAlater compared with frozen samples (interaction p < 0.01). Genes and gene modules that were significantly differently expressed over time, independent of preservation method, were AURKA, PTEN, CASP3 and WOUND (p < 0.01, range of 1-day changes in log2 expression:-0.05 to 0.10), while the expression of ESR1 and ERBB2 was unaffected. Those that were significantly influenced by sample preservation method, adjusted for time, were genes ESR1, PLAU, VEGF, PIK3CA, PTEN and gene modules GENE21 and GENE70 was (P < 0.01, range: −0.61 to 0.16). Using PAM50, 8 (61%) samples were classified at least once as a different subtype over time compared to baseline, and 4 (30%) samples fell in a different Genomic Grade Index (GGI) class risk (high/low) at least once over time. Conclusions: In this study, frozen samples were significantly less degraded over time compared with samples stored in RNAlater. Preservation method had a stronger influence on genes and gene modules values compared with prolonged transport duration. In international clinical trials we recommend freezing samples to assure more stable gene expression analysis and limit transport duration to less than 3 days. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-08.

Abstract Background: A significant proportion of breast cancer (BC) patients (pts) develop multiple synchronous unilateral breast tumors, also referred to as multifocal BC (MBC), which represent a diagnostic and therapeutic challenge. Here, we aimed first to better define the incidence of MBCs and then to compare different foci from ductal MBCs in a global analysis using genomic, transcriptomic and epigenomic data. Methods: The incidence of MBCs was sought via a systematic query of all pathology reports between 2000 and 2010 within the Institut Bordet. The biological characterization of MBCs focused on 5 ductal MBCs for which the foci did not differ in terms of grade, hormonal receptors and HER2 status, since this is the case for the majority of MBCs. It involved the identification of somatic rearrangements (Rs), transcriptomic and epigenomic profiling in 2 foci from each pt, via low-coverage whole genome sequencing, HG133 Plus 2.0 Chips and Infinium Methylation 450K arrays respectively. Genomic Rs were validated using an orthogonal sequencing platform. Results: MBC is a frequent finding since it concerns 24% (1410/5811) of primary BCs. When investigating the genomics of the 5 MBCs, we observed that the number of Rs varied in each pt, with 3 pts having few (<10) Rs whilst the other 2 pts showed > 100Rs with a particular tandem-duplication phenotype. All pts had Rs common to both foci, suggesting a shared genetic background. Strikingly, in 4 pts, we observed a branched evolutionary pattern since private Rs were present in each focus. In contrast, we observed a linear evolutionary pattern in the remaining pt, since private Rs were only found in 1 of the 2 foci. Two pts were genetically very similar with ≥75% common Rs. Although the transcriptomic profiles looked very similar between the 2 foci, significant epigenomic differences were observed in 1 of the 2 pts investigated. Two other pts had 50% common Rs. In one pt there were only minor differences in transcriptomic and methylation patterns between the 2 foci. Contrastingly, in the second pt, DNA methylation patterns were dramatically discordant between the foci, suggesting that genomic, transcriptomic and epigenomic patterns are not necessarily correlated. Only in 1 pt of the 5 investigated, the 2 foci were extremely different, with only 14% of common Rs. These genetic differences were associated with dramatic differences in methylation and transcriptomic profiles. Interestingly, for 4 pts some of the identified rearrangements were found in parts of tumor-adjacent histologically normal breast tissue. Conclusions: Today, the College of American Pathologists recommends analysing more than 1 focus from MBC if they differ in histology and grading (Lester 2009). Here, we demonstrated for the first time that different lesions from MBC, which concerns ¼ of the ductal BC population, can differ at the (epi)genetic and transcriptomic level even when the foci present similar histology, grading, hormonal and HER2 status. Since the number of genetic alterations with potential clinical utility is rapidly growing due to increasing numbers of targeted therapies, these findings suggest that interrogating only the largest lesion might not be sufficient for adequate management of MBCs. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S6-2.

Abstract Background: Invasive lobular carcinoma (ILC) is the second most common breast cancer (BC) histotype, after invasive ductal carcinoma (IDC), representing ∼10-15% of the global BC population. The vast majority of ILC are estrogen receptor (ER)-positive. PIK3CA has been reported to be the most frequently mutated gene in ER-positive BC. However, until now PIK3CA mutations have mainly been described in IDC. In this study, we sought to characterize the type, prevalence, intra-patient (pt) mutational heterogeneity, and the association with transcriptomic phenotype and clinical outcome of PIK3CA mutations in ILC. Methods: Gene expression data (HG-U133 Plus2.0) was generated from frozen samples of 116 ILC (primary tumors). For the same series, PIK3CA mutation profiling (Sequenom) was performed on at least 2 formalin-fixed paraffin embedded (FFPE) tissue blocks of the primary tumor for 102 cases, and on the only available block for the remaining 14 cases. Matched local recurrence (LR) and multiple metastatic (M) samples were also characterized for 3 and 8 pts respectively. For the transcriptomic and survival analyses, we evaluated the association between PIK3CA mutation status and gene signatures recapitulating important biological processes, and invasive disease-free & overall survival, respectively. Results: PIK3CA mutations were detected in the primary tumor of 40/116 (35%) ILC pts: 14 (35%) mutations in the helical domain and 25 (62.5%) in the kinase domain. PIK3CA analysis in different blocks from the same primary tumors revealed a mutational heterogeneity in 7/102 (7%) cases. There was a discordance between primary and LR samples in 1/3 patients with LR, with a mutation reported in the primary but in not the LR. In 3/8 pts for which M samples were available, we observed intra-pt heterogeneity. In the first pt, a mutation in the kinase domain was reported only in 1/2 investigated blocks from the primary tumor, and in only 1/2 liver Ms. In the second pt, a mutation in the helical domain was found in the primary tumor and in the LR but not in the pleura M. In the third pt, although no mutation was observed in the primary tumor, a mutation in the helical domain was found in the M of the epiploon, but not in the Ms from the diaphragm or the ovary. Transcriptomic analyses revealed that primary tumors with a PIK3CA mutation in the helical domain were associated with increased levels of 2 stroma signatures (Bonferroni p-val: PLAU sign -Desmedt 2008–0.042 and DCN sign- Farmer 2009 – 0.048) compared to tumors without mutation. The survival analyses did not reveal any significant difference in survival between pts with and without a PIK3CA mutation. Conclusions: This study represents the largest series described so far which investigated PIK3CA mutations in ILC. The incidence of PIK3CA mutations reported here is similar to the one reported for IDC. The investigation of multiple tumor samples per pt revealed intra-tumor as well as primary/recurrence heterogeneity. To get a definitive answer on the prognostic role of PIK3CA mutation in ILC, we are currently performing a PIK3CA mutation profiling study (Sequenom) on a series of ∼1000 consecutive ILC with long-term follow up (Iorfida et al. 2012); results will be available by SABCS2012. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-05-03.

The records of five patients who received local irradiation for incompletely excised or inoperable glomus tumour were retrospectively reviewed. Age ranged from 25 to 46 years. There were two female and three male patients. Two patients who were solely treated by radiation therapy remain clinically disease free at 30 and 42 months respectively. Of the remaining three patients, where radiotherapy was delivered post operatively, one achieved complete remission and is well and alive at 60 months, whereas the other two patients were lost to follow up at 22 and 26 months respectively after showing an early clinical improvement. We advocate the efficiency of radiation therapy as primary treatment in the management of primary glomus tumour.

Extramedullary haematopoiesis associated with thalassaemia leading to spinal cord compression is an extremely rare event in the course of the disease. The efficacy of radiation therapy is advocated in the management of such a complication. Two patients with thalassaemia, who had presented with spinal cord compression, were successfully treated by a modest dose of local radiotherapy. In one of the patients, however, radiotherapy was resorted to after an initial decompressive laminectomy and partial removal of the intraspinal haematopoietic mass proved unsuccessful. The other patient was managed solely by radiation therapy.

An interesting manifestation of carcinoma of the esophagus, hitherto undescribed is reported. The patient at the time of diagnosis had presented with an esophagocutaneous fistula. He was treated by feeding jejunostomy and local palliative radiotherapy and showed good clinical improvement. The extreme rarity of such a presentation is highlighted.

Primary melanoma of the meninges, a rare CNS tumor, is presented. Criteria for diagnosing a primary CNS melanoma are elucidated. Literature is reviewed in this context. The histogenesis of tumor, problem of occult primary melanoma and the role of CT scan and CSF cytology in early diagnosis have also been highlighted.

Five cases of primary lymphoma of the brain are reported and the literature reviewed. The diverse clinical spectrum at presentation and variable response to therapy are few of its unique features. The literature is reviewed briefly and the importance of diagnosis at an early stage and prompt institution of therapy in form of radiation therapy and chemotherapy when indicated is stressed.

In 1986, 50 patients with stages II and III carcinoma of the cervix were entered into this prospective randomized study. Twenty-five cases (Group I) were treated only by radical radiation whereas remaining 25 cases (Group II) received local hyperthermia in addition to radical radiation. Hyperthermia was delivered by intracavitary brachyhyperthermia approach using an endotract applicator. Both the groups were followed up for a minimum period of 18 months. Group II patients achieved better local control (14 out of 20 evaluable cases) than the Group I patients (11 out of 22 evaluable cases). A disturbing observation was the increased incidence of distant metastasis in Group II (4 out of 23 cases) as compared to Group I (1 out of 23 cases), though most of them remained disease free locally. The increasing use of hyperthermia in the management of various cancers needs to be reviewed in this context.

A rare case of primary plasmacytoma of the stomach is reported. The diagnostic value of gastroscopy leading to non-surgical management is highlighted. Radiation therapy is an effective alternative mode of treatment and should be employed as the primary modality of therapy.

A retrospective analysis was performed of 250 cases of carcinoma of the maxillary antrum seen over a 10 years period (1975–1984). 98.24% patients were seen in T3 and T4 stages (UICC 1985). 40.7% patients presented with clinically palpable nodes. 42.9% patients were treated by radical radiation and 18.6% by a combination of preoperative radiation followed by surgery. Rest 38.5% patients were treated with palliative intention. Three year disease free survival was 39.58% with radiation alone and 51.91% with combined modality treatment which includes patients salvaged by surgery. Failures were mainly at the local site, 75.86% with radiation alone and 60% with combined modality treatment. Combined modality treatment, preferably preoperative radiation followed by radical surgery, gives the best results in the management of carcinoma of the maxillary antrum.

Computed tomography (CT) has been suggested as an alternative to surgery for predicting the stage and resectability of primary gastric lymphoma. It is our policy to perform CT scan on all patients and the decision to explore is based upon CT findings. We reviewed our experience to see if any changes were warranted. 12 patients with primary gastric lymphoma underwent CT scanning. The CT findings were compared with the pathological findings available for 10 patients. CT correctly staged 90 per cent of the patients and it obviated unnecessary exploratory surgery in 42 per cent of the patients. It permitted the planning of non-excisional therapy in 75 per cent of the patients and was useful as an indicator of response to therapy and as a follow up measure.

Primary fallopian tube carcinoma is a rare neoplasm. It may be postulated that postoperative loco-regional radiotherapy is a valuable adjunctive in the overall management of the tubal carcinoma. However, we can't advocate the efficacy of this approach authentically due to a very small number of cases. A prospective controlled and randomized trial with larger patient number may yield some definite conclusions regarding it's optimal management.

Embryonal rhabdomyosarcoma of the vagina in adults per se is uncommon and pregnancy complicated by such tumors is extremely rare. We wish to report one case of vaginal embryonal rhabdomyosarcoma, diagnosed at the age of 23 years who was carrying at the time of initial presentation. The problem of management of rhabdomyosarcoma in adults, particularly when they are associated with pregnancy is discussed. We advocate the use of multiagent chemotherapy and pelvic radiotherapy in its overall management.

The clinicopathological features of 12 cases of primary lymphoma of the stomach are reviewed, and the efficacy of various treatment modalities is evaluated. The presenting symptomatology and the radiological signs were found to be similar to those of gastric carcinoma. Gastroscopy and computed tomography (CT) proved to be effective diagnostic and staging tools. There were two patients of stage I, seven patients of stage II, and three patients of stage IV. Multimodal therapy involving judiciously combined surgery, radiation therapy, and chemotherapy produced early complete remission in all the nine patients with localized disease, and all of them are disease‐free at a minimum follow‐up period of 12 months. These results are irrespective of whether surgery formed part of the treatment regimen. The relevant literature is reviewed, and the present status of the efficacy of nonsurgical therapy is evaluated.

Occurrence of skeletal metastases in carcinoma of uterine cervix is infrequent. There are only a few cases who present wits osseous involvement at the time of initial diagnosis. Appearance of skeletal metastases in carcinoma uterine cervix is an ominous sign of progression of disease. Screening studies for detection of occult skeletal metastases in asymptomatic patients are not recommended.

An extremely rare case of asymptomatic squamous cell carcinoma of the esophagus metastatic to the spine leading to paraplegia is reported. The rarity of such an event signifies the relationship between hypercalcaemia and carcinoma of the esophagus. Its occurrence as a second silent primary neoplasm and its association with a negative esophagogram is highlighted.