Characterization and clinical impact of the tumor microenvironment in post-transplant aggressive B-cell lymphomas.

Post-transplant lymphoproliferative disorders (PTLD) are iatrogenic immune deficiency-associated lymphoid/plasmacytic proliferations developing due to immunosuppression in solid organ or hematopoietic stem cell allograft patients. PTLD are characterized by abnormal proliferation of lymphoid cells and have a heterogeneous clinical behavior. We profiled expression of >700 tumor microenvironment (TME)-related genes in 75 post-transplant aggressive B-cell lymphomas (PTABCL). Epstein-Barr virus (EBV)-positive PT-ABCL clustered together and were enriched for type I interferon pathway and antiviral-response genes. Additionally, a cytotoxicity gene signature associated with EBV-positivity and favorable overall survival (OS) (hazard ratio =0.61; P=0.019). In silico immunophenotyping revealed two subgroups with distinct immune cell compositions. The inflamed subgroup with higher proportions of immune cells had better outcome compared to noninflamed subgroup (median OS >200.0 vs. 15.2 months; P=0.006). In multivariable analysis with EBV status, International Prognostic Index, and rituximab-containing treatment, inflamed TME remained as an independent predictor for favorable outcome. We also compared TME between post-transplant and immunocompetent host diffuse large B-cell lymphomas (n=75) and discovered that the proportions of T cells were lower in PT-diffuse large B-cell lymphomas. In conclusion, we provide a comprehensive phenotypic characterization of PT-ABCL, highlighting the importance of immune cell composition of TME in determining the clinical behavior and prognosis of PT-ABCL.

Repeatability of tumour hypoxia imaging using [18F]EF5 PET/CT in head and neck cancer.

PURPOSE: Hypoxia contributes to radiotherapy resistance and more aggressive behaviour of several types of cancer. This study was designed to evaluate the repeatability of intratumour uptake of the hypoxia tracer [18F]EF5 in paired PET/CT scans. METHODS: Ten patients with newly diagnosed head and neck cancer (HNC) received three static PET/CT scans before chemoradiotherapy: two with [18F]EF5 a median of 7 days apart and one with [18F]FDG. Metabolically active primary tumour volumes were defined in [18F]FDG images and transferred to co-registered [18F]EF5 images for repeatability analysis. A tumour-to-muscle uptake ratio (TMR) of 1.5 at 3 h from injection of [18F]EF5 was used as a threshold representing hypoxic tissue. RESULTS: In 10 paired [18F]EF5 PET/CT image sets, SUVmean, SUVmax, and TMR showed a good correlation with the intraclass correlation coefficients of 0.81, 0.85, and 0.87, respectively. The relative coefficients of repeatability for these parameters were 15%, 17%, and 10%, respectively. Fractional hypoxic volumes of the tumours in the repeated scans had a high correlation using the Spearman rank correlation test (r = 0.94). In a voxel-by-voxel TMR analysis between the repeated scans, the mean of Pearson correlation coefficients of individual patients was 0.65. The mean (± SD) difference of TMR in the pooled data set was 0.03 ± 0.20. CONCLUSION: Pretreatment [18F]EF5 PET/CT within one week shows high repeatability and is feasible for the guiding of hypoxia-targeted treatment interventions in HNC.

Somatostatin receptor expression in lymphomas: a source of false diagnosis of neuroendocrine tumor at 68Ga-DOTANOC PET/CT imaging.

BACKGROUND: 68Ga-DOTANOC PET/CT is routinely used to image neuroendocrine tumors (NETs). A case of lymphoma initially thought to be NET based on a positive 68Ga-DOTANOC PET/CT was recently seen at our institution. This prompted us to determine prospectively somatostatin receptor (SSTR) status in patients with lymphoma by immunohistochemical analysis of SSTR subtypes 2, 3 and 5 (SSTR2,3,5) and 68Ga-DOTANOC PET/CT imaging. MATERIAL AND METHODS: Twenty-one patients with newly diagnosed lymphoma were referred to 68Ga-DOTANOC and FDG PET/CT prior to any treatment. Tracer uptake was evaluated visually by two nuclear medicine specialists. Maximum standardized uptake values (SUVmax) were determined from 14 nodal and two extranodal regions with highest uptake in each patient. Lesions were then graded with Deauville score (1-5) on FDG PET/CT and modified Krenning score (0-4) on 68Ga-DOTANOC PET/CT, respectively. SSTR2,3,5 status was analyzed from routine biopsies of lymphomatous tissue and matched to corresponding PET/CT findings. RESULTS: About 20/21 patients had FDG-positive lymphoma (Deauville score ≥3). Uptake of 68Ga-DOTANOC was regarded as positive if Krenning score was ≥2 and resulted in 13/21 (62%) patients having 68Ga-DOTANOC-positive lymphomas. The highest uptake of 68Ga-DOTANOC was seen in Hodgkin's lymphoma of nodular sclerosis subtype and in diffuse large B-cell lymphoma (SUVmax median 9.8 and 9.7, respectively). Both cases showed strong SSTR2 immunopositivity in tumor cells. Some patients had SSTR2 immunopositivity predominantly in endothelial and dendritic cells and follicular centers of lymph nodes contributing to a positive PET/CT with probably low tumor-specific uptake. SSTR3 and SSTR5 were negative in most lymphoma subtypes. CONCLUSIONS: According to this pilot study, 68Ga-DOTANOC PET/CT is positive in some lymphoma subtypes which express SSTRs. These tumors present a potential risk of being misinterpreted as NETs if a representative tumor sample is not available. Lymphomas with high expression of SSTRs may be amenable to treatments targeting these receptors.

Epidemiological and treatment-related factors contribute to improved outcome of oropharyngeal squamous cell carcinoma in Finland.

BACKGROUND: Treatment for oropharyngeal squamous cell carcinoma (OPSCC) has changed, as the proportion of human papilloma virus (HPV)-related disease has increased. We evaluated nationwide information on its management and outcome during the treatment paradigm change period. METHODS: We included all patients diagnosed and treated for OPSCC at the five Finnish university hospitals from 2000 to 2009. Patient records and pathology registries provided the clinicopathological data. p16 staining was performed on primary tumor samples of patients who had received treatment with curative intent. RESULTS: A total of 674 patients were diagnosed and treated for OPSCC and the incidence increased along the study period. Of the evaluable tumors 58.5% were p16-positive and the number of p16-positive tumors increased along the years. The treatment was given with curative intent for 600 patients and it was completed in 564. Of them, 47.9% underwent primary surgery and 52.1% received definitive oncological treatment. Also, the treatment protocol changed towards a more oncological approach. Among patients treated with curative intent the five-year overall, disease-specific and disease-free survival rates were 60.1, 71.5 and 57.0%. In multivariate analysis, p16-positivity seemed to relate to reduced disease mortality in lateral and anterior-wall disease. Depending on primary tumor localization, also sex, classes T3-4, presence of regional metastasis and radiotherapy modality had an association with disease mortality. CONCLUSION: The incidence of p16-positive OPSCC and delivery of definitive oncological treatment increased in Finland during the study period. An improved survival outcome compared with the previous nationwide investigation was observed in this subset of patients.

Sudden Unexpected Death From Unusually Large Primary Cardiac B-cell Lymphoma.

Primary cardiac lymphomas represent approximately 1% to 2% of primary cardiac neoplasms and 5% of malignant cardiac neoplasms. Here we present a case of sudden unexpected death of a middle-aged male resulting from an unusually large cardiac B-cell lymphoma. The neoplasm infiltrated the myocardium of the right atrium and ventricle and, to a lesser extent, the wall of the left atrium and pulmonary trunk. Extensive infiltration of the heart by the primary cardiac lymphoma, combined with the complete lack of symptoms, makes this case unusual.

Type and location of tumor-infiltrating macrophages and lymphatic vessels predict survival of colorectal cancer patients.

The type of tumor-infiltrating macrophages may be decisive in tumor immunity, lymphangiogenesis and in the clinical outcome of cancer. Here, we elucidated the prognostic significance of lymphatic vessels, different types of macrophages and the balance between different macrophage types in colorectal cancer. We analyzed the impact of density, type and location of macrophages on the clinical behavior of 159 primary colorectal carcinomas using CD68 as a pan-macrophage marker and CLEVER-1/Stabilin-1 as a marker for regulatory/suppressive macrophages. Podoplanin was used as a pan-lymphatic vessel marker. A high number of CLEVER-1/Stabilin-1(+) peritumoral macrophages positively correlated with survival (p = 0.04). However, in more advanced disease (Stage IV), the patients with a high number of peritumoral or intratumoral CLEVER-1/Stabilin-1(+) macrophages had a shorter disease-specific survival (p = 0.05, and p = 0.008, respectively). Moreover, a low number of suppressive intratumoral CLEVER-1/Stabilin-1(+) macrophages among high numbers of CD68(+) macrophages correlated with a low number of distant recurrences (p = 0.01) and to fewer disease relapses exclusively in the liver as well (p = 0.006). A high number of intratumoral lymphatics correlated with poor survival (p = 0.03). The results of this work suggest that the type of macrophages, number of lymphatic vessels and their location contribute to the clinical behavior of colorectal cancer in a disease stage-specific manner.

Epidemiological Study of p16 Incidence in Head and Neck Squamous Cell Carcinoma 2005-2015 in a Representative Northern European Population.

The incidence of human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCC) has increased globally. Our research goal was to study HNSCC incidence in a representative Northern European population and evaluate the utility of the HPV surrogate marker p16 in clinical decision-making. All new HNSCC patients diagnosed and treated in Southwest Finland from 2005-2015 (n = 1033) were identified and analyzed. During the follow-up period, the incidence of oropharyngeal (OPSCC) and oral cavity squamous cell carcinoma (OSCC) increased, while the incidence of laryngeal squamous cell carcinoma (LSCC) decreased. This clinical cohort was used to generate a population-validated tissue microarray (PV-TMA) archive for p16 analyses. The incidence of p16 positivity in HNSCC and OPSCC increased in southwest Finland between 2005 and 2015. p16 positivity was mainly found in the oropharynx and was a significant factor for improved survival. p16-positive OPSCC patients had a better prognosis, regardless of treatment modality. All HNSCC patients benefited from a combination of chemotherapy and radiotherapy, regardless of p16 expression. Our study reaffirms that p16 expression offers a prognostic biomarker in OPSCC and could potentially be used in cancer treatment stratification. Focusing on p16 testing for only OPSCC might be the most cost-effective approach in clinical practice.

Structure-Activity Relationship of 3-Methylcytidine-5'-α,ß-methylenediphosphates as CD73 Inhibitors.

We recently reported N4-substituted 3-methylcytidine-5'-α,ß-methylenediphosphates as CD73 inhibitors, potentially useful in cancer immunotherapy. We now expand the structure-activity relationship of pyrimidine nucleotides as human CD73 inhibitors. 4-Chloro (MRS4598 16; Ki = 0.673 nM) and 4-iodo (MRS4620 18; Ki = 0.436 nM) substitution of the N4-benzyloxy group decreased Ki by ∼20-fold. Primary alkylamine derivatives coupled through a p-amido group with a varying methylene chain length (24 and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. X-ray structures of hCD73 with two inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between the C-terminal and N-terminal domains) as N4-benzyl groups in adenine inhibitors. Molecular dynamics identified stabilizing interactions and predicted conformational diversity. Thus, by N4-benzyloxy substitution, we have greatly enhanced the inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in tumor tissues in situ.

Somatostatin Receptors and Chemokine Receptor CXCR4 in Lymphomas: A Histopathological Review of Six Lymphoma Subtypes.

BACKGROUND: Somatostatin receptors (SSTR) and chemokine receptor CXCR4 are expressed in lymphomas, while the abundance is known to be heterogeneous in different subtypes of lymphomas. Targeting tumor cells expressing these receptors might add to therapeutic opportunities while radiolabeled ligands for both imaging and therapy have been developed. The aim of this study was to establish SSTR subtype 2, 3 and 5 and also CXCR4 status immunohistochemically in six different lymphoma subtypes: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), mucosa-associated marginal B-cell lymphoma (MALT), Hodgkin lymphoma (HL) and peripheral T-cell lymphoma (PTCL). MATERIAL AND METHODS: This study included a total of 103 lymphoma patients (24 DLBCL, 22 FL, 18 HL, 9 MALT, 20 MCL and 10 PTCL) diagnosed in the Southwest hospital district of Finland during 2010-2019. SSTR 2, 3 and 5 and CXCR4 expression was analyzed immunohistochemically (IHC) in lymphoma samples obtained from local archival Biobank tissue repository. Immunopositivity of each receptor was scored on a four-point scale accounting for staining intensity and proportion of positively stained tumor cells. RESULTS: Of different SSTR subtypes SSTR2 immunopositivity was most common and seen predominantly at the cell membrane of the malignant cells in 46-56% of DLBCL, HL and FL. CXCR4 co-expression was frequently present in these cases. SSTR3 and SSTR5 IHC were negative in DLBCL and FL but in HL SSTR expression was more heterogenous and SSTR3 and SSTR5 positivity was found in cytoplasm in 35% and 25% of cases. 2/4 blastoid MCL variants and one pleomorphic MCL variant had positive CXCR4 IHC whilst all other MCL cases (85%) were negative for all receptors. 30% (n=3) of the PTCL patients had positive SSTR5 IHC and CXCR4. MALT lymphomas were negative for all receptors. CONCLUSION: SSTR2 and CXCR4 are found in DLBCL, FL and HL and co-expression of these receptors is common. Although in general expression of SSTRs and CXCR4 is heterogenous and very low in some subtypes such as MCL and MALT there are also patients with abundant expression. The latter are candidates for trials studying SSTR2 and/or CXCR4 based treatments in the future.

Recurrence of head and neck squamous cell carcinoma in relation to high-risk treatment volume.

BACKGROUND: Locoregional recurrence remains a major cause of failure in head and neck squamous cell carcinoma (HNSCC). Human papilloma virus (HPV)-associated HNSCCs generally have a good prognosis but may recur even after standard photon radiotherapy (RT). Another incentive in observing patterns of recurrence is increased use of highly conformal techniques such as proton therapy. We therefore studied geographic distribution of recurrent tumors in relation to the high-risk treatment volume in a cohort of patients with HNSCC receiving combined modality therapy. METHODS: Medical records of 508 patients diagnosed with HNSCC in 2010-2015 were reviewed. We identified a subgroup that had local and/or regional recurrence at hybrid positron emission tomography (PET)/computed tomography (CT) and/or magnetic resonance imaging (MRI). We adapted p16 as a surrogate marker for HPV-positivity and only patients with known p16 status were eligible for a detailed analysis where recurrent tumor was copied on the planning CT and the dose received by the recurrent tumor volume was determined using dose-volume histograms. RESULTS: Twenty-five patients who had received either cisplatin (n = 23) or cetuximab-enhanced (n = 2) RT were identified. 31 locoregional recurrent tumors were detected among 18 p16 negative and 7 p16 positive patients. Of recurrent tumors 14 (45%) were classified as in-field, 5 (16%) as marginal miss, and 12 (39%) as true miss. p16 positive patients had 4 in-field, 2 marginal, and 1 true miss. By contrast, p16 negative patients had 10 in-field, 3 marginal, and 11 true miss recurrences. CONCLUSIONS: Both p16 positive and negative HNSCC recur in high-risk treatment volume despite the common view of high radiosensitivity of the former. Biomarkers predicting radioresistance should be characterized in p16 positive tumors before widely embarking on de-escalated CRT protocols. Another concern is how to decrease the number of true or marginal misses in p16 negative cases despite multimodality imaging-based target delineation.

Cdk5 regulates the organization of Nestin and its association with p35.

The intermediate filament protein nestin is characterized by its specific expression during the development of neuronal and myogenic tissues. We identify nestin as a novel in vivo target for cdk5 and p35 kinase, a critical signaling determinant in development. Two cdk5-specific phosphorylation sites on nestin, Thr-1495 and Thr-316, were established, the latter of which was used as a marker for cdk5-specific phosphorylation in vivo. Ectopic expression of cdk5 and p35 in central nervous system progenitor cells and in myogenic precursor cells induced elevated phosphorylation and reorganization of nestin. The kinetics of nestin expression corresponded to elevated expression and activation of cdk5 during differentiation of myoblast cell cultures and during regeneration of skeletal muscle. In the myoblasts, a disassembly-linked phosphorylation of Thr-316 indicated active phosphorylation of nestin by cdk5. Moreover, cdk5 occurred in physical association with nestin. Inhibition of cdk5 activity-either by transfection with dominant-negative cdk5 or by using a specific cdk5 inhibitor-blocked myoblast differentiation and phosphorylation of nestin at Thr-316, and this inhibition markedly disturbed the organization of nestin. Interestingly, the interaction between p35, the cdk5 activator, and nestin appeared to be regulated by cdk5. In differentiating myoblasts, p35 was not complexed with nestin phosphorylated at Thr-316, and inhibition of cdk5 activity during differentiation induced a marked association of p35 with nestin. These results demonstrate that there is a continuous turnover of cdk5 and p35 activity on a scaffold formed by nestin. This association is likely to affect the organization and operation of both cdk5 and nestin during development.

Muscle injuries: optimising recovery.

Muscle injuries are one of the most common traumas occurring in sports. Despite their clinical importance, there are only a few clinical studies on the treatment of muscle injuries. Lack of clinical studies is most probably attributable to the fact that there is not only a high heterogeneity in the severity of injuries, but also the injuries take place in different muscles, making it very demanding to carry out clinical trials. Accordingly, the current treatment principles of muscle injuries have either been derived from experimental studies or been tested empirically only. Clinically, first aid for muscle injuries follows the RICE (Rest, Ice, Compression and Elevation) principle. The objective of RICE is to stop the injury-induced bleeding into the muscle tissue and thereby minimise the extent of the injury. Clinical examination should be carried out immediately after the injury and 5-7 days after the initial trauma, at which point the severity of the injury can be assessed more reliably. At that time, a more detailed characterisation of the injury can be made using imaging diagnostic modalities (ultrasound or MRI) if desired. The treatment of injured skeletal muscle should be carried out by immediate immobilisation of the injured muscle (clinically, relative immobility/avoidance of muscle contractions). However, the duration of immobilisation should be limited to a period sufficient to produce a scar of sufficient strength to bear the forces induced by remobilisation without re-rupture and the return to activity (mobilisation) should then be started gradually within the limits of pain. Early return to activity is needed to optimise the regeneration of healing muscle and recovery of the flexibility and strength of the injured skeletal muscle to pre-injury levels. The rehabilitation programme should be built around progressive agility and trunk stabilisation exercises, as these exercises seem to yield better outcome for injured skeletal muscle than programmes based exclusively on stretching and strengthening of the injured muscle.

Tumoral Expression of CD44 and HIF1α Predict Stage I Oral Cavity Squamous Cell Carcinoma Outcome.

OBJECTIVES/HYPOTHESIS: No biomarkers are used to estimate the prognosis in oral cavity squamous cell carcinoma (OSCC). In our previously published work, we have reported the prognostic value of CD44 and hypoxia inducible factor (HIF)-1α in patients with stage I disease. STUDY DESIGN: In this study, we tested our previous observations in a larger cohort. We also studied the predictive value of common lymphatic endothelial and vascular endothelial receptor (CLEVER)-1 in this material. METHODS: CD44, HIF1α, and CLEVER-1 were immunohistochemically analyzed in paraffin-embedded tissue material of stage I OSCC patients treated at three Finnish university hospitals. Microscopy results were correlated with OSCC outcome. RESULTS: As in our pilot study, the CD44lowHIF1αhigh signature was associated with poorer disease-free survival. Clear correlations between CLEVER-1 expression and clinical outcome were not evident. CONCLUSION: Our results suggest that immunohistochemistry of CD44 and HIF1α may be useful in identification of patients with poor prognoses. These parameters could be used to select the optimal treatment modalities for stage I OSCC patients. LEVEL OF EVIDENCE: 2b.

Transected myofibres may remain permanently divided in two parts.

During regeneration of transected myofibres a scar is formed between their stumps. Myofibres restore their tendon-muscle-tendon continuity and contractile function by attaching to the scar with new myotendinous junctions. The scar contracts with time, and thereby the stumps are pulled close to each other. During early regeneration, myoblasts and myotubes can fuse with the surviving parts of the transected myofibres. However, it is not known whether it is possible that the opposite stumps could eventually fuse to reunite the divided parts of the transected fibres. In this study, we show in rat that even after 12 months the stumps remain attached to the separating scar by myotendinous junctions without showing definite fusion of the stumps. We conclude that transected myofibres probably remain permanently divided in two consecutive tendon-muscle-tendon units.

Restoration of myofiber continuity after transection injury in the rat soleus.

In a shearing type of muscle injury, scar formation prevents restoration of myofiber continuity and the transected myofibers may become permanently divided into two separate myofibers. We have analysed whether the injured myofiber stumps can fuse and continuity of the transected fibers be re-established, if the stumps are surgically closely apposed immediately after injury. 55 rat soleus muscles were transected, after which the epimysium was carefully sutured and the leg was immobilised for seven days. The animals were sacrificed at 2, 5, 7, 10, 14 and 25 days after surgery. All muscles were analysed by light and electron microscopy as well as by immunohistochemistry. Mechanical strength was also measured at day 10 and 25. We observed that suturing reduced the extent of the intervening scar and accelerated healing. More importantly our results indicate that fusion of the stumps and thus restoration of myofiber continuity, is possible after myofiber transection injury.

Prognostic markers in stage I oral cavity squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: Early-stage oral squamous cell carcinoma (OSCC) treatment is based on anatomic location, clinical TNM staging, and histological grade. It is a heterogeneous disease group. Classification of patients with OSCC by immunohistochemical analysis of established oncoproteins and evaluate disease course was our primary objective. Characterization of stage I OSCC patients in Southwest Finland was our secondary objective. STUDY DESIGN: Immunohistochemical analysis of tumor specimens and retrospective analysis of patient data of the patient treated in Turku University Hospital for T1N0M0 OSCC during the years 2000-2004. METHODS: Paraffin-embedded tumor specimens from 35 OSCC patients were collected and analyzed for HIF-1α, CD44, p16, Ki67, and podoplanin by immunohistochemistry and correlated with clinical findings. RESULTS: Tumoral CD44 and HIF1-α expression levels, in combination, predicted 5-year disease-free survival. Reduced expression of CD44 and elevated expression of HIF1-α is associated with the lowest probability of disease-free survival compared to the population as a whole (P < .001 in Kaplan-Meier analysis). Patients with grade I tumors demonstrated improved disease-specific survival compared to those with grade II tumors (P = .027). No association was seen between p16 expression, Ki67 labeling index, or podoplanin expression and prognosis in our 35 specimens. CONCLUSIONS: HIF-1α and CD44 immunohistochemical detection could potentially serve as a prognostic tool in therapy selection for early-stage OSCC. LEVEL OF EVIDENCE: 2b.

Proximal hamstring tendinopathy: results of surgical management and histopathologic findings.

BACKGROUND: Tendon disorders are common problems in sports and are known to be difficult to treat. Only limited information is available concerning treatment of proximal hamstring tendinopathy. To the authors' knowledge, no histopathologic findings of proximal hamstring tendinosis have been published. HYPOTHESIS: Surgery (semimembranosus tenotomy and exploration of the sciatic nerve) is an effective treatment for proximal hamstring tendinopathy. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: A total of 103 cases of proximal hamstring tendinopathy in athletes (58 men, 32 women; 13 bilateral operations) with surgical treatment were included. The cases were retrospectively analyzed, and a 4-category rating system was used to evaluate the overall result. At the follow-up, the patients were asked about possible symptoms and their return to sports. Biopsy samples from 15 of the operated tendons were taken and analyzed by a pathologist. RESULTS: The average follow-up was 49 months (range, 12-156 months). The result was evaluated to be excellent in 62 cases, good in 30, fair in 5, and poor in 6. After surgery, 80 of the 90 patients were able to return to the same level of sporting activity as before the onset of the symptoms. This took a mean of 5 months (range, 2-12 months). Typical morphologic findings of tendinosis were found in all biopsy specimens. CONCLUSION: Given the good functional outcome and low complication rate, the authors present surgical treatment as a valuable option in proximal hamstring tendinopathy if conservative treatment fails.